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2022
Hess-Rieger, J. ; Unger, K. ; Maihoefer, C. ; Schuettrumpf, L. ; Weber, P. ; Marschner, S. ; Wintergerst, L. ; Pflugradt, U. ; Baumeister, P. ; Walch, A.K. ; Woischke, C. ; Kirchner, T. ; Werner, M. ; Soerensen, K. ; Baumann, M. ; Tinhofer, I. ; Combs, S.E. ; Debus, J. ; Schaefer, H. ; Krause, M. ; Linge, A. ; von der Gruen, J. ; Stuschke, M. ; Zips, D. ; Canis, M. ; Lauber, K. ; Ganswindt, U. ; Henke, M. ; Zitzelsberger, H. ; Belka, C.
Cancers 14:3745 (2022)
Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD-L1 expression/PD-1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.
Wissenschaftlicher Artikel
Scientific Article
Kreutzer, L. ; Weber, P. ; Heider, T. ; Heikenwaelder, M. ; Riedl, T. ; Baumeister, P. ; Klauschen, F. ; Belka, C. ; Walch, A.K. ; Zitzelsberger, H. ; Hess-Rieger, J. ; Unger, K.
Lab. Invest., DOI: 10.1038/s41374-022-00829-0 (2022)
Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows spatial analysis of proteins, metabolites, or small molecules from tissue sections. Here, we present the simultaneous generation and analysis of MALDI-MSI, whole-exome sequencing (WES), and RNA-sequencing data from the same formalin-fixed paraffin-embedded (FFPE) tissue sections. Genomic DNA and total RNA were extracted from (i) untreated, (ii) hematoxylin-eosin (HE) stained, and (iii) MALDI-MSI-analyzed FFPE tissue sections from three head and neck squamous cell carcinomas. MALDI-MSI data were generated by a time-of-flight analyzer prior to preprocessing and visualization. WES data were generated using a low-input protocol followed by detection of single-nucleotide variants (SNVs), tumor mutational burden, and mutational signatures. The transcriptome was determined using 3’-RNA sequencing and was examined for similarities and differences between processing stages. All data met the commonly accepted quality criteria. Besides SNVs commonly identified between differently processed tissues, FFPE-typical artifactual variants were detected. Tumor mutational burden was in the same range for tissues from the same patient and mutational signatures were highly overlapping. Transcriptome profiles showed high levels of correlation. Our data demonstrate that simultaneous molecular profiling of MALDI-MSI-processed FFPE tissue sections at the transcriptome and exome levels is feasible and reliable.
Wissenschaftlicher Artikel
Scientific Article
Hui, B. ; Lu, C. ; Li, H. ; Hao, X. ; Liu, H. ; Zhuo, D. ; Wang, Q. ; Li, Z. ; Liu, L. ; Wang, X. ; Gu, Y. ; Tang, W.
Int. J. Biol. Sci. 18, 5230-5240 (2022)
Checkpoint immunotherapy is capable of unleashing T cells for controlling tumor, whereas it is destroyed by immunosuppressive myeloid cell. Apoprotein E (APOE) refers to a ligand in terms of the members of low-density lipoprotein (LDL) receptor family for mediating Apoprotein B-involving atherogenic lipoprotein clearance. Besides, tumor-infiltration macrophage can express APOE. The present study reported Apoe-/- mice to exhibit higher resistance toward the development of three types of carcinomas as compared with mice with wild type and to have greater responses to αPD-1 (anti-PD-1) immunotherapy. Moreover, treatment by exploiting APOE inhibitor (COG 133TFA, αAPOE) was capable of curbing tumor development and fostering regression if in combination of αPD-1. According to single-cell RNA sequencing (scRNA-seq), Apoe deletion was correlated with the decline of C1QC+ and CCR2+ macrophage within tumor infiltration, and mass spectrometry results noticeably showed down-regulated the number of M2 macrophages as well. Furthermore, APOE expression in cancer patients resistant to αPD-1 treatment significantly exceeded that in the sensitive group. For this reason, APOE is likely to be targeted for modifying tumor macrophage infiltrate and augmenting checkpoint immunotherapy.
Wissenschaftlicher Artikel
Scientific Article
Erlmeier, F. ; Sun, N. ; Shen, J. ; Feuchtinger, A. ; Buck, A. ; Prade, V.M. ; Kunzke, T. ; Schraml, P. ; Moch, H. ; Autenrieth, M. ; Weichert, W. ; Hartmann, A. ; Walch, A.K.
Cancers 14:1763 (2022)
High mass resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a suitable method for biomarker detection for several tumor entities. Renal cell carcinoma (RCC) is the seventh most common cancer type and accounts for more than 80% of all renal tumors. Prognostic biomarkers for RCC are still missing. Therefore, we analyzed a large, multicenter cohort including the three most common RCC subtypes (clear cell RCC (ccRCC), papillary RCC (pRCC) and chromophobe RCC (chRCC)) by MALDI for prognostic biomarker detection. MALDI-Fourier-transform ion cyclotron resonance (FT-ICR)-MSI analysis was performed for renal carcinoma tissue sections from 782 patients. SPACiAL pipeline was integrated for automated co-registration of histological and molecular features. Kaplan–Meier analyses with overall survival as endpoint were executed to determine the metabolic features associated with clinical outcome. We detected several pathways and metabolites with prognostic power for RCC in general and also for different RCC subtypes.
Wissenschaftlicher Artikel
Scientific Article
Wang, Q. ; Sun, N. ; Kunzke, T. ; Buck, A. ; Shen, J. ; Prade, V.M. ; Stöckl, B. ; Wang, J. ; Feuchtinger, A. ; Walch, A.K.
Histochem. Cell Biol. 157, 595–605 (2022)
Matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) imaging mass spectrometry (MS) is a powerful technology used to analyze metabolites in various tissues. However, it faces significant challenges in studying adipose tissues. Poor matrix distribution and crystallization caused by excess liquid lipids on the surface of tissue sections hamper m/z species detection, an adverse effect that particularly presents in lipid-rich white adipose tissue (WAT). In this study, we integrated a simple and low-cost preparation step into the existing MALDI-FTICR imaging MS pipeline. The new method—referred to as filter paper application—is characterized by an easy sample handling and high reproducibility. The aforementioned filter paper is placed onto the tissue prior to matrix application in order to remove the layer of excess liquid lipids. Consequently, MALDI-FTICR imaging MS detection was significantly improved, resulting in a higher number of detected m/z species and higher ion intensities. After analyzing various durations of filter paper application, 30 s was found to be optimal, resulting in the detection of more than 3700 m/z species. Apart from the most common lipids found in WAT, other molecules involved in various metabolic pathways were detected, including nucleotides, carbohydrates, and amino acids. Our study is the first to propose a solution to a specific limitation of MALDI-FTICR imaging MS in investigating lipid-rich WAT. The filter paper approach can be performed quickly and is particularly effective for achieving uniform matrix distribution on fresh frozen WAT while maintaining tissue integrity. It thus helps to gain insight into the metabolism in WAT.
Wissenschaftlicher Artikel
Scientific Article
Ebert, K. ; Haffner, I. ; Zwingenberger, G. ; Keller, S. ; Raimúndez, E. ; Geffers, R. ; Wirtz, R. ; Barbaria, E. ; Hollerieth, V. ; Arnold, R. ; Walch, A.K. ; Hasenauer, J. ; Maier, D. ; Lordick, F. ; Luber, B.
BMC Cancer 22:254 (2022)
BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.
Wissenschaftlicher Artikel
Scientific Article
Ravi, V.M. ; Will, P. ; Kueckelhaus, J. ; Sun, N. ; Joseph, K. ; Salié, H. ; Vollmer, L. ; Kuliesiute, U. ; von Ehr, J. ; Benotmane, J.K. ; Neidert, N. ; Follo, M. ; Scherer, F. ; Goeldner, J.M. ; Behringer, S.P. ; Franco, P. ; Khiat, M. ; Zhang, J. ; Hofmann, U.G. ; Fung, C. ; Ricklefs, F.L. ; Lamszus, K. ; Boerries, M. ; Ku, M.C. ; Beck, J. ; Sankowski, R. ; Schwabenland, M. ; Prinz, M. ; Schüller, U. ; Killmer, S. ; Bengsch, B. ; Walch, A.K. ; Delev, D. ; Schnell, O. ; Heiland, D.H.
Cancer Cell 40, 639-655.e13 (2022)
Glioblastomas are malignant tumors of the central nervous system hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. The source of dynamic reorganization within the spatial context of these tumors remains elusive. Here, we characterized glioblastomas by spatially resolved transcriptomics, metabolomics, and proteomics. By deciphering regionally shared transcriptional programs across patients, we infer that glioblastoma is organized by spatial segregation of lineage states and adapts to inflammatory and/or metabolic stimuli, reminiscent of the reactive transformation in mature astrocytes. Integration of metabolic imaging and imaging mass cytometry uncovered locoregional tumor-host interdependence, resulting in spatially exclusive adaptive transcriptional programs. Inferring copy-number alterations emphasizes a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. A model of glioblastoma stem cells implanted into human and rodent neocortical tissue mimicking various environments confirmed that transcriptional states originate from dynamic adaptation to various environments.
Wissenschaftlicher Artikel
Scientific Article
Tang, W. ; Zhou, Y. ; Zhao, H. ; Sun, G. ; Rong, D. ; Li, Z. ; Hu, M. ; Han, L.F. ; He, X. ; Zhao, S. ; Chen, X. ; Yuan, H. ; Chen, S. ; Wang, Q. ; Gu, J. ; Wang, X. ; Song, J.
Ann. Transl. Med. 10:625 (2022)
Background: Anesthesia, nerve block, therapeutic injections, and biopsies all require an acupuncture intervention. However, traditional two-dimensional (2D) ultrasound-guided needle puncture is often challenging and therefore requires the use of three-dimensional (3D) ultrasound images to accurately identify and evaluate the patient’s anatomical structure. Methods: In this study, a 3D multi-modal intelligent intervention system using electromagnetic navigation for real-time positioning and ultrasound images was described. A total of 190 cases requiring puncture were randomly divided into control (conventional 2D ultrasound instrument) and experimental (novel 3D ultrasound imedis9000) groups. The advantages and disadvantages of the two puncture methods were prospectively analyzed in the 190 cases, and the feasibility of electromagnetic navigation real-time positioning was compared to ultrasound imaging. Results: This study included 190 cases from two centers that required puncture treatment and were randomly assigned to the control (conventional 2D ultrasound instrument; n=95) or the experimental (novel 3D ultrasound imedis9000; n=95) groups. Percutaneous vascular puncture, percutaneous biopsy, percutaneous bile duct puncture, thoracic paravertebral nerve block, and sciatic nerve block operations were performed separately. The results indicated that the puncture time and number of trials in the experimental group were significantly lower than those in the control group. No significant difference was identified in the basic vital signs between the two groups before and after surgery. The success rate of the novel 3D ultrasound imedis9000 was 100%, and the success rate of the conventional 2D ultrasound instrument was 95.7%. Furthermore, the results also showed that the novel 3D ultrasound imedis9000 and the matching coaxial positioning channel puncture needle had low pain, good toughness and strength, and great convenience. Conclusions: The new 3D multi-modal intelligent intervention system using electromagnetic navigation real-time positioning and ultrasound images has significant advantages compared with conventional 2D ultrasound in terms of puncture time, number of trials, operation difficulty, and convenience, and is worthy of further promotion and use in clinics. Trial Registration: Beijing Municipal Drug Administration, 20190015.
Wissenschaftlicher Artikel
Scientific Article
Shen, J. ; Sun, N. ; Zens, P. ; Kunzke, T. ; Buck, A. ; Prade, V.M. ; Wang, J. ; Wang, Q. ; Hu, R. ; Feuchtinger, A. ; Berezowska, S. ; Walch, A.K.
Cancer Comm. 42, 517-535 (2022)
BACKGROUND: The response to neoadjuvant chemotherapy (NAC) differs substantially among individual patients with non-small cell lung cancer (NSCLC). Major pathological response (MPR) is a histomorphological read-out used to assess treatment response and prognosis in patients NSCLC after NAC. Although spatial metabolomics is a promising tool for evaluating metabolic phenotypes, it has not yet been utilized to assess therapy responses in patients with NSCLC. We evaluated the potential application of spatial metabolomics in cancer tissues to assess the response to NAC, using a metabolic classifier that utilizes mass spectrometry imaging combined with machine learning. METHODS: Resected NSCLC tissue specimens obtained after NAC (n = 88) were subjected to high-resolution mass spectrometry, and these data were used to develop an approach for assessing the response to NAC in patients with NSCLC. The specificities of the generated tumor cell and stroma classifiers were validated by applying this approach to a cohort of biologically matched chemotherapy-naïve patients with NSCLC (n = 85). RESULTS: The developed tumor cell metabolic classifier stratified patients into different prognostic groups with 81.6% accuracy, whereas the stroma metabolic classifier displayed 78.4% accuracy. By contrast, the accuracies of MPR and TNM staging for stratification were 62.5% and 54.1%, respectively. The combination of metabolic and MPR classifiers showed slightly lower accuracy than either individual metabolic classifier. In multivariate analysis, metabolic classifiers were the only independent prognostic factors identified (tumor: P = 0.001, hazards ratio [HR] = 3.823, 95% confidence interval [CI] = 1.716-8.514; stroma: P = 0.049, HR = 2.180, 95% CI = 1.004-4.737), whereas MPR (P = 0.804; HR = 0.913; 95% CI = 0.445-1.874) and TNM staging (P = 0.078; HR = 1.223; 95% CI = 0.977-1.550) were not independent prognostic factors. Using Kaplan-Meier survival analyses, both tumor and stroma metabolic classifiers were able to further stratify patients as NAC responders (P < 0.001) and non-responders (P < 0.001). CONCLUSIONS: Our findings indicate that the metabolic constitutions of both tumor cells and the stroma are valuable additions to the classical histomorphology-based assessment of tumor response.
Wissenschaftlicher Artikel
Scientific Article
Prade, V.M. ; Sun, N. ; Shen, J. ; Feuchtinger, A. ; Kunzke, T. ; Buck, A. ; Schraml, P. ; Moch, H. ; Schwamborn, K. ; Autenrieth, M. ; Gschwend, J.E. ; Erlmeier, F. ; Hartmann, A. ; Walch, A.K.
Clin. Transl. Med. 12:e666 (2022)
Letter to the Editor
Letter to the Editor
Paul, T. ; Ledderose, S. ; Bartsch, H. ; Sun, N. ; Soliman, S. ; Märkl, B. ; Ruf, V. ; Herms, J. ; Stern, M. ; Keppler, O.T. ; Delbridge, C. ; Müller, S. ; Piontek, G. ; Kimoto, Y.S. ; Schreiber, F. ; Williams, T.A. ; Neumann, J. ; Knösel, T. ; Schulz, H. ; Spallek, R. ; Graw, M. ; Kirchner, T. ; Walch, A.K. ; Rudelius, M.
Nat. Commun. 13:1589 (2022)
Progressive respiratory failure and hyperinflammatory response is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. Despite mounting evidence of disruption of the hypothalamus-pituitary-adrenal axis in COVID-19, relatively little is known about the tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to adrenal glands and associated changes. Here we demonstrate adrenal viral tropism and replication in COVID-19 patients. Adrenal glands showed inflammation accompanied by inflammatory cell death. Histopathologic analysis revealed widespread microthrombosis and severe adrenal injury. In addition, activation of the glycerophospholipid metabolism and reduction of cortisone intensities were characteristic for COVID-19 specimens. In conclusion, our autopsy series suggests that SARS-CoV-2 facilitates the induction of adrenalitis. Given the central role of adrenal glands in immunoregulation and taking into account the significant adrenal injury observed, monitoring of developing adrenal insufficiency might be essential in acute SARS-CoV-2 infection and during recovery.
Wissenschaftlicher Artikel
Scientific Article
Loft, A. ; Schmidt, S.F. ; Caratti, G. ; Stifel, U. ; Havelund, J.F. ; Sekar, R. ; Kwon, Y. ; Sulaj, A. ; Chow, K.K. ; Alfaro, A.J. ; Schwarzmayr, T. ; Rittig, N. ; Svart, M. ; Tsokanos, F.-F. ; Maida, A. ; Blutke, A. ; Feuchtinger, A. ; Møller, N. ; Blüher, M. ; Nawroth, P. ; Szendrödi, J. ; Færgeman, N.J. ; Zeigerer, A. ; Tuckermann, J. ; Herzig, S.
Cell Metab. 34, 473-486.e9 (2022)
Fasting metabolism and immunity are tightly linked; however, it is largely unknown how immune cells contribute to metabolic homeostasis during fasting in healthy subjects. Here, we combined cell-type-resolved genomics and computational approaches to map crosstalk between hepatocytes and liver macrophages during fasting. We identified the glucocorticoid receptor (GR) as a key driver of fasting-induced reprogramming of the macrophage secretome including fasting-suppressed cytokines and showed that lack of macrophage GR impaired induction of ketogenesis during fasting as well as endotoxemia. Mechanistically, macrophage GR suppressed the expression of tumor necrosis factor (TNF) and promoted nuclear translocation of hepatocyte GR to activate a fat oxidation/ketogenesis-related gene program, cooperatively induced by GR and peroxisome proliferator-activated receptor alpha (PPARα) in hepatocytes. Together, our results demonstrate how resident liver macrophages directly influence ketogenesis in hepatocytes, thereby also outlining a strategy by which the immune system can set the metabolic tone during inflammatory disease and infection.
Wissenschaftlicher Artikel
Scientific Article
Wang, J. ; Kunzke, T. ; Prade, V.M. ; Shen, J. ; Buck, A. ; Feuchtinger, A. ; Haffner, I. ; Luber, B. ; Liu, D.H.W. ; Langer, R. ; Lordick, F. ; Sun, N. ; Walch, A.K.
Clin. Cancer Res. 28, 2865-2877 (2022)
PURPOSE: Current systems of gastric cancer (GC) molecular classification include genomic, molecular, and morphological features. GC classification based on tissue metabolomics remains lacking. This study aimed to define metabolically distinct GC subtypes and identify their clinicopathological and molecular characteristics. EXPERIMENTAL DESIGN: Spatial metabolomics by high mass resolution imaging mass spectrometry was performed in 362 GC patients. K-means clustering was used to define tumor and stroma-related subtypes based on tissue metabolites. The identified subtypes were linked with clinicopathological characteristics, molecular features, and metabolic signatures. Responses to trastuzumab treatment were investigated across the subtypes by introducing an independent patient cohort with HER2-positive GC from a multicenter observational study. RESULTS: Three tumor- and three stroma-specific subtypes with distinct tissue metabolite patterns were identified. Tumor-specific subtype T1(HER2+MIB+CD3+) positively correlated with HER2, MIB1, DEFA-1, CD3, CD8, FOXP3, but negatively correlated with MMR. Tumor-specific subtype T2(HER2-MIB-CD3-) negatively correlated with HER2, MIB1, CD3, FOXP3, but positively correlated with MMR. Tumor-specific subtype T3(pEGFR+) positively correlated with pEGFR. Patients with tumor subtype T1(HER2+MIB+CD3+) had elevated nucleotide levels, enhanced DNA metabolism, and a better prognosis than T2(HER2-MIB-CD3-) and T3(pEGFR+). An independent validation cohort confirmed that the T1 subtype benefited from trastuzumab therapy. Stroma-specific subtypes had no association with clinicopathological characteristics, however linked to distinct metabolic pathways and molecular features. CONCLUSIONS: Patient subtypes derived by tissue-based spatial metabolomics are a valuable addition to existing GC molecular classification systems. Metabolic differences between the subtypes and their associations with molecular features could provide a valuable tool to aid in selecting specific treatment approaches.
Wissenschaftlicher Artikel
Scientific Article
Sato, S. ; Dyar, K.A. ; Treebak, J.T. ; Jepsen, S.L. ; Ehrlich, A.M. ; Ashcroft, S.P. ; Trost, K. ; Kunzke, T. ; Prade, V.M. ; Small, L. ; Basse, A.L. ; Schönke, M. ; Chen, S. ; Samad, M. ; Baldi, P. ; Barrès, R. ; Walch, A.K. ; Moritz, T. ; Holst, J.J. ; Lutter, D. ; Zierath, J.R. ; Sassone-Corsi, P.
Cell Metab. 34, 329-345.e8 (2022)
Tissue sensitivity and response to exercise vary according to the time of day and alignment of circadian clocks, but the optimal exercise time to elicit a desired metabolic outcome is not fully defined. To understand how tissues independently and collectively respond to timed exercise, we applied a systems biology approach. We mapped and compared global metabolite responses of seven different mouse tissues and serum after an acute exercise bout performed at different times of the day. Comparative analyses of intra- and inter-tissue metabolite dynamics, including temporal profiling and blood sampling across liver and hindlimb muscles, uncovered an unbiased view of local and systemic metabolic responses to exercise unique to time of day. This comprehensive atlas of exercise metabolism provides clarity and physiological context regarding the production and distribution of canonical and novel time-dependent exerkine metabolites, such as 2-hydroxybutyrate (2-HB), and reveals insight into the health-promoting benefits of exercise on metabolism.
Wissenschaftlicher Artikel
Scientific Article
Weber, P. ; Künstner, A. ; Hess-Rieger, J. ; Unger, K. ; Marschner, S. ; Idel, C. ; Ribbat-Idel, J. ; Walz, C. ; Rietzler, S. ; Valeanu, L. ; Herkommer, T. ; Kreutzer, L. ; Klymenko, O. ; Kirchner, T. ; Ganswindt, U. ; Walch, A.K. ; Sterr, M. ; Lickert, H. ; Canis, M. ; Rades, D. ; Perner, S. ; Berriel Diaz, M. ; Herzig, S. ; Wollenberg, B. ; Busch, H. ; Zitzelsberger, H.
Clin. Cancer Res. 28, 1038-1052 (2022)
PURPOSE: The genetic relatedness between primary and recurrent head and neck squamous cell carcinomas (HNSCC) reflects the extent of heterogeneity and therapy-driven selection of tumor subpopulations. Yet, current treatment of recurrent HNSCC ignores the molecular characteristics of therapy-resistant tumor populations. EXPERIMENTAL DESIGN: From 150 tumors, 74 primary HNSCCs were RNA-sequenced and 38 matched primary/recurrent tumor pairs were both, whole-exome and RNA-sequenced. Transcriptome analysis determined the predominant classical (CL), basal (BA) and inflamed-mesenchymal (IMS) transcriptional subtypes according to an established classification. Genomic alterations and clonal compositions of tumors were evaluated from whole-exome data. RESULTS: While CL and IMS subtypes were more common in primary HNSCC with low recurrence rates, the BA subtype was more prevalent and stable in recurrent tumors. The BA subtype was associated with a transcriptional signature of partial epithelial-to-mesenchymal transition (p-emt) and early recurrence. In 44% of matched cases, the dominant subtype changed from primary to recurrent tumors, preferably from IMS to BA or CL. Gene set enrichment analysis identified upregulation of Hypoxia, p-emt and radiation resistance signatures and downregulation of tumor inflammation in recurrences compared to index tumors. A relevant subset of primary/recurrent tumor pairs presented no evidence for a common clonal origin. CONCLUSIONS: Our study showed a high degree of genetic and transcriptional heterogeneity between primary/recurrent tumors, suggesting therapy-related selection of a transcriptional subtype with characteristics unfavorable for therapy. We conclude that therapy decisions should be based on genetic and transcriptional characteristics of recurrences rather than primary tumors to enable optimally tailored treatment strategies.
Wissenschaftlicher Artikel
Scientific Article
2021
Liu, N. ; O'Connor, P. ; Gujrati, V. ; Gorpas, D. ; Glasl, S. ; Blutke, A. ; Walch, A.K. ; Kleigrewe, K. ; Sattler, M. ; Plettenburg, O. ; Ntziachristos, V.
In: (European Conference on Biomedical Optics, 20–24 June 2021, Munich Germany). 2021. DOI: 10.1117/12.2615998 ( ; 11923)
CR760, a croconaine dye with excellent optical properties, was synthesized in a single step and subsequently nano-formulated for optoacoustic imaging and photothermal therapy of cancer.
Li, Z. ; Sun, G. ; Sun, G. ; Cheng, Y. ; Wu, L. ; Wang, Q. ; Lv, C. ; Zhou, Y. ; Xia, Y. ; Tang, W.
Front. Oncol. 11:771335 (2021)
The occurrence and development of cancer are closely related to the immune escape of tumor cells and immune tolerance. Unlike previous surgical, chemotherapy, radiotherapy and targeted therapy, tumor immunotherapy is a therapeutic strategy that uses various means to stimulate and enhance the immune function of the body, and ultimately achieves the goal of controlling tumor cells.With the in-depth understanding of tumor immune escape mechanism and tumor microenvironment, and the in-depth study of tumor immunotherapy, immune checkpoint inhibitors represented by Programmed Death 1/Programmed cell Death-Ligand 1(PD-1/PD-L1) inhibitors are becoming increasingly significant in cancer medication treatment. employ a variety of ways to avoid detection by the immune system, a single strategy is not more effective in overcoming tumor immune evasion and metastasis. Combining different immune agents or other drugs can effectively address situations where immunotherapy is not efficacious, thereby increasing the chances of success and alternative access to alternative immunotherapy. Immune combination therapies for cancer have become a hot topic in cancer treatment today. In this paper, several combination therapeutic modalities of PD1/PD-L1 inhibitors are systematically reviewed. Finally, an analysis and outlook are provided in the context of the recent advances in combination therapy with PD1/PD-L1 inhibitors and the pressing issues in this field.
Review
Review
Sun, N. ; Trajkovic-Arsic, M. ; Li, F. ; Wu, Y. ; Münch, C. ; Kunzke, T. ; Feuchtinger, A. ; Steiger, K. ; Schlitter, A.M. ; Weichert, W. ; Esposito, I. ; Siveke, J.T. ; Walch, A.K.
EJNMMI Res. 11:120 (2021)
Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies to date. The impressively developed stroma that surrounds and modulates the behavior of cancer cells is one of the main factors regulating the PDAC growth, metastasis and therapy resistance. Here, we postulate that stromal and cancer cell compartments differentiate in protein/lipid glycosylation patterns and analyze differences in glycan fragments in those compartments with clinicopathologic correlates. Results: We analyzed native glycan fragments in 109 human FFPE PDAC samples using high mass resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometric imaging (MALDI-FT-ICR-MSI). Our method allows detection of native glycan fragments without previous digestion with PNGase or any other biochemical reaction. With this method, 8 and 18 native glycans were identified as uniquely expressed in only stromal or only cancer cell compartment, respectively. Kaplan–Meier survival model identified glycan fragments that are expressed in cancer cell or stromal compartment and significantly associated with patient outcome. Among cancer cell region-specific glycans, 10 predicted better and 6 worse patient survival. In the stroma, 1 glycan predicted good and 4 poor patient survival. Using factor analysis as a dimension reduction method, we were able to group the identified glycans in 2 factors. Multivariate analysis revealed that these factors can be used as independent survival prognostic elements with regard to the established Union for International Cancer Control (UICC) classification both in tumor and stroma regions. Conclusion: Our method allows in situ detection of naturally occurring glycans in FFPE samples of human PDAC tissue and highlights the differences among glycans found in stromal and cancer cell compartment offering a basis for further exploration on the role of specific glycans in cancer–stroma communication.
Wissenschaftlicher Artikel
Scientific Article
Zhao, L. ; Chen, F. ; Quitt, O. ; Festag, M. ; Ringelhan, M. ; Wisskirchen, K. ; Festag, J. ; Yakovleva, L. ; Sureau, C. ; Bohne, F. ; Aichler, M. ; Bruss, V. ; Shevtsov, M. ; van de Klundert, M. ; Momburg, F. ; Möhl, B.S. ; Protzer, U.
Cell. Microbiol. 23:e13399 (2021)
Hepatitis B virus (HBV) infection is a major health threat causing 880,000 deaths each year. Available therapies control viral replication, but do not cure HBV leaving patients at risk to develop hepatocellular carcinoma. Here we show that HBV envelope proteins (HBs) - besides their integration into endosomal membranes - become embedded in the plasma membrane where they can be targeted by redirected T-cells. HBs was detected on the surface of HBV-infected cells, in livers of mice replicating HBV and in HBV-induced hepatocellular carcinoma. Staining with HBs-specific recombinant antibody MoMab recognizing a conformational epitope indicated that membrane-associated HBs remains correctly folded in HBV-replicating cells in cell culture and in livers of HBV-transgenic mice in vivo. MoMab coated onto superparamagnetic iron oxide nanoparticles allowed to detect membrane-associated HBs after HBV infection by electron microscopy in distinct stretches of the hepatocyte plasma membrane. Last not least we demonstrate that HBs located to the cell surface allows therapeutic targeting of HBV-positive cells by T-cells either engrafted with a chimeric antigen receptor or redirected by bispecific, T-cell engager antibodies. This article is protected by copyright. All rights reserved.
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Scientific Article
Yuan, S. ; Liao, G. ; Zhang, M. ; Zhu, Y. ; Xiao, W. ; Wang, K. ; Li, C. ; Jia, C. ; Sun, N. ; Walch, A.K. ; Gao, D. ; Xu, P. ; Deng, Q. ; Zhang, J. ; Wang, H. ; Hu, R.
Cell Discov. 7:105 (2021)
Hepatitis B Virus (HBV) constitutes a major threat to global public health. Current understanding of HBV-host interaction is yet limited. Here, ribosome profiling, quantitative mass spectrometry and RNA-sequencing were conducted on a recently established HBV replication system, through which we identified multiomic differentially expressed genes (DEGs) that HBV orchestrated to remodel host proteostasis networks. Our multiomics interrogation revealed that HBV induced significant changes in both transcription and translation of 35 canonical genes including PPP1R15A, PGAM5 and SIRT6, as well as the expression of at least 15 non-canonical open reading frames (ncORFs) including ncPON2 and ncGRWD1, thus revealing an extra coding potential of human genome. Overexpression of these five genes but not the enzymatically deficient SIRT6 mutants suppressed HBV replication while knockdown of SIRT6 had opposite effect. Furthermore, the expression of SIRT6 was down-regulated in patients, cells or animal models of HBV infection. Mechanistic study further indicated that SIRT6 directly binds to mini-chromosome and deacetylates histone H3 lysine 9 (H3K9ac) and histone H3 lysine 56 (H3K56ac), and chemical activation of endogenous SIRT6 with MDL800 suppressed HBV infection in vitro and in vivo. By generating the first multiomics landscape of host-HBV interaction, our work is thus opening a new avenue to facilitate therapeutic development against HBV infection.
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Buck, A. ; Prade, V.M. ; Kunzke, T. ; Feuchtinger, A. ; Kröll, D. ; Feith, M. ; Dislich, B. ; Balluff, B. ; Langer, R. ; Walch, A.K.
J. Pathol., DOI: 10.1002/path.5828 (2021)
The response to neoadjuvant therapy can vary widely between individual patients. Histopathological tumor regression grading (TRG) is a strong factor for treatment response and survival prognosis of esophageal adenocarcinoma (EAC) patients following neoadjuvant treatment and surgery. However, TRG systems are usually based on the estimation of residual tumor but do not consider stromal or metabolic changes after treatment. Spatial metabolomics analysis is a powerful tool for molecular tissue phenotyping but has not been used so far in the context of neoadjuvant treatment of esophageal cancer. We used imaging mass spectrometry to assess the potential of spatial metabolomics on tumor and stroma tissue for evaluating therapy response of neoadjuvant-treated EAC patients. With an accuracy of 89.7%, the binary classifier trained on spatial tumor metabolite data proved to be superior for stratifying patients when compared to histopathological response assessment which had an accuracy of 70.5%. Sensitivities and specificities for the poor and favorable survival patient groups ranged from 84.9 to 93.3% using the metabolic classifier and from 62.2 to 78.1% using TRG. The tumor classifier was the only significant prognostic factor (HR 3.38, 95% CI = 1.40-8.12, P = 0.007) when adjusted for clinicopathological parameters such as TRG (HR 1.01, 95% CI = 0.67-1.53, P = 0.968) or stromal classifier (HR 1.856, 95% CI = 0.81-4.25, P = 0.143). The classifier even allowed to further stratify patients within the TRG1-3 categories. The underlying mechanisms of response to treatment has been figured out through network analysis. In summary, metabolic response evaluation outperformed histopathological response evaluation in our study with regard to prognostic stratification. This finding indicates that the metabolic constitution of tumor may have a greater impact on patient survival than the quantity of residual tumor cells or the stroma. This article is protected by copyright. All rights reserved.
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Scientific Article
Burkhardt, R. ; Gora, T. ; Fingerle, A.A. ; Sauter, A.P. ; Meurer, F. ; Gassert, F.T. ; Dobiasch, S. ; Schilling, D. ; Feuchtinger, A. ; Walch, A.K. ; Multhoff, G. ; Herzen, J. ; Noel, P.B. ; Rummeny, E.J. ; Combs, S.E. ; Schmid, T.E. ; Pfeiffer, F. ; Wilkens, J.J.
Phys. Imag. Radiat. Oncology 20, 11-16 (2021)
Background and Purpose: Radiotherapy of thoracic tumours can lead to side effects in the lung, which may benefit from early diagnosis. We investigated the potential of X-ray dark-field computed tomography by a proof-of-principle murine study in a clinically relevant radiotherapeutic setting aiming at the detection of radiation-induced lung damage. Material and Methods: Six mice were irradiated with 20 Gy to the entire right lung. Together with five unirradiated control mice, they were imaged using computed tomography with absorption and dark-field contrast before and 16 weeks post irradiation. Mean pixel values for the right and left lung were calculated for both contrasts, and the right-to-left-ratio R of these means was compared. Radiologists also assessed the tomograms acquired 16 weeks post irradiation. Sensitivity, specificity, inter- and intra-reader accuracy were evaluated. Results: In absorption contrast the group-average of R showed no increase in the control group and increased by 7% (p = 0.005) in the irradiated group. In dark-field contrast, it increased by 2% in the control group and by 14% (p = 0.005) in the irradiated group. Specificity was 100% for both contrasts but sensitivity was almost four times higher using dark-field tomography. Two cases were missed by absorption tomography but were detected by dark-field tomography. Conclusions: The applicability of X-ray dark-field computed tomography for the detection of radiation-induced lung damage was demonstrated in a pre-clinical mouse model. The presented results illustrate the differences between dark-field and absorption contrast and show that dark-field tomography could be advantageous in future clinical settings.
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Scientific Article
Kunzke, T. ; Prade, V.M. ; Buck, A. ; Sun, N. ; Feuchtinger, A. ; Matzka, M. ; Fernandez, I.E. ; Wuyts, W.A. ; Ackermann, M. ; Jonigk, D. ; Aichler, M. ; Schmid, R.A. ; Eickelberg, O. ; Berezowska, S. ; Walch, A.K.
Cancer Res. 81, 5862-5875 (2021)
Asymptomatic anthracosis is the accumulation of black carbon particles in adult human lungs. It is a common occurrence, but the pathophysiological significance of anthracosis is debatable. Using in situ high mass resolution matrix-assisted laser desorption/ionization (MALDI) fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry imaging analysis, we discovered noxious carbon-bound exogenous compounds, such as polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines, or aromatic amines, in a series of 330 lung cancer patients in highly variable and unique patterns. The characteristic nature of carbon-bound exogenous compound had a strong association with patient outcome, tumor progression, the tumor immune microenvironment, PD-L1 expression, and DNA damage. Spatial correlation network analyses revealed substantial differences in the metabolome of tumor cells compared to tumor stroma depending on carbon-bound exogenous compounds. Overall, the bioactive pool of exogenous compounds is associated with several changes in lung cancer pathophysiology and correlates with patient outcome. Given the high prevalence of anthracosis in the lungs of adult humans, future work should investigate the role of carbon-bound exogenous compounds in lung carcinogenesis and lung cancer therapy.
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Kunzke, T. ; Hölzl, F.T. ; Prade, V.M. ; Buck, A. ; Huber, K. ; Feuchtinger, A. ; Ebert, K. ; Zwingenberger, G. ; Geffers, R. ; Hauck, S.M. ; Haffner, I. ; Luber, B. ; Lordick, F. ; Walch, A.K.
Clin. Transl. Med. 11:e547 (2021)
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Scientific Article
Oppenländer, L. ; Palit, S. ; Stemmer, K. ; Greisle, T. ; Sterr, M. ; Salinno, C. ; Bastidas-Ponce, A. ; Feuchtinger, A. ; Böttcher, A. ; Ansarullah ; Theis, F.J. ; Lickert, H.
Mol. Metab. 54:101330 (2021)
While the effectiveness of bariatric surgery in restoring β-cell function has been described in type-2 diabetes (T2D) patients and animal models for years, the mechanistic underpinnings are largely unknown. The possibility of vertical sleeve gastrectomy (VSG) to rescue a clinically-relevant, late-stage T2D condition and to promote β-cell recovery has not been investigated on a single-cell level. Nevertheless, characterization of the heterogeneity and functional states of β-cells after VSG is a fundamental step to understand mechanisms of glycaemic recovery and to ultimately develop alternative, less-invasive therapies. Here, we report that VSG was superior to calorie restriction in late-stage T2D and rapidly restored normoglycaemia in morbidly obese and overt diabetic db/db mice. Single-cell profiling of islets of Langerhans showed that VSG induced distinct, intrinsic changes in the β-cell transcriptome, but not in that of α-, δ-, and PP-cells. VSG triggered fast β-cell redifferentiation and functional improvement within only two weeks of intervention, which is not seen upon calorie restriction. Furthermore, VSG expanded β-cell area by means of redifferentiation and by creating a proliferation competent β-cell state. Collectively, our study reveals the superiority of VSG in the remission of far-progressed T2D and presents paths of β-cell regeneration and molecular pathways underlying the glycaemic benefits of VSG.
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Scientific Article
Aliluev, A. ; Tritschler, S. ; Sterr, M. ; Oppenländer, L. ; Hinterdobler, J. ; Greisle, T. ; Irmler, M. ; Beckers, J. ; Sun, N. ; Walch, A.K. ; Stemmer, K. ; Kindt, A. ; Krumsiek, J. ; Tschöp, M.H. ; Luecken, M. ; Theis, F.J. ; Lickert, H. ; Böttcher, A.
Nat. Metab. 3, 1202-1216 (2021)
Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.
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Scientific Article
Huang, S. ; Blutke, A. ; Feuchtinger, A. ; Klemm, U. ; Zachariah Tom, R. ; Hofmann, S.M. ; Stiel, A.C. ; Ntziachristos, V.
EMBO Mol. Med. 13:e13490 (2021)
The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label-free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ’s function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.
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Scientific Article
Chhabra, N.F. ; Amend, A.-L. ; Bastidas-Ponce, A. ; Sabrautzki, S. ; Tarquis Medina, M. ; Sachs, S. ; Rubey, M. ; Lorenz-Depiereux, B. ; Feuchtinger, A. ; Bakhti, M. ; Lickert, H. ; Przemeck, G.K.H. ; Hrabě de Angelis, M.
Mol. Metab. 54:101334 (2021)
OBJECTIVE: Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to carry a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. METHODS: We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. RESULTS: Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although, no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP as well as PDI family member PDIA4, however without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa and Slc2a2 along with increased expression of α-cell markers, Mafb and glucagon was observed in adult mice, suggesting loss of β-cell identity. CONCLUSIONS: The data demonstrates that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.
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Scientific Article
Maier, J.P. ; Kueckelhaus, J. ; Behringer, S.P. ; Garrelfs, N. ; Will, P. ; Sun, N. ; von Ehr, J. ; Goeldner, J.M. ; Pfeifer, D. ; Follo, M. ; Hannibal, L. ; Walch, A.K. ; Hofmann, U.G. ; Beck, J. ; Heiland, D.H. ; Schnell, O.
Cell Death Dis. 12:723 (2021)
Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.
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Scientific Article
Loft, A. ; Alfaro, A.J. ; Schmidt, S.F. ; Pedersen, F.B. ; Terkelsen, M.K. ; Puglia, M. ; Chow, K.K. ; Feuchtinger, A. ; Troullinaki, M. ; Maida, A. ; Wolff, G. ; Sakurai, M. ; Berutti, R. ; Ekim Üstünel, B. ; Nawroth, P.P. ; Ravnskjaer, K. ; Diaz, M.B. ; Blagoev, B. ; Herzig, S.
Cell Metab. 33, 1685-1700.e9 (2021)
Liver fibrosis is a strong predictor of long-term mortality in individuals with metabolic-associated fatty liver disease; yet, the mechanisms underlying the progression from the comparatively benign fatty liver state to advanced non-alcoholic steatohepatitis (NASH) and liver fibrosis are incompletely understood. Using cell-type-resolved genomics, we show that comprehensive alterations in hepatocyte genomic and transcriptional settings during NASH progression, led to a loss of hepatocyte identity. The hepatocyte reprogramming was under tight cooperative control of a network of fibrosis-activated transcription factors, as exemplified by the transcription factor Elf-3 (ELF3) and zinc finger protein GLIS2 (GLIS2). Indeed, ELF3- and GLIS2-controlled fibrosis-dependent hepatokine genes targeting disease-associated hepatic stellate cell gene programs. Thus, interconnected transcription factor networks not only promoted hepatocyte dysfunction but also directed the intra-hepatic crosstalk necessary for NASH and fibrosis progression, implying that molecular "hub-centered" targeting strategies are superior to existing mono-target approaches as currently used in NASH therapy.
Wissenschaftlicher Artikel
Scientific Article
Murakami, M. ; Sun, N. ; Greunke, C. ; Feuchtinger, A. ; Kircher, S. ; Deutschbein, T. ; Papathomas, T. ; Bechmann, N. ; Wallace, P.W. ; Peitzsch, M. ; Korpershoek, E. ; Friemel, J. ; Gimenez Roqueplo, A.P. ; Robledo, M. ; Timmers, H.J. ; Canu, L. ; Weber, A. ; de Krijger, R.R. ; Fassnacht, M. ; Knösel, T. ; Kirchner, T. ; Reincke, M. ; Walch, A.K. ; Kroiss, M. ; Beuschlein, F.
Eur. J. Endocrinol. 185, 179-191 (2021)
OBJECTIVE: Within the past decade, important genetic drivers of pheochromocytoma and paraganglioma (PPGLs) development have been identified. The pathophysiological mechanism that translate these alterations into functional autonomy and potentially malignant behavior have not been elucidated in detail. Here we used MALDI-mass spectrometry imaging (MALDI-MSI) of formalin-fixed paraffin-embedded tissue specimens to comprehensively characterize the metabolic profiles of PPGLs. DESIGN AND METHODS: MALDI-MSI was conducted in 344 PPGLs and results correlated with genetic and phenotypic information. We experimentally silenced genetic drivers by siRNA in PC12 cells to confirm their metabolic impact in vitro. RESULTS: Tissue abundance of kynurenine pathway metabolites such as xanthurenic acid was significantly lower (P = 5.06E-11) in the pseudohypoxia pathway cluster 1 compared to PPGLs of the kinase-driven PPGLs cluster 2. Lower abundance of xanthurenic acid was associated with shorter metastasis-free survival (log-rank tests P = 7.96E-06) and identified as a risk factor for metastasis independent of the genetic status (hazard ratio, 32.6, P = 0.002). Knock-down of Sdhb and Vhl in an in vitro model demonstrated that inositol metabolism and sialic acids were similarly modulated as in tumors of the respective cluster. CONCLUSIONS: The present study has identified distinct tissue metabolomic profiles of PPGLs in relation to tumor genotypes. In addition, we revealed significantly altered metabolites in the kynurenine pathway in metastatic PPGLs, which can aid in the prediction of its malignant potential. However, further validation studies will be required to confirm our findings.
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Scientific Article
Georgiadi, A. ; Lopez Salazar, V. ; El-Merahbi, R. ; Karikari, R.A. ; Ma, X. ; Mourao, A. ; Klepac, K. ; Bühler, L. ; Alfaro, A.J. ; Kaczmarek, I. ; Linford, A. ; Bosma, M. ; Shilkova, O. ; Ritvos, O. ; Nakamura, N. ; Hirose, S. ; Lassi, M. ; Teperino, R. ; Machado, J. ; Scheideler, M. ; Dietrich, A. ; Geerlof, A. ; Feuchtinger, A. ; Blutke, A. ; Fischer, K. ; Müller, T.D. ; Kessler, K. ; Schöneberg, T. ; Thor, D. ; Hornemann, S. ; Kruse, M. ; Nawroth, P.P. ; Pivovarova-Ramich, O. ; Pfeiffer, A.F.H. ; Sattler, M. ; Blüher, M. ; Herzig, S.
Nat. Commun. 12:2999 (2021)
The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.
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Scientific Article
Giroud, M. ; Tsokanos, F.-F. ; Caratti, G. ; Kotschi, S. ; Khani, S. ; Jouffe, C. ; Vogl, E.S. ; Irmler, M. ; Glantschnig, C. ; Gil Lozano, M. ; Haß, D. ; Khan, A.A. ; Rios Garcia, M. ; Mattijssen, F. ; Maida, A. ; Tews, D. ; Fischer-Posovszky, P. ; Feuchtinger, A. ; Virtanen, K.A. ; Beckers, J. ; Wabitsch, M. ; Uhlenhaut, N.H. ; Blüher, M. ; Tuckermann, J. ; Scheideler, M. ; Bartelt, A. ; Herzig, S.
Diabetologia 64, 1850-1865 (2021)
Aims/hypothesis: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. Methods: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2 ) and performed a large panel of metabolic tests. Results: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR–HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. Conclusions/interpretation: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. Data availability: Array data have been submitted to the GEO database at NCBI (GSE148699). Graphical abstract: [Figure not available: see fulltext.] AdipoqCre
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Scientific Article
Liu, N. ; Gujrati, V. ; Malekzadeh-Najafabadi, J. ; Werner, J.P,F. ; Klemm, U. ; Tang, L. ; Chen, Z. ; Prakash, J. ; Huang, Y. ; Stiel, A.-C. ; Mettenleiter, G. ; Aichler, M. ; Blutke, A. ; Walch, A.K. ; Kleigrewe, K. ; Razansky, D. ; Sattler, M. ; Ntziachristos, V.
Photoacoustics 22:100263 (2021)
Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.
Wissenschaftlicher Artikel
Scientific Article
Orth, M. ; Albrecht, V. ; Seidl, K. ; Kinzel, L. ; Unger, K. ; Hess-Rieger, J. ; Kreutzer, L. ; Sun, N. ; Stegen, B. ; Nieto, A. ; Maas, J. ; Winssinger, N. ; Friedl, A.A. ; Walch, A.K. ; Belka, C. ; Zitzelsberger, H. ; Niyazi, M. ; Lauber, K.
Front. Oncol. 11:612354 (2021)
Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.
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Haffner, I. ; Schierle, K. ; Raimúndez, E. ; Geier, B. ; Maier, D. ; Hasenauer, J. ; Luber, B. ; Walch, A.K. ; Kolbe, K. ; Riera Knorrenschild, J. ; Kretzschmar, A. ; Rau, B. ; Fischer von Weikersthal, L. ; Ahlborn, M. ; Siegler, G. ; Fuxius, S. ; Decker, T. ; Wittekind, C. ; Lordick, F.
J. Clin. Oncol. 39, 1468-1478 (2021)
PURPOSE: Trastuzumab is the only approved targeted drug for first-line treatment of human epidermal growth factor receptor 2-positive (HER2+) metastatic gastric cancer (mGC). However, not all patients respond and most eventually progress. The multicenter VARIANZ study aimed to investigate the background of response and resistance to trastuzumab in mGC. METHODS: Patients receiving medical treatment for mGC were prospectively recruited in 35 German sites and followed for up to 48 months. HER2 status was assessed centrally by immunohistochemistry and chromogenic in situ hybridization. In addition, HER2 gene expression was assessed using qPCR. RESULTS: Five hundred forty-eight patients were enrolled, and 77 had HER2+ mGC by central assessment (14.1%). A high deviation rate of 22.7% between central and local test results was seen. Patients who received trastuzumab for centrally confirmed HER2+ mGC (central HER2+/local HER2+) lived significantly longer as compared with patients who received trastuzumab for local HER2+ but central HER2- mGC (20.5 months, n = 60 v 10.9 months, n = 65; hazard ratio, 0.42; 95% CI, 8.2 to 14.4; P < .001). In the centrally confirmed cohort, significantly more tumor cells stained HER2+ than in the unconfirmed cohort, and the HER2 amplification ratio was significantly higher. A minimum of 40% HER2+ tumor cells and a HER2 amplification ratio of ≥ 3.0 were calculated as optimized thresholds for predicting benefit from trastuzumab. CONCLUSION: Significant discrepancies in HER2 assessment of mGC were found in tumor specimens with intermediate HER2 expression. Borderline HER2 positivity and heterogeneity of HER2 expression should be considered as resistance factors for HER2-targeting treatment of mGC. HER2 thresholds should be reconsidered. Detailed reports with quantification of HER2 expression and amplification levels may improve selection of patients for HER2-directed treatment.
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Liu, N. ; O'Connor, P. ; Gujrati, V. ; Gorpas, D. ; Glasl, S. ; Blutke, A. ; Walch, A.K. ; Kleigrewe, K. ; Sattler, M. ; Plettenburg, O. ; Ntziachristos, V.
Adv. Healthc. Mater. 10:e2002115 (2021)
Near-infrared (NIR) light absorbing theranostic agents can integrate optoacoustic imaging and photothermal therapy for effective personalized precision medicine. However, most of these agents face the challenges of unstable optical properties, material-associated toxicity, and nonbiodegradability, all of which limit their biomedical application. Several croconaine-based organic agents able to overcome some of these limitations have been recently reported, but these suffer from complicated multistep synthesis protocols. Herein, the use of CR760, a croconaine dye with excellent optical properties, is reported for nanoparticle formulation and subsequent optoacoustic imaging and photothermal therapy. Importantly, CR760 can be conveniently prepared in a single step from commercially available materials. Furthermore, CR760 can be covalently attached, via a polyethylene glycol linker, to the αvβ3 integrin ligand c(RGDyC), resulting in self-assembled nanoparticles (NPs) with cancer-targeting capability. Such CR760RGD-NPs exhibit strong NIR absorption, high photostability, high optoacoustic generation efficiency, and active tumor-targeting, making them ideal candidates for optoacoustic imaging. Due to favorable electron transfer, CR760RGD-NPs display a 45.37% photothermal conversion efficiency thereby rendering them additionally useful for photothermal therapy. Targeted tumor elimination, biosafety, and biocompatibility are demonstrated in a 4T1 murine breast tumor model. This work points to the use of CR760RGD-NPs as a promising nanoagent for NIR-based cancer phototheranostics.
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Ansarullah ; Jain, C. ; Far, F.F. ; Homberg, S. ; Wißmiller, K. ; von Hahn, F. ; Raducanu, A. ; Schirge, S. ; Sterr, M. ; Bilekova, S. ; Siehler, J. ; Wiener, J. ; Oppenländer, L. ; Morshedi, A. ; Bastidas-Ponce, A. ; Collden, G. ; Irmler, M. ; Beckers, J. ; Feuchtinger, A. ; Grzybek, M. ; Ahlbrecht, C. ; Feederle, R. ; Plettenburg, O. ; Müller, T.D. ; Meier, M. ; Tschöp, M.H. ; Coskun, Ü. ; Lickert, H.
Nature 592:E1 (2021)
In this Article, the affiliations for author Ünal Coskun were incorrect. They should be ‘German Center for Diabetes Research (DZD), Neuherberg, Germany’, ‘Paul Langerhans Institute Dresden of Helmholtz Center Munich, Technical University Dresden, Dresden, Germany’ and ‘Institute for Clinical Chemistry and Laboratory Medicine, Faculty of Medicine and University Clinic Carl Gustav Carus, Technical University Dresden, Dresden, Germany’ (affiliations 2, 10 and 14, respectively), and not ‘Department of Microsystems Engineering (IMTEK), University of Freiburg, Freiburg, Germany’ (affiliation 5). The original Article has been corrected online.
Stauffer, E. ; Weber, P. ; Heider, T. ; Dalke, C. ; Blutke, A. ; Walch, A.K. ; Burgstaller, G. ; Brix, N. ; Lauber, K. ; Zitzelsberger, H. ; Unger, K. ; Selmansberger, M.
Endocr. Relat. Cancer 28, 213-224 (2021)
Thyroid carcinoma incidence rates in western societies are among the fastest rising, compared to all malignant tumors over the past two decades. While risk factors such as age and exposure to ionizing radiation are known, early-state carcinogenic processes or pre-lesions are poorly understood or unknown. This study aims at the identification and characterization of early-state radiation-associated neoplastic processes by histologic and transcriptomic analyses of thyroid tissues derived from a mouse model. Comprehensive histological examination of 246 thyroids (164 exposed, 82 non-exposed) was carried out. Proliferative and normal tissues from exposed cases and normal tissue from non-exposed cases were collected by laser-capture microdissection, followed by RNAseq transcriptomic profiling using a low input 3`-library preparation protocol, differential gene expression analysis and functional association by Gene Set Enrichment Analysis. Nine exposed samples exhibited proliferative lesions, while none of the non-exposed samples showed histological abnormalities, indicating an association of ionizing radiation exposure with histological abnormalities. Activated immune response signaling and deregulated metabolic processes were observed in irradiated tissue with normal histology compared to normal tissue from non-exposed samples. Proliferative lesions compared to corresponding normal tissues showed enrichment for mainly proliferation-associated gene sets. Consistently, proliferative lesion samples from exposed mice showed elevated proliferation-associated signaling and deregulated metabolic processes compared to normal samples from non-exposed mice. Our findings suggest that a molecular deregulation may be detectable in histologically normal thyroid tissues and in early proliferative lesions in the frame of multi-step progression from irradiated normal tissue to tumorous lesions.
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Scientific Article
Yuan, S. ; Liao, G. ; Zhang, M. ; Zhu, Y. ; Wang, K. ; Xiao, W. ; Jia, C. ; Dong, M. ; Sun, N. ; Walch, A.K. ; Xu, P. ; Zhang, J. ; Deng, Q. ; Hu, R.
J. Hepatol. 75, 74-85 (2021)
BACKGROUND AND AIMS: Hepatitis B Virus remains to be yet unresolved global threat to human health. It remains incompletely understood how HBV self-restricts in host during most adulthood infections, and multi-omics analyses were performed to systematically interrogate into HBV-host interaction and the life cycle of HBV. METHODS: RNA-sequencing and ribosome profiling were conducted with cell-based models for HBV replication and gene expression. The novel translational events or products hereby detected were then characterized, and functionally assessed in both cell and mouse models. Moreover, quasi-species analyses of HBV subpopulations were conducted with patients at immune tolerance or activation phases, using next- or third-generation sequencing. RESULTS: We identified EnhI-SL (Enhancer I-stem loop) as a new cis element in HBV genome, and the mutations disrupting EnhI-SL were found to elevate viral polymerase expression. Furthermore, while re-discovering HpZ/P', a previously under-explored isoform of HBV polymerase, we also identified HBxZ as a novel short isoform of HBX and confirmed their existence and functionally characterized them as potent suppressors for HBV gene expression or genome replication. Mechanistically, HpZ/P' was found to repress HBV gene expression partially through interacting with, and sequestering SUPV3L1. The abundances of the HBV mutants either deficient of HpZ/P' or disrupted in EnhI-SL seemed to be diminished upon the activation of host immune system. Finally, SRSF2, a HBV-down-regulated host protein in RNA spliceosome, was found to promote the splicing of viral pre-genomic RNA and HpZ/P' biogenesis. CONCLUSION: This study has identified multiple viral self-restricting mechanisms in HBV-host interaction. Particularly, SRSF2-HpZ/P' appeared to constitute another negative feedback mechanism in controlling HBV life-cycle. Targeting host splicing machinery might thus represent a yet under-explored strategy to intervene into HBV-host interaction.
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Theobalt, N. ; Hofmann, I. ; Fiedler, S. ; Renner, S. ; Dhom, G. ; Feuchtinger, A. ; Walch, A.K. ; Hrabě de Angelis, M. ; Wolf, E. ; Wanke, R. ; Blutke, A.
PLoS ONE 16:e0248594 (2021)
In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species.
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Scientific Article
Ruiz Ojeda, F.J. ; Wang, J. ; Bäcker, T. ; Krueger, M. ; Zamani, S. ; Rosowski, S. ; Gruber, T. ; Onogi, Y. ; Feuchtinger, A. ; Schulz, T.J. ; Fässler, R. ; Müller, T.D. ; García-Cáceres, C. ; Meier, M. ; Blüher, M. ; Ussar, S.
Mol. Metab. 45:101147 (2021)
Objective: Reorganization of the extracellular matrix is a prerequisite for healthy adipose tissue expansion, whereas fibrosis is a key feature of adipose dysfunction and inflammation. However, very little is known about the direct effects of impaired cell–matrix interaction in adipocyte function and insulin sensitivity. The objective of this study was to determine whether integrin activity can regulate insulin sensitivity in adipocytes and thereby systemic metabolism. Methods: We characterized integrin activity in adipose tissue and its consequences on whole-body metabolism using adipose-selective deletion of β1 integrin (Itgb1adipo-cre) and Kindlin-2 (Kind2adipo-cre) in mice. Results: We demonstrate that integrin signaling regulates white adipocyte insulin action and systemic metabolism. Consequently, loss of adipose integrin activity, similar to loss of adipose insulin receptors, results in a lipodystrophy-like phenotype and systemic insulin resistance. However, brown adipose tissue of Kind2adipo-cre and Itgb1adipo-cre mice is chronically hyperactivated and has increased substrate delivery, reduced endothelial basement membrane thickness, and increased endothelial vesicular transport. Conclusions: Thus, we establish integrin-extracellular matrix interactions as key regulators of white and brown adipose tissue function and whole-body metabolism.
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Scientific Article
Ansarullah ; Jain, C. ; Far, F.F. ; Homberg, S. ; Wissmiller, K. ; Gräfin von Hahn, F. ; Raducanu, A. ; Schirge, S. ; Sterr, M. ; Bilekova, S. ; Siehler, J. ; Wiener, J. ; Oppenländer, L. ; Morshedi, A. ; Bastidas-Ponce, A. ; Collden, G. ; Irmler, M. ; Beckers, J. ; Feuchtinger, A. ; Grzybek, M. ; Ahlbrecht, C. ; Feederle, R. ; Plettenburg, O. ; Müller, T.D. ; Meier, M. ; Tschöp, M.H. ; Coskun, Ü. ; Lickert, H.
Nature 590, 326–331 (2021)
Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic β-cells causes overt diabetes in mice; thus, therapies that sensitize β-cells to insulin may protect patients with diabetes against β-cell failure1–3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse β-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir−/−) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir−/− mice showed an increase in the activation of INSR–IGF1R in Iir−/− pancreatic tissue, resulting in an increase in the proliferation and mass of β-cells. Similarly, inducible β-cell-specific Iir−/− knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR–IGF1R and increased proliferation of β-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR–IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR–IGF1R in β-cells. Together, our findings show that inceptor shields insulin-producing β-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR–IGF1R sensitization and diabetes therapy.
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Scientific Article
Bühler, L. ; Maida, A. ; Vogl, E.S. ; Georgiadi, A. ; Takacs, A. ; Kluth, O. ; Schürmann, A. ; Feuchtinger, A. ; von Toerne, C. ; Tsokanos, F.-F. ; Klepac, K. ; Wolff, G. ; Sakurai, M. ; Ekim Üstünel, B. ; Nawroth, P.P. ; Herzig, S.
Life Sci. All. 4:e202000898 (2021)
Members of the lipocalin protein family serve as biomarkers for kidney disease and acute phase inflammatory reactions, and are under preclinical development for the diagnosis and therapy of allergies. However, none of the lipocalin family members has made the step into clinical development, mostly due to their complex biological activity and the lack of in-depth mechanistic knowledge. Here, we show that the hepatokine lipocalin 13 (LCN13) triggers glucose-dependent insulin secretion and cell proliferation of primary mouse islets. However, inhibition of endogenous LCN13 expression in lean mice did not alter glucose and lipid homeostasis. Enhanced hepatic secretion of LCN13 in either diet-induced or genetic obesity led to no discernible impact on systemic glucose and lipid metabolism, neither in preventive nor therapeutic setting. Of note, loss or forced LCN13 hepatic secretion did not trigger any compensatory regulation of related lipocalin family members. Together, these data are in stark contrast to the suggested gluco-regulatory and therapeutic role of LCN13 in obesity, and imply complex regulatory steps in LCN13 biology at the organismic level mitigating its principal insulinotropic effects.
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Scientific Article
Zhang, Q. ; Delessa, C.T. ; Augustin, R. ; Bakhti, M. ; Collden, G. ; Drucker, D.J. ; Feuchtinger, A. ; García-Cáceres, C. ; Grandl, G. ; Harger, A. ; Herzig, S. ; Hofmann, S.M. ; Holleman, C.L. ; Jastroch, M. ; Keipert, S. ; Kleinert, M. ; Knerr, P.J. ; Kulaj, K. ; Legutko, B. ; Lickert, H. ; Liu, X. ; Luippold, G. ; Lutter, D. ; Malogajski, E. ; Tarquis Medina, M. ; Mowery, S.A. ; Blutke, A. ; Perez-Tilve, D. ; Salinno, C. ; Sehrer, L. ; DiMarchi, R.D. ; Tschöp, M.H. ; Stemmer, K. ; Finan, B. ; Wolfrum, C. ; Müller, T.D.
Cell Metab. 33, 833-844.e5 (2021)
Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.
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Scientific Article
Schinke, H. ; Heider, T. ; Herkommer, T. ; Simon, F. ; Blancke Soares, A. ; Kranz, G. ; Samaga, D. ; Dajka, L. ; Feuchtinger, A. ; Walch, A.K. ; Valeanu, L. ; Walz, C. ; Kirchner, T. ; Canis, M. ; Baumeister, P. ; Belka, C. ; Maihöfer, C. ; Marschner, S. ; Pflugradt, U. ; Ganswindt, U. ; Hess-Rieger, J. ; Zitzelsberger, H. ; Gires, O.
Mol. Oncol. 15, 1040-1053 (2021)
Head and neck squamous cell carcinomas (HNSCCs) have poor clinical outcome owing to therapy resistance and frequent recurrences that are among others attributable to tumor cells in partial epithelial-to-mesenchymal transition (pEMT). We compared side-by-side software-based and visual quantification of immunohistochemistry (IHC) staining of epithelial marker EpCAM and EMT regulator Slug in n = 102 primary HNSCC to assess optimal analysis protocols. IHC scores incorporated expression levels and percentages of positive cells. Digital and visual evaluation of membrane-associated EpCAM yielded correlating scorings, whereas visual evaluation of nuclear Slug resulted in significantly higher overall scores. Multivariable Cox proportional hazard analysis defined the median EpCAM expression levels resulting from visual quantification as an independent prognostic factor of overall survival. Slug expression levels resulting from digital quantification were an independent prognostic factor of recurrence-free survival, locoregional recurrence-free survival, and disease-specific survival. Hence, we propose to use visual assessment for the membrane-associated EpCAM protein, whereas nuclear protein Slug assessment was more accurate following digital measurement.
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Scientific Article
Napieralski, R. ; Schricker, G. ; Auer, G. ; Aubele, M. ; Perkins, J. ; Magdolen, V. ; Ulm, K. ; Hamann, M. ; Walch, A.K. ; Weichert, W. ; Kiehle, M. ; Weichert, O.G.
Breast Care 16, 523-531 (2021)
Background: PITX2 DNA methylation has been shown to predict outcomes in high-risk breast cancer patients after anthracycline-based chemotherapy. To determine its prognostic versus predictive value, the impact of PITX2 DNA methylation on outcomes was studied in an untreated cohort vs. an anthracycline-treated triple-negative breast cancer (TNBC) cohort. Material and Methods: The percent DNA methylation ratio (PMR) of paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time PCR test. Patient samples of routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue and clinical data from 144 TNBC patients of 2 independent cohorts (i.e., 66 untreated patients and 78 patients treated with anthracycline-based chemotherapy) were analyzed. Results: The risk of 5- and 10-year overall survival (OS) increased continuously with rising PITX2 DNA methylation in the anthracycline-treated population, but it increased only slightly during 10-year follow-up time in the untreated patient population. PITX2 DNA methylation with a PMR cutoff of 2 did not show significance for poor vs. good outcomes (OS) in the untreated patient cohort (HR = 1.55; p = 0.259). In contrast, the PITX2 PMR cutoff of 2 identified patients with poor (PMR >2) vs. good (PMR ≤2) outcomes (OS) with statistical significance in the anthracycline-treated cohort (HR = 3.96; p = 0.011). The results in the subgroup of patients who did receive anthracyclines only (no taxanes) confirmed this finding (HR = 5.71; p = 0.014). Conclusion: In this hypothesis-generating study PITX2 DNA methylation demonstrated predominantly predictive value in anthracycline treatment in TNBC patients. The risk of poor outcome (OS) correlates with increasing PITX2 DNA methylation.
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Karlina, R. ; Lutter, D. ; Miok, V. ; Fischer, D.S. ; Altun, I. ; Schöttl, T. ; Schorpp, K.K. ; Israel, A. ; Cero, C. ; Johnson, J.W. ; Kapser-Fischer, I. ; Böttcher, A. ; Keipert, S. ; Feuchtinger, A. ; Graf, E. ; Strom, T.M. ; Walch, A.K. ; Lickert, H. ; Walzthoeni, T. ; Heinig, M. ; Theis, F.J. ; García-Cáceres, C. ; Cypess, A.M. ; Ussar, S.
Life Sci. All. 4:e202000924 (2021)
Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. Although increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Recently, UCP1 high and low expressing brown adipocytes were identified, but a developmental origin of these subtypes has not been studied. To obtain more insights into brown preadipocyte heterogeneity, we use single-cell RNA sequencing of the BAT stromal vascular fraction of C57/BL6 mice and characterize brown preadipocyte and adipocyte clonal cell lines. Statistical analysis of gene expression profiles from brown preadipocyte and adipocyte clones identify markers distinguishing brown adipocyte subtypes. We confirm the presence of distinct brown adipocyte populations in vivo using the markers EIF5, TCF25, and BIN1. We also demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that BIN1 marks dormant brown adipocytes. The existence of multiple brown adipocyte subtypes suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct functions in thermogenesis and the regulation of whole body energy homeostasis.
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Burkhardt, R. ; Gora, T. ; Fingerle, A.A. ; Sauter, A.P. ; Meurer, F. ; Umkehrer, S. ; Von Teuffenbach, M. ; Kampfer, S. ; Schilling, D. ; Feuchtinger, A. ; Walch, A.K. ; Rummeny, E. ; Combs, S.E. ; Schmid, T.E. ; Pfeiffer, F. ; Wilkens, J.J. ; Herzen, J.
Eur. Radiol. 31, 4175–4183 (2021)
Objective: Assessing the advantage of x-ray dark-field contrast over x-ray transmission contrast in radiography for the detection of developing radiation-induced lung damage in mice. Methods: Two groups of female C57BL/6 mice (irradiated and control) were imaged obtaining both contrasts monthly for 28 weeks post irradiation. Six mice received 20 Gy of irradiation to the entire right lung sparing the left lung. The control group of six mice was not irradiated. A total of 88 radiographs of both contrasts were evaluated for both groups based on average values for two regions of interest, covering (irradiated) right lung and healthy left lung. The ratio of these average values, R, was distinguished between healthy and damaged lungs for both contrasts. The time-point when deviations of R from healthy lung exceeded 3σ was determined and compared among contrasts. The Wilcoxon-Mann-Whitney test was used to test against the null hypothesis that there is no difference between both groups. A selection of 32 radiographs was assessed by radiologists. Sensitivity and specificity were determined in order to compare the diagnostic potential of both contrasts. Inter-reader and intra-reader accuracy were rated with Cohen’s kappa. Results: Radiation-induced morphological changes of lung tissue caused deviations from the control group that were measured on average 10 weeks earlier with x-ray dark-field contrast than with x-ray transmission contrast. Sensitivity, specificity, and accuracy doubled using dark-field radiography. Conclusion: X-ray dark-field radiography detects morphological changes of lung tissue associated with radiation-induced damage earlier than transmission radiography in a pre-clinical mouse model. Key Points: • Significant deviations from healthy lung due to irradiation were measured after 16 weeks with x-ray dark-field radiography (p = 0.004). • Significant deviations occur on average 10 weeks earlier for x-ray dark-field radiography in comparison to x-ray transmission radiography. • Sensitivity and specificity doubled when using x-ray dark-field radiography instead of x-ray transmission radiography.
Wissenschaftlicher Artikel
Scientific Article
Sachs, S. ; Niu, L. ; Geyer, P. ; Jall, S. ; Kleinert, M. ; Feuchtinger, A. ; Stemmer, K. ; Brielmeier, M. ; Finan, B. ; DiMarchi, R.D. ; Tschöp, M.H. ; Wewer Albrechtsen, N. ; Mann, M. ; Müller, T.D. ; Hofmann, S.M.
Diabetes Obes. Metab. 23, 195-207 (2021)
Aims Unimolecular peptides targeting the receptors for glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (GLP-1/GIP co-agonist) have been shown to outperform each single peptide in the treatment of obesity and cardiometabolic disease in preclinical and clinical trials. By combining physiological treatment endpoints with plasma proteomic profiling (PPP), we aimed to identify biomarkers to advance non-invasive metabolic monitoring of compound treatment success and exploration of ulterior treatment effects on an individual basis.Materials and methods We performed metabolic phenotyping along with PPP in body weight-matched male and female diet-induced obese (DIO) mice treated for 21 days with phosphate-buffered saline, single GIP and GLP-1 mono-agonists, or a GLP-1/GIP co-agonist.Results GLP-1R/GIPR co-agonism improved obesity, glucose intolerance, non-alcoholic fatty liver disease (NAFLD) and dyslipidaemia with superior efficacy in both male and female mice compared with mono-agonist treatments. PPP revealed broader changes of plasma proteins after GLP-1/GIP co-agonist compared with mono-agonist treatments in both sexes, including established and potential novel biomarkers for systemic inflammation, NAFLD and atherosclerosis. Subtle sex-specific differences have been observed in metabolic phenotyping and PPP.Conclusions We herein show that a recently developed unimolecular GLP-1/GIP co-agonist is more efficient in improving metabolic disease than either mono-agonist in both sexes. PPP led to the identification of a sex-independent protein panel with the potential to monitor non-invasively the treatment efficacies on metabolic function of this clinically advancing GLP-1/GIP co-agonist.
Wissenschaftlicher Artikel
Scientific Article
Sammer, M. ; Dombrowsky, A. ; Schauer, J. ; Oleksenko, K. ; Bicher, S. ; Schwarz, B. ; Rudigkeit, S. ; Matejka, N. ; Reindl, J. ; Bartzsch, S. ; Blutke, A. ; Feuchtinger, A. ; Combs, S.E. ; Dollinger, G. ; Schmid, T.E.
Int. J. Radiat. Oncol. Biol. Phys. 109, 76-83 (2021)
Purpose: Proton minibeam radiation therapy, a spatial fractionation concept, widens the therapeutic window. By reducing normal tissue toxicities, it allows a temporally fractionated regime with high daily doses. However, an array shift between daily fractions can affect the tissue-sparing effect by decreasing the total peak-to-valley dose ratio. Therefore, combining temporal fractions with spatial fractionation raises questions about the impact of daily applied dose modulations, reirradiation accuracies, and total dose modulations. Methods and Materials: Healthy mouse ear pinnae were irradiated with 4 daily fractions of 30 Gy mean dose, applying proton pencil minibeams (pMB) of Gaussian σ = 222 μm in 3 different schemes: a 16 pMB array with a center-to-center distance of 1.8 mm irradiated the same position in all sessions (FS1) or was shifted by 0.9 mm to never hit the previously irradiated tissue in each session (FS2), or a 64 pMB array with a center-to-center distance of 0.9 mm irradiated the same position in all sessions (FS3), resulting in the same total dose distribution as FS2. Reirradiation positioning and its accuracy were obtained from image guidance using the unique vessel structure of ears. Acute toxicities (swelling, erythema, and desquamation) were evaluated for 153 days after the first fraction. Late toxicities (fibrous tissue, inflammation) were analyzed on day 153. Results: Reirradiation of highly dose-modulated arrays at a positioning accuracy of 110 ± 52 μm induced the least severe acute and late toxicities. A shift of the same array in FS2 led to significantly inducted acute toxicities, a higher otitis score, and a slight increase in fibrous tissue. FS3 led to the strongest increase in acute and late toxicities. Conclusions: The highest normal-tissue sparing is achieved after accurate reirradiation of a highly dose modulated pMB array, although high positioning accuracies are challenging in a clinical environment. Nevertheless, the same integral dose applied in highly dose-modulated fractions is superior to low daily dose-modulated fractions.
Wissenschaftlicher Artikel
Scientific Article
2020
Fiedler, S. ; Wünnemann, H. ; Hofmann, I. ; Theobalt, N. ; Feuchtinger, A. ; Walch, A.K. ; Schwaiger, J. ; Wanke, R. ; Blutke, A.
PLoS ONE 15:e0243462 (2020)
Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300–2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.
Wissenschaftlicher Artikel
Scientific Article
Wang, Q. ; Yang, L. ; Fan, Y. ; Tang, W. ; Sun, H. ; Xu, Z. ; Zhou, J. ; Zhang, Y. ; Zhu, B. ; Cao, X.
Front. Cell Dev. Biol. 8:570305 (2020)
Circular RNA (circRNA) exhibits a covalently closed circular conformation and is structurally stable. Nevertheless, the precise effects exerted by circRNA in esophageal squamous cell carcinoma (ESCC) remains uncertain. circRNA was ascertained by a human circRNA array study and was confirmed by the quantification of reverse transcriptase polymerase reactions. A luciferase reporter, fluorescence in situ hybridization experiment was exploited to explore the interaction between circ-ZDHHC5 and miR-217. The function of circ-ZDHHC5 was determined by siRNA-mediated knockout of circ-ZDHHC5 in in vitro proliferation, migration, and invasion. circ-ZDHHC5, rather than linear ZDHHC5 mRNA, rose in the tissues of patients with ESCC, plasma, and ESCC cell lines in comparison with normal controls. Knockdown of circ-ZDHHC5 inhibited tumorigenesis in ESCC cells, and the co-transfection of si-circ-ZDHHC5 and miR-217 mimics further enhanced the above effect. Noticeably, the present study showed that circ-ZDHHC5 was an miR-217 sponge that modulated the expression of zinc finger E-box binding homeobox 1 (ZEB1), further facilitating ESCC tumorigenesis. As revealed by this study, circ-ZDHHC5 can act as a new potential circular biomarker for detecting ESCC. It provides a novel perceptivity for the treatment of ESCC suggesting that circ-ZDHHC5 could impact on ESCC progression by sponging miR-217 with ZEB1.
Wissenschaftlicher Artikel
Scientific Article
Papathomas, T. ; Tzortzakakis, A. ; Sun, N. ; Erlmeier, F. ; Feuchtinger, A. ; Trpkov, K. ; Bazarova, A. ; Arvanitis, A. ; Wang, W. ; Bozoky, B. ; Kokaraki, G. ; Axelsson, R. ; Walch, A.K.
Eu. Urol. Open Sci. 22, 88-96 (2020)
Background: Definite noninvasive characterisation of renal tumours positive on 99mTc-sestamibi single photon emission computed tomography/computed tomography (SPECT/CT) examination including renal oncocytomas (ROs), hybrid oncocytic chromophobe tumours (HOCTs), and chromophobe renal cell carcinoma (chRCC) is currently not feasible. Objective: To investigate whether combined 99mTc-sestamibi SPECT/CT and in situ metabolomic profiling can accurately characterise renal tumours exhibiting 99mTc-sestamibi uptake. Design, setting, and participants: A tissue microarray analysis of 33 tumour samples from 28 patients was used to investigate whether their in situ metabolomic status correlates with their features on 99mTc-sestamibi SPECT/CT examination. In order to validate emerging data, an independent cohort comprising 117 tumours was subjected to matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI MSI). Outcome measurements and statistical analysis: MALDI MSI data analysis and image generation were facilitated by FlexImaging v. 4.2, while k-means analysis by SCiLS Lab software followed by R-package CARRoT analysis was used for assessing the highest predictive power in the differential of RO versus chRCC. Heatmap-based clustering, sparse partial least-squares discriminant analysis, and volcano plots were created with MetaboAnalyst 3.0. Results and limitations: We identified a discriminatory metabolomic signature for 99mTc-sestamibi SPECT/CT–positive Birt-Hogg-Dubè–associated HOCTs versus other renal oncocytic tumours. Metabolomic differences were also evident between 99mTc-sestamibi–positive and 99mTc-sestamibi–negative chRCCs, prompting additional expert review; two of three 99mTc-sestamibi–positive chRCCs were reclassified as low-grade oncocytic tumours (LOTs). Differences were identified between distal-derived tumours from those of proximal tubule origin, including differences between ROs and chRCCs. Conclusions: The current study expands the spectrum of 99mTc-sestamibi SPECT/CT–positive renal tumours, encompassing ROs, HOCTs, LOTs, and chRCCs, and supports the feasibility of in situ metabolomic profiling in the diagnostics and classification of renal tumours. Patient summary: For preoperative evaluation of solid renal tumours, 99mTc-sestamibi single photon emission computed tomography/computed tomography (SPECT/CT) is a novel examination method. To increase diagnostic accuracy, we propose that 99mTc-sestamibi–positive renal tumours should be biopsied and followed by a combined histometabolomic analysis.
Wissenschaftlicher Artikel
Scientific Article
Vohra, T. ; Kemter, E. ; Sun, N. ; Dobenecker, B. ; Hinrichs, A. ; Burrello, J. ; Gomez-Sanchez, E.P. ; Gomez-Sanchez, C.E. ; Wang, J. ; Kinker, I.S. ; Teupser, D. ; Fischer, K. ; Schnieke, A. ; Peitzsch, M. ; Eisenhofer, G. ; Walch, A.K. ; Reincke, M. ; Wolf, E. ; Williams, T.A.
Hypertension 76, 1769-1777 (2020)
Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (P=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (P<0.001) and an almost 3-fold upregulation of CYP11B (P<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.
Wissenschaftlicher Artikel
Scientific Article
Wu, F. ; Cheng, Y. ; Wu, L. ; Zhang, W. ; Zheng, W. ; Wang, Q. ; Cao, H. ; Pan, X. ; Tang, W.
Front. Oncol. 10:575037 (2020)
The metabolic reprogramming of cancer tissue has higher metabolic activity than surrounding tissues. At the same time, the local infiltration of immunosuppressive cells is also significantly increased, resulting in a significant decrease in tumor immunity. During the progression of cancer cells, immunosuppressive tumor microenvironment is formed around the tumor due to their metabolic reprogramming. In addition, it is the changes in metabolic patterns that make tumor cells resistant to certain drugs, impeding cancer treatment. This article reviews the mechanisms of immune escape caused by metabolic reprogramming, and aims to provide new ideas for clinical tumor immunotherapy combined with metabolic intervention for tumor treatment.
Review
Review
Zettler, S. ; Renner, S. ; Kemter, E. ; Hinrichs, A. ; Klymiuk, N. ; Backman, M. ; Riedel, E.O. ; Mueller, C. ; Streckel, E. ; Braun-Reichhart, C. ; Martins, A.S. ; Kurome, M. ; Keßler, B. ; Zakhartchenko, V. ; Flenkenthaler, F. ; Arnold, G.J. ; Fröhlich, T. ; Blum, H. ; Blutke, A. ; Wanke, R. ; Wolf, E.
Anim. Reprod. 17:e20200064 (2020)
The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes. Findings in rodent models are consequently often difficult to extrapolate to human clinical trials. To overcome this 'translational gap', we and other groups are developing porcine disease models. Pigs share many anatomical and physiological traits with humans and thus hold great promise as translational animal models. Importantly, the toolbox for genetic engineering of pigs is rapidly expanding. Human disease mechanisms and targets can therefore be reproduced in pigs on a molecular level, resulting in precise and predictive porcine (PPP) models. In this short review, we summarize our work on the development of genetically (pre)diabetic pig models and how they have been used to study disease mechanisms and test therapeutic strategies. This includes the generation of reporter pigs for studying beta-cell maturation and physiology. Furthermore, genetically engineered pigs are promising donors of pancreatic islets for xenotransplantation. In summary, genetically tailored pig models have become an important link in the chain of translational diabetes and metabolic research.
Review
Review
Chhabra, N.F. ; Amarie, O.V. ; Wu, M. ; Amend, A.-L. ; Rubey, M. ; Gradinger, D. ; Irmler, M. ; Beckers, J. ; Rathkolb, B. ; Wolf, E. ; Feuchtinger, A. ; Huypens, P. ; Teperino, R. ; Rozman, J. ; Przemeck, G.K.H. ; Hrabě de Angelis, M.
Comm. Biol. 3:628 (2020)
The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep-wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6(Leca2) mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite beta cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for beta cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6. Nirav Chhabra et al. characterize adult mice carrying a homozygous mutation in Pax6 that was identified in a patient with foveal hypoplasia. They find that the Pax6 point mutation has pleiotropic effects, including defects in the mouse retinal structures, loss of the optic nerve, changes in energy metabolism and circadian rhythms, and dysregulation of genes expressed in the pancreas.
Wissenschaftlicher Artikel
Scientific Article
Azimzadeh, O. ; Azizova, T. ; Merl-Pham, J. ; Blutke, A. ; Moseeva, M. ; Zubkova, O. ; Anastasov, N. ; Feuchtinger, A. ; Hauck, S.M. ; Atkinson, M.J. ; Tapio, S.
Int. J. Mol. Sci. 21:6832 (2020)
Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.
Wissenschaftlicher Artikel
Scientific Article
Neef, S.K. ; Winter, S. ; Hofmann, U. ; Mürdter, T.E. ; Schaeffeler, E. ; Horn, H. ; Buck, A. ; Walch, A.K. ; Hennenlotter, J. ; Ott, G. ; Fend, F. ; Bedke, J. ; Schwab, M. ; Haag, M.
Anal. Chim. Acta 1134, 125-135 (2020)
Formalin-fixed and paraffin-embedded (FFPE) tissue represents a valuable resource to examine cancer metabolic alterations and to identify potential markers of disease. Protocols commonly used for liquid-chromatography mass spectrometry (LC-MS)-based FFPE metabolomics have not been optimized for lipidomic analysis and pre-analytical factors, that potentially affect metabolite levels, were scarcely investigated. We here demonstrate the assessment and optimization of sample preparation procedures for comprehensive metabolomic and lipidomic profiling in FFPE kidney tissue by LC-QTOF-MS. The optimized protocol allows improved monitoring of lipids including ceramides (Cer), glycosphingolipids (GSL) and triglycerides (TAGs) while the profiling capability for small polar molecules is maintained. Further, repeatable sample preparation (CVs < 20%) along with high analytical (CVs < 10%) and inter-day precision (CVs < 20%) is achieved. As proof of concept, we analyzed a set of clear cell renal cell carcinoma (ccRCC) and corresponding non-tumorous FFPE tissue samples, achieving phenotypic distinction. Investigation of the impact of tissue fixation time (6 h, 30 h and 54 h) on FFPE tissue metabolic profiles revealed metabolite class-dependent differences on their detection abundance. Whereas specific lipids (e.g. phosphatidylinositoles, GSLs, saturated fatty acids and saturated lyso-phosphatidytlethanolamines LPED remained largely unaffected (CVs < 20% between groups of fixation time), neutral lipids (e.g. Cer and TAGs) exhibited high variability (CVs > 80%). Strikingly, out of the lipid classes assigned as unaffected, fatty acids 18:0, 16:0 and LPE 18:0 were detectable by high-resolution MALDI-FT-ICR MS imaging in an independent cohort of ccRCC tissues (n = 64) and exhibited significant differences between tumor and non-tumor regions.
Wissenschaftlicher Artikel
Scientific Article
Blutke, A. ; Sun, N. ; Xu, Z. ; Buck, A. ; Harrison, L. ; Schriever, S.C. ; Pfluger, P.T. ; Wiles, D. ; Kunzke, T. ; Huber, K. ; Schlegel, J. ; Aichler, M. ; Feuchtinger, A. ; Matiasek, K. ; Hauck, S.M. ; Walch, A.K.
Sci. Rep. 10:14461 (2020)
Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. Multimodal combinations of LSFM with additional analyses of the identical sample help to limit the consumption of restricted specimen and reduce inter-sample variation. Here, we demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Samples of freshly dissected murine brain and of archived formalin-fixed paraffin-embedded (FFPE) human brain tissue were cleared (3DISCO). Tissue regions of interest were defined by LSFM and excised, (re)-embedded in paraffin, and sectioned. Mouse sections were coated with sinapinic acid matrix. Human brain sections were pre-digested with trypsin and coated with α-cyano-4-hydroxycinnamic acid matrix. Subsequently, sections were subjected to MALDI-time-of-flight (TOF)-MSI in mass ranges between 0.8 to 4 kDa (human tissue sections), or 2.5–25 kDa (mouse tissue sections) with a lateral resolution of 50 µm. Protein- and peptide-identities corresponding to acquired MALDI-MSI spectra were confirmed by parallel liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The spatial abundance- and intensity-patterns of established marker proteins detected by MALDI-MSI were also confirmed by immunohistochemistry.
Wissenschaftlicher Artikel
Scientific Article
Geng, X. ; Babayeva, L. ; Walch, A.K. ; Aubele, M. ; Gross, E. ; Kiechle, M. ; Bronger, H. ; Dreyer, T. ; Magdolen, V. ; Dorn, J.
Am. J. Cancer Res. 10, 1785-1792 (2020)
In normal physiology, kallikrein-related peptidase 7 (KLK7), together with other members of the kallikrein-related peptidase family, is mainly involved in skin desquamation and keratinization processes. Moreover, expression of KLK7 was shown in various tumor types to be dysregulated and to correlate to patients' survival time. However, there are contradictory reports in breast cancer whether KLK7 represents an unfavorable or favorable prognostic biomarker. In the present study, we examined the prognostic value of KLK7 protein expression in triple-negative breast cancer (TNBC), determined by immunohistochemistry (IHC). A cohort encompassing 133 TNBC specimens, present on tissue microarrays, was analyzed. For quantification of the staining intensity, an automated digital IHC image analysis algorithm was applied. In both Kaplan-Meier and univariate Cox analyses, elevated KLK7 protein levels were significantly linked with prolonged overall survival (OS). In multivariable Cox analysis, addition of KLK7 immunoreactivity scores to the base model (including the clinical parameters age, tumor size, and nodal status) demonstrated that KLK7 protein expression remained as a statistically significant, independent parameter for prolonged OS. These results strongly indicate that KLK7 is a favorable prognostic biomarker in triple negative breast cancer.
Wissenschaftlicher Artikel
Scientific Article
Turcu, A.F. ; Walch, A.K.
Lancet Diabet. Endocrinol. 8, 733-734 (2020)
Sonstiges: Meinungsartikel
Other: Opinion
Ščupáková, K. ; Dewez, F. ; Walch, A.K. ; Heeren, R.M.A. ; Balluff, B.
Angew. Chem.-Int. Edit. 132, 17600-17603 (2020)
The large-scale and label-free molecular characterization of single cells in their natural tissue habitat remains a major challenge in molecular biology. We present a method that integrates morphometric image analysis to delineate and classify individual cells with their single-cell-specific molecular profiles. This approach provides a new means to study spatial biological processes such as cancer field effects and the relationship between morphometric and molecular features.
Wissenschaftlicher Artikel
Scientific Article
Fischer, A. ; Koopmans, T. ; Ramesh, P. ; Christ, S. ; Strunz, M. ; Wannemacher, J. ; Aichler, M. ; Feuchtinger, A. ; Walch, A.K. ; Ansari, M. ; Theis, F.J. ; Schorpp, K.K. ; Hadian, K. ; Neumann, P.A. ; Schiller, H. B. ; Rinkevich, Y.
Nat. Commun. 11:3068 (2020)
Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.
Wissenschaftlicher Artikel
Scientific Article
Niu, Z. ; Sarkar, R. ; Aichler, M. ; Wester, H. ; Yousefi, B.H. ; Reif, B.
ChemBioChem 21, 2495-2502 (2020)
Positron emission tomography (PET) tracer molecules like thioflavin T specifically recognize amyloid deposition in brain tissue by selective binding to hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The molecular basis of this interaction is, however, not well understood. We have employed magic angle spinning (MAS) solid-state NMR spectroscopy to characterize Aβ-PET tracer complexes at atomic resolution. We established a titration protocol by using bovine serum albumin as a carrier to transfer hydrophobic small molecules to Aβ(1-40) fibrillar aggregates. The same Aβ(1-40) amyloid fibril sample was employed in subsequent titrations to minimize systematic errors that potentially arise from sample preparation. In the experiments, the small molecules 13C-methylated Pittsburgh compound B (PiB) as well as a novel Aβ tracer based on a diarylbithiazole (DABTA) scaffold were employed. Classical 13C-detected as well as proton-detected spectra of protonated and perdeuterated samples with back-substituted protons, respectively, were acquired and analyzed. After titration of the tracers, chemical-shift perturbations were observed in the loop region involving residues Gly25-Lys28 and Ile32-Gly33, thus suggesting that the PET tracer molecules interact with the loop region connecting β-sheets β1 and β2 in Aβ fibrils. We found that titration of the PiB derivatives suppressed fibril polymorphism and stabilized the amyloid fibril structure.
Wissenschaftlicher Artikel
Scientific Article
Li, F. ; Feuchtinger, A. ; Walch, A.K. ; Sun, N.
Horm. Metab. Res. 52, 435-447 (2020)
The adrenal gland integrates catecholamine-producing neuroendocrine cells and steroid-producing cells with mesenchymal origin in a structured manner under one capsule and is a key regulator for vital bioactivity. In addition to adrenal-specific disease, dysregulation of adrenal hormones is associated with systemic effects, leading to undesirable metabolic and cardiovascular consequences. Mass spectrometry imaging (MSI) technique can simultaneously measure a broad range of biomolecules, including metabolites and hormones, which has enabled the study of tissue metabolic and hormone alterations in adrenal and adrenal-related diseases. Furthermore, this technique coupled with labeled immunohistochemistry staining has enabled the study of the pathophysiological adaptation of the adrenal gland under normal and abnormal conditions at different molecular levels. This review discusses the recent applications of in situ MSI in the adrenal gland. For example, the combination of formalin-fixed paraffin-embedded tissue microarray and MSI to tissues from patient cohorts has facilitated the discovery of clinically relevant prognostic biomolecules and generated promising hypotheses for new sights into physiology and pathophysiology of adrenal gland. MSI also has enabled the discovery of clinically significant tissue molecular (i. e., biomarker) and pathway changes in adrenal disease, particularly in adrenal tumors. In addition, MSI has advanced the ability to optimally identify and detect adrenal gland specific molecules. Thus, as a novel analytical methodology, MSI has provided unprecedented capabilities for in situ tissue study.
Review
Review
Bergmann, N. ; Delbridge, C. ; Gempt, J. ; Feuchtinger, A. ; Walch, A.K. ; Schirmer, L. ; Bunk, W. ; Aschenbrenner, T. ; Liesche-Starnecker, F. ; Schlegel, J.
Front. Oncol. 10:494 (2020)
Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor in adults. Despite extensive therapy the prognosis for GBM patients remains poor and the extraordinary therapy resistance has been attributed to intertumoral heterogeneity of glioblastoma. Different prognostic relevant GBM tumor subtypes have been identified based on their molecular profile. This approach, however, neglects the heterogeneity within individual tumors, that is, the intratumoral heterogeneity. Here, we detected the regional immunoreactivity by immunohistochemistry and immunofluorescence using nine different markers on resected GBM specimens (IDH wildtype, WHO grade IV). We found repetitive expression profiles, that could be classified into clusters. These clusters could then be assigned to five pathophysiologically relevant groups that reflect the previously described subclasses of GBM, including mesenchymal, classical, and proneural subtype. Our data indicate the presence of tumor differentiations and tumor subclasses that occur within individual tumors, and might therefore contribute to develop adapted, individual-based therapies.
Wissenschaftlicher Artikel
Scientific Article
Hedegger, K. ; Algül, H. ; Lesina, M. ; Blutke, A. ; Schmid, R.M. ; Schneider, M.R. ; Dahlhoff, M.
Mol. Oncol. 14, 1653-1669 (2020)
Pancreatic ductal adenocarcinoma (PDAC) will soon belong to the top three cancer killers. The only approved specific PDAC therapy targets the epidermal growth factor receptor (EGFR). Although EGFR is a crucial player in PDAC development, EGFR-based therapy is disappointing. In this study, we evaluated the role of the EGFR ligand betacellulin (BTC) in PDAC. The expression of BTC was investigated in human pancreatic cancer specimen. Then, we generated a BTC knockout mouse model by CRISPR/Cas9 technology and a BTC overexpression model. Both models were crossed with the Ptf1a(Cre/+);KRAS(G12D/+) (KC) mouse model (B-/-KC or BKC, respectively). In addition, EGFR, ERBB2, and ERBB4 were investigated by the pancreas-specific deletion of each receptor using the Cre-loxP system. Tumor initiation and progression were analyzed in all mouse lines, and the underlying molecular biology of PDAC was investigated at different time points. BTC is expressed in human and murine PDAC. B-/-KC mice showed a decelerated PDAC progression, associated with decreased EGFR activation. BKC mice developed severe PDAC with a poor survival rate. The dramatically increased BTC-mediated tumor burden was EGFR-dependent, but also ERBB4 and ERBB2 were involved in PDAC development or progression, as depletion of EGFR, ERBB2, or ERBB4 significantly improved the survival rate of BTC-mediated PDAC. BTC increases PDAC tumor burden dramatically by enhanced RAS activation. EGFR signaling, ERBB2 signaling, and ERBB4 signaling are involved in accelerated PDAC development mediated by BTC indicating that targeting the whole ERBB family, instead of a single receptor, is a promising strategy for the development of future PDAC therapies.
Wissenschaftlicher Artikel
Scientific Article
Prade, V.M. ; Kunzke, T. ; Feuchtinger, A. ; Rohm, M. ; Luber, B. ; Lordick, F. ; Buck, A. ; Walch, A.K.
Mol. Metab. 36:100953 (2020)
Background: Imaging mass spectrometry enables in situ label-free detection of thousands of metabolites from intact tissue samples. However, automated steps for multi-omics analyses and interpretation of histological images have not yet been implemented in mass spectrometry data analysis workflows. The characterization of molecular properties within cellular and histological features is done via time-consuming, nonobjective, and irreproducible definitions of regions of interest, which are often accompanied by a loss of spatial resolution due to mass spectra averaging.Methods: We developed a new imaging pipeline called Spatial Correlation Image Analysis (SPACiAL), which is a computational multimodal workflow designed to combine molecular imaging data with multiplex immunohistochemistry (IHC). SPACiAL allows comprehensive and spatially resolved in situ correlation analyses on a cellular resolution. To demonstrate the method, matrix-assisted laser desorption-ionization (MALDI) Fourier-transform ion cyclotron resonance (FTICR) imaging mass spectrometry of metabolites and multiplex IHC staining were performed on the very same tissue section of mouse pancreatic islets and on human gastric cancer tissue specimens. The SPACiAL pipeline was used to perform an automatic, semantic-based, functional tissue annotation of histological and cellular features to identify metabolic profiles. Spatial correlation networks were generated to analyze metabolic heterogeneity associated with cellular features.Results: To demonstrate the new method, the SPACiAL pipeline was used to identify metabolic signatures of alpha and beta cells within islets of Langerhans, which are cell types that are not distinguishable via morphology alone. The semantic-based, functional tissue annotation allows an unprecedented analysis of metabolic heterogeneity via the generation of spatial correlation networks. Additionally, we demonstrated intra- and intertumoral metabolic heterogeneity within HER2/neu-positive and -negative gastric tumor cells.Conclusions: We developed the SPACiAL workflow to provide IHC-guided in situ metabolomics on intact tissue sections. Diminishing the workload by automated recognition of histological and functional features, the pipeline allows comprehensive analyses of metabolic heterogeneity. The multimodality of immunohistochemical staining and extensive molecular information from imaging mass spectrometry has the advantage of increasing both the efficiency and precision for spatially resolved analyses of specific cell types. The SPACiAL method is a stepping stone for the objective analysis of high-throughput, multi-omics data from clinical research and practice that is required for diagnostics, biomarker discovery, or therapy response prediction.
Wissenschaftlicher Artikel
Scientific Article
Khaloian, S. ; Rath, E. ; Hammoudi, N. ; Gleisinger, E. ; Blutke, A. ; Giesbertz, P. ; Berger, E. ; Metwaly, A. ; Waldschmitt, N. ; Allez, M. ; Haller, D.
Gut 69, 1939-1951 (2020)
Objective Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5(+) intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.Design Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60(Delta/Delta ISC)) and CD-like ileitis (TNF Delta ARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.Results In patients with CD and TNF Delta ARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased Lgr5 expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNF Delta ARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNF Delta ARE mice-derived crypts to form organoids.Conclusion We provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.
Wissenschaftlicher Artikel
Scientific Article
Sachs, S. ; Bastidas-Ponce, A. ; Tritschler, S. ; Bakhti, M. ; Böttcher, A. ; Sánchez-Garrido, M.A. ; Tarquis Medina, M. ; Kleinert, M. ; Fischer, K. ; Jall, S. ; Harger, A. ; Bader, E. ; Roscioni, S. ; Ussar, S. ; Feuchtinger, A. ; Yesildag, B. ; Neelakandhan, A. ; Jensen, C.B. ; Cornu, M. ; Yang, B. ; Finan, B. ; DiMarchi, R.D. ; Tschöp, M.H. ; Theis, F.J. ; Hofmann, S.M. ; Müller, T.D. ; Lickert, H.
Nat. Metab. 2, 380 (2020)
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Huang, Y.M. ; Hong, X.Z. ; Shen, J. ; Huang, Y. ; Zhao, H.L.
J. Am. Geriatr. Soc. 68, 940-942 (2020)
Wissenschaftlicher Artikel
Scientific Article
Weigand, I. ; Schreiner, J. ; Röhrig, F. ; Sun, N. ; Landwehr, L.S. ; Urlaub, H. ; Kendl, S. ; Kiseljak-Vassiliades, K. ; Wierman, M.E. ; Angeli, J.P.F. ; Walch, A.K. ; Sbiera, S. ; Fassnacht, M. ; Kroiss, M.
Cell Death Dis. 11:192 (2020)
Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 x 10(-8), 8.1 x 10(-7) and 2.1 x 10(-8) M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.
Wissenschaftlicher Artikel
Scientific Article
Sun, N. ; Meyer, L.S. ; Feuchtinger, A. ; Kunzke, T. ; Knösel, T. ; Reincke, M. ; Walch, A.K. ; Williams, T.A.
Hypertension 75, 634-644 (2020)
Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.
Wissenschaftlicher Artikel
Scientific Article
Riedel, E.O. ; Hinrichs, A. ; Kemter, E. ; Dahlhoff, M. ; Backman, M. ; Rathkolb, B. ; Prehn, C. ; Adamski, J. ; Renner, S. ; Blutke, A. ; Hrabě de Angelis, M. ; Bidlingmaier, M. ; Schopohl, J. ; Arnold, G.J. ; Fröhlich, T. ; Wolf, E.
Mol. Metab. 36:100978 (2020)
Objective: The liver is a central target organ of growth hormone (GH), which stimulates the synthesis of insulin-like growth factor 1 (IGF1) and affects multiple biochemical pathways. A systematic multi-omics analysis of GH effects in the liver has not been performed. GH receptor (GHR) deficiency is a unique model for studying the consequences of lacking GH action. In this study, we used molecular profiling techniques to capture a broad spectrum of these effects in the liver of a clinically relevant large animal model for Laron syndrome.Methods: We performed holistic proteome and targeted metabolome analyses of liver samples from 6-month-old GHR-deficient (GHR-KO) pigs and GHR-expressing controls (four males, four females per group).Results: GHR deficiency resulted in an increased abundance of enzymes involved in amino acid degradation, in the urea cycle, and in the tricarboxylic acid cycle. A decreased ratio of long-chain acylcarnitines to free carnitine suggested reduced activity of carnitine palmitoyltransferase 1A and thus reduced mitochondrial import of fatty acids for beta-oxidation. Increased levels of short-chain acylcarnitines in the liver and in the circulation of GHR-KO pigs may result from impaired beta-oxidation of short-chain fatty acids or from increased degradation of specific amino acids. The concentration of mono-unsaturated glycerophosphocholines was significantly increased in the liver of GHR-KO pigs without morphological signs of steatosis, although the abundances of several proteins functionally linked to non-alcoholic fatty liver disease (fetuin B, retinol binding protein 4, several mitochondrial proteins) were increased. Moreover, GHR-deficient liver samples revealed distinct changes in the methionine and glutathione metabolic pathways, in particular, a significantly increased level of glycine N-methyltransferase and increased levels of total and free glutathione. Several proteins revealed a sex-related abundance difference in the control group but not in the GHR-KO group.Conclusions: Our integrated proteomics/targeted metabolomics study of GHR-deficient and control liver samples from a clinically relevant large animal model identified a spectrum of biological pathways that are significantly altered in the absence of GH action. Moreover, new insights into the role of GH in the sex-related specification of liver functions were provided.
Wissenschaftlicher Artikel
Scientific Article
Lohöfer, F. ; Buchholz, R. ; Glinzer, A. ; Huber, K. ; Haas, H. ; Kaissis, G. ; Feuchtinger, A. ; Aichler, M. ; Sporns, P.B. ; Höltke, C. ; Stölting, M. ; Schilling, F. ; Botnar, R.M. ; Kimm, M.A. ; Faber, C. ; Walch, A.K. ; Zernecke, A. ; Karst, U. ; Wildgruber, M.
Sci. Rep. 10:79 (2020)
Molecular imaging of atherosclerosis by Magnetic Resonance Imaging (MRI) has been impaired by a lack of validation of the specific substrate responsible for the molecular imaging signal. We therefore aimed to investigate the additive value of mass spectrometry imaging (MSI) of atherosclerosis-affine Gadofluorine P for molecular MRI of atherosclerotic plaques. Atherosclerotic Ldlr(-/-) mice were investigated by high-field MRI (7T) at different time points following injection of atherosclerosis-affine Gadofluorine P as well as at different stages of atherosclerosis formation (4, 8, 16 and 20 weeks of HFD). At each imaging time point mice were immediately sacrificed after imaging and aortas were excised for mass spectrometry imaging: Matrix Assisted Laser Desorption Ionization (MALDI) Imaging and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. Mass spectrometry imaging allowed to visualize the localization and measure the concentration of the MR imaging probe Gadofluorine P in plaque tissue ex vivo with high spatial resolution and thus adds novel and more target specific information to molecular MR imaging of atherosclerosis.
Wissenschaftlicher Artikel
Scientific Article
Schüttrumpf, L. ; Marschner, S. ; Scheu, K. ; Hess-Rieger, J. ; Rietzler, S. ; Walch, A.K. ; Baumeister, P. ; Kirchner, T. ; Ganswindt, U. ; Zitzelsberger, H. ; Belka, C. ; Maihoefer, C.
Radiat. Oncol. 15:7 (2020)
Background Definitive chemoradiotherapy (dCRT) is a standard treatment for patients with locally advanced head and neck cancer. There is a clinical need for a stratification of this prognostically heterogeneous group of tumors in order to optimize treatment of individual patients. We retrospectively reviewed all patients with head and neck squamous cell carcinoma (HNSCC) of the oral cavity, oropharynx, hypopharynx, or larynx, treated with dCRT from 09/2008 until 03/2016 at the Department of Radiation Oncology, LMU Munich. Here we report the clinical results of the cohort which represent the basis for biomarker discovery and molecular genetic research within the framework of a clinical cooperation group. Methods Patient data were collected and analyzed for outcome and treatment failures with regard to previously described and established risk factors. Results We identified 184 patients with a median follow-up of 65 months and a median age of 64 years. Patients received dCRT with a median dose of 70 Gy and simultaneous chemotherapy in 90.2% of cases, mostly mitomycin C / 5-FU in concordance with the ARO 95-06 trial. The actuarial 3-year overall survival (OS), local, locoregional and distant failure rates were 42.7, 29.8, 34.0 and 23.4%, respectively. Human papillomavirus-associated oropharynx cancer (HPVOPC) and smaller gross tumor volume were associated with significantly improved locoregional tumor control rate, disease-free survival (DFS) and OS in multivariate analysis. Additionally, lower hemoglobin levels were significantly associated with impaired DFS und OS in univariate analysis. The extent of lymph node involvement was associated with distant failure, DFS and OS. Moreover, 92 patients (50%) of our cohort have been treated in concordance with the ARO 95-06 study, corroborating the results of this study. Conclusion Our cohort is a large unselected monocentric cohort of HNSCC patients treated with dCRT. Tumor control rates and survival rates compare favorably with the results of previously published reports. The clinical data, together with the available tumor samples from biopsies, will allow translational research based on molecular genetic analyses.
Wissenschaftlicher Artikel
Scientific Article
Renner, S. ; Blutke, A. ; Clauss, S. ; Deeg, C.A. ; Kemter, E. ; Merkus, D. ; Wanke, R. ; Wolf, E.
Cell Tissue Res. 380, 341-378 (2020)
The worldwide prevalence of diabetes mellitus and obesity is rapidly increasing not only in adults but also in children and adolescents. Diabetes is associated with macrovascular complications increasing the risk for cardiovascular disease and stroke, as well as microvascular complications leading to diabetic nephropathy, retinopathy and neuropathy. Animal models are essential for studying disease mechanisms and for developing and testing diagnostic procedures and therapeutic strategies. Rodent models are most widely used but have limitations in translational research. Porcine models have the potential to bridge the gap between basic studies and clinical trials in human patients. This article provides an overview of concepts for the development of porcine models for diabetes and obesity research, with a focus on genetically engineered models. Diabetes-associated ocular, cardiovascular and renal alterations observed in diabetic pig models are summarized and their similarities with complications in diabetic patients are discussed. Systematic multi-organ biobanking of porcine models of diabetes and obesity and molecular profiling of representative tissue samples on different levels, e.g., on the transcriptome, proteome, or metabolome level, is proposed as a strategy for discovering tissue-specific pathomechanisms and their molecular key drivers using systems biology tools. This is exemplified by a recent study providing multi-omics insights into functional changes of the liver in a transgenic pig model for insulin-deficient diabetes mellitus. Collectively, these approaches will provide a better understanding of organ crosstalk in diabetes mellitus and eventually reveal new molecular targets for the prevention, early diagnosis and treatment of diabetes mellitus and its associated complications.
Review
Review
Sachs, S. ; Bastidas-Ponce, A. ; Tritschler, S. ; Bakhti, M. ; Böttcher, A. ; Sánchez-Garrido, M.A. ; Tarquis Medina, M. ; Kleinert, M. ; Fischer, K. ; Jall, S. ; Harger, A. ; Bader, E. ; Roscioni, S. ; Ussar, S. ; Feuchtinger, A. ; Yesildag, B. ; Neelakandhan, A. ; Jensen, C.B. ; Cornu, M. ; Yang, B. ; Finan, B. ; DiMarchi, R.D. ; Tschöp, M.H. ; Theis, F.J. ; Hofmann, S.M. ; Müller, T.D. ; Lickert, H.
Nat. Metab. 2, 192-209 (2020)
Dedifferentiation of insulin-secreting β cells in the islets of Langerhans has been proposed to be a major mechanism of β-cell dysfunction. Whether dedifferentiated β cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study β-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with β-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in β cells and restores maturation and function for diabetes remission. Additional β-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases β-cell survival and regeneration. GLP-1-oestrogen also protects human β cells against cytokine-induced dysfunction. This study not only describes mechanisms of β-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated β cells for diabetes remission.
Wissenschaftlicher Artikel
Scientific Article
Hofmann, I. ; Kemter, E. ; Theobalt, N. ; Fiedler, S. ; Bidlingmaier, M. ; Hinrichs, A. ; Aichler, M. ; Burkhardt, K. ; Klymiuk, N. ; Wolf, E. ; Wanke, R. ; Blutke, A.
Growth Horm. IGF Res. 51, 6-16 (2020)
Objective: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model.Methods: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods.Results: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls.Conclusion: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.
Wissenschaftlicher Artikel
Scientific Article
Kunzke, T. ; Buck, A. ; Prade, V.M. ; Feuchtinger, A. ; Prokopchuk, O. ; Martignoni, M.E. ; Heisz, S. ; Hauner, H. ; Janssen, K.P. ; Walch, A.K. ; Aichler, M.
J. Cachexia Sarcopenia Muscle 11, 226-240 (2020)
Background Cachexia is the direct cause of at least 20% of cancer-associated deaths. Muscle wasting in skeletal muscle results in weakness, immobility, and death secondary to impaired respiratory muscle function. Muscle proteins are massively degraded in cachexia; nevertheless, the molecular mechanisms related to this process are poorly understood. Previous studies have reported conflicting results regarding the amino acid abundances in cachectic skeletal muscle tissues. There is a clear need to identify the molecular processes of muscle metabolism in the context of cachexia, especially how different types of molecules are involved in the muscle wasting process. Methods New in situ -omics techniques were used to produce a more comprehensive picture of amino acid metabolism in cachectic muscles by determining the quantities of amino acids, proteins, and cellular metabolites. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging, we determined the in situ concentrations of amino acids and proteins, as well as energy and other cellular metabolites, in skeletal muscle tissues from genetic mouse cancer models (n = 21) and from patients with cancer (n = 6). Combined results from three individual MALDI mass spectrometry imaging methods were obtained and interpreted. Immunohistochemistry staining for mitochondrial proteins and myosin heavy chain expression, digital image analysis, and transmission electron microscopy complemented the MALDI mass spectrometry imaging results. Results Metabolic derangements in cachectic mouse muscle tissues were detected, with significantly increased quantities of lysine, arginine, proline, and tyrosine (P = 0.0037, P = 0.0048, P = 0.0430, and P = 0.0357, respectively) and significantly reduced quantities of glutamate and aspartate (P = 0.0008 and P = 0.0124). Human skeletal muscle tissues revealed similar tendencies. A majority of altered amino acids were released by the breakdown of proteins involved in oxidative phosphorylation. Decreased energy charge was observed in cachectic muscle tissues (P = 0.0101), which was related to the breakdown of specific proteins. Additionally, expression of the cationic amino acid transporter CAT1 was significantly decreased in the mitochondria of cachectic mouse muscles (P = 0.0133); this decrease may play an important role in the alterations of cationic amino acid metabolism and decreased quantity of glutamate observed in cachexia. Conclusions Our results suggest that mitochondrial dysfunction has a substantial influence on amino acid metabolism in cachectic skeletal muscles, which appears to be triggered by diminished CAT1 expression, as well as the degradation of mitochondrial proteins. These findings provide new insights into the pathobiochemistry of muscle wasting.
Wissenschaftlicher Artikel
Scientific Article
Dombrowsky, A. ; Burger, K. ; Porth, A.K. ; Stein, M. ; Dierolf, M. ; Günther, B. ; Achterhold, K. ; Gleich, B. ; Feuchtinger, A. ; Bartzsch, S. ; Beyreuther, E. ; Combs, S.E. ; Pfeiffer, F. ; Wilkens, J.J. ; Schmid, T.E.
Radiat. Environ. Biophys. 59, 111-120 (2020)
Microbeam radiation therapy (MRT), a preclinical form of spatially fractionated radiotherapy, uses an array of microbeams of hard synchrotron X-ray radiation. Recently, compact synchrotron X-ray sources got more attention as they provide essential prerequisites for the translation of MRT into clinics while overcoming the limited access to synchrotron facilities. At the Munich compact light source (MuCLS), one of these novel compact X-ray facilities, a proof of principle experiment was conducted applying MRT to a xenograft tumor mouse model. First, subcutaneous tumors derived from the established squamous carcinoma cell line FaDu were irradiated at a conventional X-ray tube using broadbeam geometry to determine a suitable dose range for the tumor growth delay. For irradiations at the MuCLS, FaDu tumors were irradiated with broadbeam and microbeam irradiation at integral doses of either 3 Gy or 5 Gy and tumor growth delay was measured. Microbeams had a width of 50 µm and a center-to-center distance of 350 µm with peak doses of either 21 Gy or 35 Gy. A dose rate of up to 5 Gy/min was delivered to the tumor. Both doses and modalities delayed the tumor growth compared to a sham-irradiated tumor. The irradiated area and microbeam pattern were verified by staining of the DNA double-strand break marker γH2AX. This study demonstrates for the first time that MRT can be successfully performed in vivo at compact inverse Compton sources.
Wissenschaftlicher Artikel
Scientific Article
Altamura, S. ; Vegi, N.M. ; Hoppe, P.S. ; Schroeder, T. ; Aichler, M. ; Walch, A.K. ; Okreglicka, K. ; Hültner, L. ; Schneider, M. ; Ladinig, C. ; Kuklik-Roos, C. ; Mysliwietz, J. ; Janik, D. ; Neff, F. ; Rathkolb, B. ; Hrabě de Angelis, M. ; Buske, C. ; da Silva, A.R. ; Muedder, K. ; Conrad, M. ; Ganz, T. ; Kopf, M. ; Muckenthaler, M.U. ; Bornkamm, G.W.
Haematologica 105, 937-950 (2020)
Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing . oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the ation of mitochondria but is now known to occur through mitophary Yet, ggenetic ablation of the Alox15 gene in mice failed to provice evidence or this hyppothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullaty erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.
Wissenschaftlicher Artikel
Scientific Article
2019
Kreutzer, L. ; Aichler, M. ; Walch, A.K.
In: Fundamentals and Applications of Fourier Transform Mass Spectrometry. 2019. 253-279
This chapter introduces the in situ investigation on metabolomics in cancer tissues by high-resolution mass spectrometry imaging. Metabolomics is a rapidly increasing field, since the detection of biochemical processes can improve the diagnostic, therapeutic treatment prediction, and prognosis in diseases such as cancer. By analyzing metabolic alterations in cancer tissues, insights into the pathway regulations and the resulting clinical outcome can be obtained and associated. In recent years, especially high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was an emerging technique in the analysis of metabolite molecular data and their spatial distribution in tissues. The main intentions in the combination of metabolomics with MALDI MSI are the discovery of molecular biomarkers and metabolic pathways altered in tumors, as well as therapy response prediction and method development. Therefore, increasing numbers of studies were published recently, investigating the optimization of the MSI methods, overcoming the current difficulties and studying the role of clinical translation.
Baumann, P. ; Schriever, S.C. ; Kullmann, S. ; Zimprich, A. ; Feuchtinger, A. ; Amarie, O.V. ; Peter, A. ; Walch, A.K. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Wurst, W. ; Tschöp, M.H. ; Heni, M. ; Hölter, S.M. ; Pfluger, P.T.
Sci. Rep. 9:19483 (2019)
Dual-specificity phosphatase 8 (Dusp8) acts as physiological inhibitor for the MAPKs Jnk, Erk and p38 which are involved in regulating multiple CNS processes. While Dusp8 expression levels are high in limbic areas such as the hippocampus, the functional role of Dusp8 in hippocampus morphology, MAPK-signaling, neurogenesis and apoptosis as well as in behavior are still unclear. It is of particular interest whether human carriers of a DUSP8 allelic variant show similar hippocampal alterations to mice. Addressing these questions using Dusp8WT and KO mouse littermates, we found that KOs suffered from mildly impaired spatial learning, increased locomotor activity and elevated anxiety. Cell proliferation, apoptosis and p38 and Jnk phosphorylation were unaffected, but phospho-Erk levels were higher in hippocampi of the KOs. Consistent with a decreased hippocampus size in Dusp8 KO mice, we found reduced volumes of the hippocampal subregions subiculum and CA4 in humans carrying the DUSP8 allelic variant SNP rs2334499:C > T. Overall, aberrations in morphology and behavior in Dusp8 KO mice and a decrease in hippocampal volume of SNP rs2334499:C > T carriers point to a novel, translationally relevant role of Dusp8 in hippocampus function that warrants further studies on the role of Dusp8 within the limbic network.
Wissenschaftlicher Artikel
Scientific Article
Hladik, D. ; Buratovic, S. ; von Toerne, C. ; Azimzadeh, O. ; Subedi, P. ; Philipp, J. ; Winkler, S. ; Feuchtinger, A. ; Samson, E. ; Hauck, S.M. ; Stenerlöw, B. ; Eriksson, P. ; Atkinson, M.J. ; Tapio, S.
Int. J. Mol. Sci. 20:2103 (2019)
In children, ketamine sedation is often used during radiological procedures. Combined exposure of ketamine and radiation at doses that alone did not affect learning and memory induced permanent cognitive impairment in mice. The aim of this study was to elucidate the mechanism behind this adverse outcome. Neonatal male NMRI mice were administered ketamine (7.5 mg kg(-1)) and irradiated (whole-body, 100 mGy or 200 mGy, Cs-137) one hour after ketamine exposure on postnatal day 10. The control mice were injected with saline and sham-irradiated. The hippocampi were analyzed using label-free proteomics, immunoblotting, and Golgi staining of CA1 neurons six months after treatment. Mice co-exposed to ketamine and low-dose radiation showed alterations in hippocampal proteins related to neuronal shaping and synaptic plasticity. The expression of brain-derived neurotrophic factor, activity-regulated cytoskeleton-associated protein, and postsynaptic density protein 95 were significantly altered only after the combined treatment (100 mGy or 200 mGy combined with ketamine, respectively). Increased numbers of basal dendrites and branching were observed only after the co-exposure, thereby constituting a possible reason for the displayed alterations in behavior. These data suggest that the risk of radiation-induced neurotoxic effects in the pediatric population may be underestimated if based only on the radiation dose.
Wissenschaftlicher Artikel
Scientific Article
Correa-Gallegos, D. ; Jiang, D. ; Christ, S. ; Ramesh, P. ; Ye, H. ; Wannemacher, J. ; Kalgudde Gopal, S. ; Yu, Q. ; Aichler, M. ; Walch, A.K. ; Mirastschijski, U. ; Volz, T. ; Rinkevich, Y.
Nature 576, 287-292 (2019)
Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism(1-5). Here we show that skin scars originate from prefabricated matrix in the subcutaneous fascia. Fate mapping and live imaging revealed that fascia fibroblasts rise to the skin surface after wounding, dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages and peripheral nerves, to form the provisional matrix. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in defective scars, whereas placing an impermeable film beneath the skin-preventing fascia fibroblasts from migrating upwards-led to chronic open wounds. Thus, fascia contains a specialized prefabricated kit of sentry fibroblasts, embedded within a movable sealant, that preassemble together diverse cell types and matrix components needed to heal wounds. Our findings suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.
Wissenschaftlicher Artikel
Scientific Article
Yang, L. ; Gradl, R. ; Dierolf, M. ; Möller, W. ; Kutschke, D. ; Feuchtinger, A. ; Hehn, L. ; Donnelley, M. ; Günther, B. ; Achterhold, K. ; Walch, A.K. ; Stöger, T. ; Razansky, D. ; Pfeiffer, F. ; Morgan, K.S. ; Schmid, O.
Small 15:1904112 (2019)
Targeted delivery of nanomedicine/nanoparticles (NM/NPs) to the site of disease (e.g., the tumor or lung injury) is of vital importance for improved therapeutic efficacy. Multimodal imaging platforms provide powerful tools for monitoring delivery and tissue distribution of drugs and NM/NPs. This study introduces a preclinical imaging platform combining X-ray (two modes) and fluorescence imaging (three modes) techniques for time-resolved in vivo and spatially resolved ex vivo visualization of mouse lungs during pulmonary NP delivery. Liquid mixtures of iodine (contrast agent for X-ray) and/or (nano)particles (X-ray absorbing and/or fluorescent) are delivered to different regions of the lung via intratracheal instillation, nasal aspiration, and ventilator-assisted aerosol inhalation. It is demonstrated that in vivo propagation-based phase-contrast X-ray imaging elucidates the dynamic process of pulmonary NP delivery, while ex vivo fluorescence imaging (e.g., tissue-cleared light sheet fluorescence microscopy) reveals the quantitative 3D drug/particle distribution throughout the entire lung with cellular resolution. The novel and complementary information from this imaging platform unveils the dynamics and mechanisms of pulmonary NM/NP delivery and deposition for each of the delivery routes, which provides guidance on optimizing pulmonary delivery techniques and novel-designed NM for targeting and efficacy.
Wissenschaftlicher Artikel
Scientific Article
Seitz, S. ; Kwon, Y. ; Hartleben, G. ; Jülg, J. ; Sekar, R. ; Krahmer, N. ; Najafi, B. ; Loft, A. ; Gancheva, S. ; Stemmer, K. ; Feuchtinger, A. ; Hrabě de Angelis, M. ; Müller, T.D. ; Mann, M. ; Blüher, M. ; Roden, M. ; Berriel Diaz, M. ; Behrends, C. ; Gilleron, J. ; Herzig, S. ; Zeigerer, A.
Nat. Metab. 1, 1009-1026 (2019)
Non-alcoholic fatty liver disease (NAFLD) represents a key feature of obesity-related type 2 diabetes with increasing prevalence worldwide. To our knowledge, no treatment options are available to date, paving the way for more severe liver damage, including cirrhosis and hepatocellular carcinoma. Here, we show an unexpected function for an intracellular trafficking regulator, the small Rab GTPase Rab24, in mitochondrial fission and activation, which has an immediate impact on hepatic and systemic energy homeostasis. RAB24 is highly upregulated in the livers of obese patients with NAFLD and positively correlates with increased body fat in humans. Liver-selective inhibition of Rab24 increases autophagic flux and mitochondrial connectivity, leading to a strong improvement in hepatic steatosis and a reduction in serum glucose and cholesterol levels in obese mice. Our study highlights a potential therapeutic application of trafficking regulators, such as RAB24, for NAFLD and establishes a conceptual functional connection between intracellular transport and systemic metabolic dysfunction.
Wissenschaftlicher Artikel
Scientific Article
Stutte, S. ; Ruf, J. ; Kugler, I. ; Ishikawa-Ankerhold, H. ; Blutke, A. ; Marconi, P. ; Maeda, T. ; Kaisho, T. ; Krug, A. ; Lauterbach, H. ; Colonna, M. ; von Andrian, U. ; Brocker, T.
Eur. J. Immunol. 49, 69-70 (2019)
Meeting abstract
Meeting abstract
Arndt, D. ; Fux, R. ; Blutke, A. ; Schwaiger, J. ; El-Matbouli, M. ; Sutter, G. ; Langenmayer, M.C.
Pathogens 8:177 (2019)
For many years, brown trout (Salmo trutta fario) mortalities within the pre-alpine Isar River in Germany were reported by the Bavarian Fisheries Association (Landesfischereiverband Bayern e.V.) and local recreational anglers during August and September. Moribund fish seemed to be affected by proliferative darkening syndrome (PDS). In addition, proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae was discussed. To investigate this phenomenon, the present field study monitored brown trout mortalities by daily river inspection in 2017 and 2018. Moribund brown trout (n = 31) were collected and examined using histology, immunohistochemistry, qPCR, and quantitative stereology. Our investigations identified 29 (93.5%) brown trout affected by PKD. Four brown trout (12.9%) displayed combined hepatic and splenic lesions fitting the pathology of PDS. The piscine orthoreovirus 3, suspected as causative agent of PDS, was not detectable in any of the samples. Quantitative stereological analysis of the kidneys revealed a significant increase of the renal tissue volumes with interstitial inflammation and hematopoietic hyperplasia in PKD-affected fish as compared to healthy brown trout. The identified T. bryosalmonae strain was classified as part of the North American clade by phylogenetical analysis. This study highlights PKD and PDS as contributing factors to recurrent autumnal brown trout mortalities.
Wissenschaftlicher Artikel
Scientific Article
Sammer, M. ; Teiluf, K. ; Girst, S. ; Greubel, C. ; Reindl, J. ; Ilicic, K. ; Walsh, D.W.M. ; Aichler, M. ; Walch, A.K. ; Combs, S.E. ; Wilkens, J.J. ; Dollinger, G. ; Schmid, T.E.
PLoS ONE 14:e0221454 (2019)
Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams >= 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Sub-millimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation.
Wissenschaftlicher Artikel
Scientific Article
Wagner, A. ; Hofmeister, O. ; Rolland, S.G. ; Maiser, A. ; Aasumets, K. ; Schmitt, S. ; Schorpp, K.K. ; Feuchtinger, A. ; Hadian, K. ; Schneider, S. ; Zischka, H. ; Leonhardt, H. ; Conradt, B. ; Gerhold, J.M. ; Wolf, A.
J. Cell Sci. 132:jcs223891 (2019)
The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m(3)C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N-1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f(5)C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM-microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.
Wissenschaftlicher Artikel
Scientific Article
Murakami, M. ; Rhayem, Y. ; Kunzke, T. ; Sun, N. ; Feuchtinger, A. ; Ludwig, P. ; Strom, T.M. ; Gomez-Sanchez, C. ; Knösel, T. ; Kirchner, T. ; Williams, T.A. ; Reincke, M. ; Walch, A.K. ; Beuschlein, F.
JCI insight 4, DOI: 10.1172/jci.insight.130356 (2019)
Recent genetic examinations and multisteroid profiles have provided the basis for subclassification of aldosterone-producing adenomas (APAs). The objective of the current study was to produce a comprehensive, high-resolution mass spectrometry imaging (MSI) map of APAs in relation to morphometry, immunohistochemical profiles, mutational status, and clinical outcome. The study cohort comprised 136 patients with unilateral primary aldosteronism. Matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI was conducted, and metabolite profiles were analyzed with genotype/phenotype information, including digital image analysis from morphometry and IHC of steroidogenic enzymes. Distinct molecular signatures between KCNJ5- and CACNA1D-mutated APAs with significant differences of 137 metabolites, including metabolites of purine metabolism and steroidogenesis, were observed. Intratumor concentration of 18-oxocortisol and 18-hydroxycortisol were inversely correlated with the staining intensity of CYP11B1. Lower staining intensity of CYP11B1 and higher levels of 18-oxocortisol were associated with a higher probability of complete clinical success after surgery. The present study demonstrates distinct metabolomic profiles of APAs in relation to tumor genotype. In addition, we reveal an inverse correlation between cortisol derivatives and CYP11B1 and the impact of 18-oxocortisol and CYP11B1 on clinical outcome, which provides unprecedented insights into the pathophysiology, clinical features, and steroidogenesis of APAs.
Wissenschaftlicher Artikel
Scientific Article
Sun, N. ; Kunzke, T. ; Sbiera, S. ; Kircher, S. ; Feuchtinger, A. ; Aichler, M. ; Herterich, S. ; Ronchi, C.L. ; Weigand, I. ; Schlegel, N. ; Waldmann, J. ; Fragoso, M.C.V. ; Whitsett, T.G. ; Gill, A.J. ; Fassnacht, M. ; Walch, A.K. ; Kroiss, M.
Clin. Chem. 65, 1276-1286 (2019)
BACKGROUND: Adrenocortical carcinoma (ACC) is a rare tumor with variable prognosis even within the same tumor stage. Cancer-related sex hormones and their sulfated metabolites in body fluids can be used as tumor markers. The role of steroid sulfation in ACC has not yet been studied. MALDI mass spectrometry imaging (MALDI-MSI) is a novel tool for tissue-based chemical phenotyping.METHODS: We performed phenotyping of formalin-fixed, paraffin-embedded tissue samples from 72 ACC by MALDI-MSI at a metabolomics level.RESULTS: Tumoral steroid hormone metabolites-estradiol sulfate [hazard ratio (HR) 0.26; 95% CI, 0.10-0.69; P = 0.005] and estrone 3-sulfate (HR 0.22; 95% CI, 0.07-0.63; P = 0.003)-were significantly associated with prognosis in Kaplan-Meier analyses and after multivariable adjustment for age, tumor stage, and sex (HR 0.29; 95% CI, 0.11-0.79; P= 0.015 and HR 0.30; 95% CI, 0.10-0.91; P = 0.033, respectively). Expression of sulfotransferase SULT2A1 was associated with prognosis to a similar extent and was validated to be a prognostic factor in two published data sets. We discovered the presence of estradiol-17 beta 3,17-disulfate (E2S2) in a subset of tumors with particularly poor overall survival. Electron microscopy revealed novel membrane-delimited organelles in only these tumors. By applying duster analyses of metabolomic data, 3 sulfation-related phenotypes exhibited specific metabolic features unrelated to steroid metabolism.CONCLUSIONS: MALDI-MSI provides novel insights into the pathophysiology of ACC. Steroid hormone sulfation may be used for prognostication and treatment stratification. Sulfation-related metabolic reprogramming may be of relevance also in conditions beyond the rare ACC and can be directly investigated by the use of MALDI-MSI.
Wissenschaftlicher Artikel
Scientific Article
Siebert, C. ; Ciato, D. ; Murakami, M. ; Frei-Stuber, L. ; Perez-Rivas, L.G. ; Monteserin-Garcia, J.L. ; Noelting, S. ; Maurer, J. ; Feuchtinger, A. ; Walch, A.K. ; Haak, H.R. ; Bertherat, J. ; Mannelli, M. ; Fassnacht, M. ; Korpershoek, E. ; Reincke, M. ; Stalla, G.K. ; Hantel, C. ; Beuschlein, F.
Front. Endocrin. 10:487 (2019)
Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC.Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines.Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90 beta. Within a cohort of ACC patients, HSP90 beta expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2-and AKT-pathways by luminespib and ganetespib treatment.Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC.
Wissenschaftlicher Artikel
Scientific Article
Renner, S. ; Martins, A.S. ; Streckel, E. ; Braun-Reichhart, C. ; Backman, M. ; Prehn, C. ; Klymiuk, N. ; Bähr, A. ; Blutke, A. ; Landbrecht-Schessl, C. ; Wünsch, A. ; Kessler, B. ; Kurome, M. ; Hinrichs, A. ; Koopmans, S.J. ; Krebs, S. ; Kemter, E. ; Rathkolb, B. ; Nagashima, H. ; Blum, H. ; Ritzmann, M. ; Wanke, R. ; Aigner, B. ; Adamski, J. ; Hrabě de Angelis, M. ; Wolf, E.
Dis. Model. Mech. 12:dmm039156 (2019)
Alongside the obesity epidemic, the prevalence of maternal diabetes is rising worldwide, and adverse effects on fetal development and metabolic disturbances in the offspring's later life have been described. To clarify whether metabolic programming effects are due to mild maternal hyperglycemia without confounding obesity, we investigated wild-type offspring of INSC93S transgenic pigs, which are a novel genetically modified large-animal model expressing mutant insulin (INS) C93S in pancreatic beta-cells. This mutation results in impaired glucose tolerance, mild fasting hyperglycemia and insulin resistance during late pregnancy. Compared with offspring from wildtype sows, piglets from hyperglycemic mothers showed impaired glucose tolerance and insulin resistance (homeostatic model assessment of insulin resistance: +3-fold in males; +4.4-fold in females) prior to colostrum uptake. Targeted metabolomics in the fasting and insulin-stimulated state revealed distinct alterations in the plasma metabolic profile of piglets from hyperglycemic mothers. They showed increased levels of acylcarnitines, gluconeogenic precursors such as alanine, phospholipids (in particular lysophosphatidylcholines) and a-aminoadipic acid, a potential biomarker for type 2 diabetes. These observations indicate that mild gestational hyperglycemia can cause impaired glucose tolerance, insulin resistance and associated metabolic alterations in neonatal offspring of a large-animal model born at a developmental maturation status comparable to human babies.
Wissenschaftlicher Artikel
Scientific Article
Rehm, S.R.T. ; Smirnova, N.F. ; Morrone, C. ; Götzfried, J. ; Feuchtinger, A. ; Pedersen, J. ; Korkmaz, B. ; Yildirim, A.Ö. ; Jenne, D.
Sci. Rep. 9:9925 (2019)
Neutrophil serine proteases (NSPs), like proteinase 3 (PR3) and neutrophil elastase (NE) are implicated in ischemia-reperfusion responses after lung transplantation (LTx). Cathepsin C (CatC) acts as the key regulator of NSP maturation during biosynthesis. We hypothesized that CatC inhibitors would reduce vascular breakdown and inflammation during reperfusion in pretreated lung transplant recipients by blocking NSP maturation in the bone marrow. An orthotopic LTx model in mice was used to mimic the induction of an ischemia-reperfusion response after 18 h cold storage of the graft and LTx. Recipient mice were treated subcutaneously with a chemical CatC inhibitor (ICatC) for 10 days prior to LTx. We examined the effect of the ICatC treatment by measuring the gas exchange function of the left lung graft, protein content, neutrophil numbers and NSP activities in the bone marrow 4 h after reperfusion. Pre-operative ICatC treatment of the recipient mice improved early graft function and lead to the disappearance of active NSP protein in the transplanted lung. NSP activities were also substantially reduced in bone marrow neutrophils. Preemptive NSP reduction by CatC inhibition may prove to be a viable and effective approach to reduce immediate ischemia reperfusion responses after LTx.
Wissenschaftlicher Artikel
Scientific Article
Yang, L. ; Gradl, R. ; Feuchtinger, A. ; Morgan, K.S. ; Kutschke, D. ; Stöger, T. ; Razansky, D. ; Walch, A.K. ; Pfeiffer, F. ; Schmidt, O.
J. Aerosol Med. Pulm. Drug Deliv. 32, A12-A12 (2019)
Meeting abstract
Meeting abstract
Sigmund, F. ; Pettinger, S. ; Kube, M. ; Schneider, F. ; Schifferer, M. ; Schneider, S. ; Efremova, M.V. ; Pujol-Martí, J. ; Aichler, M. ; Walch, A.K. ; Misgeld, T. ; Dietz, H. ; Westmeyer, G.G.
ACS Nano 13, 8114-8123 (2019)
Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of similar to 43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.
Wissenschaftlicher Artikel
Scientific Article
Schneider, A. ; Kurz, S. ; Manske, K. ; Janas, M.K. ; Heikenwälder, M. ; Misgeld, T. ; Aichler, M. ; Weissmann, S.F. ; Zischka, H. ; Knolle, P. ; Wohlleber, D.
Sci. Rep. 9:8492 (2019)
Mitochondria are key for cellular metabolism and signalling processes during viral infection. We report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. Resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. Thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies.
Wissenschaftlicher Artikel
Scientific Article
Wintergerst, L. ; Selmansberger, M. ; Maihoefer, C. ; Schuettrumpf, L. ; Walch, A.K. ; Wilke, C. ; Pitea, A. ; Woischke, C. ; Baumeister, P. ; Kirchner, T. ; Belka, C. ; Ganswindt, U. ; Zitzelsberger, H. ; Unger, K. ; Hess-Rieger, J.
Strahlenther. Onkol. 195, S24-S25 (2019)
Meeting abstract
Meeting abstract
Hess-Rieger, J. ; Unger, K. ; Maihoefer, C. ; Schuettrumpf, L. ; Schneider, L. ; Heider, T. ; Weber, P. ; Marschner, S. ; Braselmann, H. ; Kuger, S. ; Pflugradt, U. ; Baumeister, P. ; Walch, A.K. ; Woischke, C. ; Kirchner, T. ; Werner, M. ; Werner, K. ; Baumann, M. ; Budach, V. ; Combs, S.E. ; Debus, J. ; Grosu, A.-L. ; Krause, M. ; Roedel, C. ; Stuschke, M. ; Zips, D. ; Zitzelsberger, H. ; Ganswindt, U. ; Henke, M. ; Belka, C.
Strahlenther. Onkol. 195, S25-S25 (2019)
Meeting abstract
Meeting abstract
Wiesner, M. ; Glawischnig, W. ; Lutzmann, I. ; Grimm, F. ; Blutke, A.
Wien. Tierärztl. Mschr. 106, 109-115 (2019)
Captive primates are susceptible to infection with cestodes of the family of Taeniidae. This report describes the infection of a five-year-old female ring tailed-lemur (Lemur catta) in Salzburg Zoo (Salzburg, Austria) with Taenia crassiceps. Necropsy revealed extensive amounts of organized and free cysts in the thoracic cavity, completely encasing and compressing the lungs and the heart. Infection probably occurred by oral uptake of Taenia crassiceps eggs from faeces of red fox (Vulpes vulpes) within the zoo or in the surrounding park, where the lemurs roam freely. Two foxes shot in the vicinity of the zoo were confirmed to have an intestinal infection with Taenia crassiceps. The simultaneous detection of Taenia crassiceps tapeworms in a natural definite host (fox) and of their metacestodes in an accidental intermediate host provides evidence of an autochthonous infection. Compared to other zoo primates, ring-tailed lemurs (Lemur catta) seem to be highly susceptible to infection with Taenia crassiceps. Therapy and preventive methods are discussed.
Wissenschaftlicher Artikel
Scientific Article
Huber, K. ; Kunzke, T. ; Buck, A. ; Langer, R. ; Luber, B. ; Feuchtinger, A. ; Walch, A.K.
Lab. Invest. 99, 1535-1546 (2019)
Multimodal tissue analyses that combine two or more detection technologies provide synergistic value compared to single methods and are employed increasingly in the field of tissue-based diagnostics and research. Here, we report a technical pipeline that describes a combined approach of HER2/CEP17 fluorescence in situ hybridization (FISH) analysis with MALDI imaging on the very same section of formalin-fixed and paraffin-embedded (FFPE) tissue. FFPE biopsies and a tissue microarray of human gastroesophageal adenocarcinoma were analyzed by MALDI imaging. Subsequently, the very same section was hybridized by HER2/CEP17 FISH. We found that tissue morphology of both, the biopsies and the tissue microarray, was unaffected by MALDI imaging and the HER2 and CEP17 FISH signals were analyzable. In comparison with FISH analysis of samples without MALDI imaging, we observed no difference in terms of fluorescence signal intensity and gene copy number. Our combined approach revealed adenosine monophosphate, measured by MALDI imaging, as a prognostic marker. HER2 amplification, which was detected by FISH, is a stratifier between good and poor patient prognosis. By integrating both stratification parameters on the basis of our combined approach, we were able to strikingly improve the prognostic effect. Combining molecules detected by MALDI imaging with the gene copy number detected by HER2/CEP17 FISH, we found a synergistic effect, which enhances patient prognosis. This study shows that our combined approach allows the detection of genetic and metabolic properties from one very same FFPE tissue section, which are specific for HER2 and hence suitable for prognosis. Furthermore, this synergism might be useful for response prediction in tumors.
Wissenschaftlicher Artikel
Scientific Article
von Gamm, M. ; Schaub, A. ; Jones, A. ; Wolf, C. ; Behrens, G. ; Lichti, J. ; Essig, K. ; Macht, A. ; Pircher, J. ; Ehrlich, A. ; Davari, K. ; Chauhan, D. ; Busch, B. ; Wurst, W. ; Feederle, R. ; Feuchtinger, A. ; Tschöp, M.H. ; Friedel, C.C. ; Hauck, S.M. ; Sattler, M. ; Geerlof, A. ; Hornung, V. ; Heissmeyer, V. ; Schulz, C. ; Heikenwalder, M. ; Glasmacher, E.
J. Exp. Med. 216, 1700-1723 (2019)
The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3-deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell-specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a (Regnase-1), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages.
Wissenschaftlicher Artikel
Scientific Article
Backman, M. ; Flenkenthaler, F. ; Blutke, A. ; Dahlhoff, M. ; Ländström, E. ; Renner, S. ; Philippou-Massier, J. ; Krebs, S. ; Rathkolb, B. ; Prehn, C. ; Grzybek, M. ; Coskun, Ü. ; Rothe, M. ; Adamski, J. ; Hrabě de Angelis, M. ; Wanke, R. ; Fröhlich, T. ; Arnold, G.J. ; Blum, H. ; Wolf, E.
Mol. Metab. 26, 30-44 (2019)
Objective: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically.Methods: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics.Results: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples.Conclusions: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets. (C) 2019 The Authors. Published by Elsevier GmbH.
Wissenschaftlicher Artikel
Scientific Article
Dombrowsky, A. ; Schauer, J. ; Sammer, M. ; Blutke, A. ; Walsh, D.W.M. ; Schwarz, B. ; Bartzsch, S. ; Feuchtinger, A. ; Reindl, J. ; Combs, S.E. ; Dollinger, G. ; Schmid, T.E.
Cancers 11:727 (2019)
The use of different scoring systems for radiation-induced toxicity limits comparability between studies. We examined dose-dependent tissue alterations following hypofractionated X-ray irradiation and evaluated their use as scoring criteria. Four dose fractions (0, 5, 10, 20, 30 Gy/fraction) were applied daily to ear pinnae. Acute effects (ear thickness, erythema, desquamation) were monitored for 92 days after fraction 1. Late effects (chronic inflammation, fibrosis) and the presence of transforming growth factor beta 1 (TGF beta 1)-expressing cells were quantified on day 92. The maximum ear thickness displayed a significant positive correlation with fractional dose. Increased ear thickness and erythema occurred simultaneously, followed by desquamation from day 10 onwards. A significant dose-dependency was observed for the severity of erythema, but not for desquamation. After 4 x 20 and 4 x 30 Gy, inflammation was significantly increased on day 92, whereas fibrosis and the abundance of TGF beta 1-expressing cells were only marginally increased after 4 x 30 Gy. Ear thickness significantly correlated with the severity of inflammation and fibrosis on day 92, but not with the number of TGF beta 1-expressing cells. Fibrosis correlated significantly with inflammation and fractional dose. In conclusion, the parameter of ear thickness can be used as an objective, numerical and dose-dependent quantification criterion to characterize the severity of acute toxicity and allow for the prediction of late effects.
Wissenschaftlicher Artikel
Scientific Article
Bechmann, N. ; Poser, I. ; Seifert, V. ; Greunke, C. ; Ullrich, M. ; Qin, N. ; Walch, A.K. ; Peitzsch, M. ; Robledo, M. ; Pacak, K. ; Pietzsch, J. ; Richter, S. ; Eisenhofer, G.
Cancers 11:594 (2019)
Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2 alpha (MPC and MTT) or expressing both Hif1 alpha and Hif2 alpha (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hifla- and Hif2 alpha-driven effects we expressed Hif2 alpha in MTT and MPC-mCherry cells (naturally lacking Hif2 alpha). Presence of Hif2 alpha resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1 alpha in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2 alpha under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIf1 alpha. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2 alpha-mediated phosphorylation of TH (permanent status).
Wissenschaftlicher Artikel
Scientific Article
Sperling, S. ; Fiedler, P. ; Lechner, M. ; Pollithy, A. ; Ehrenberg, S. ; Schiefer, A.I. ; Kenner, L. ; Feuchtinger, A. ; Kühn, R. ; Swinerd, G. ; Schmidt-Supprian, M. ; Strobl, L.J. ; Zimber-Strobl, U.
Blood 133, 2597-2609 (2019)
CD30 is expressed on a variety of B-cell lymphomas, such as Hodgkin lymphoma, primary effusion lymphoma, and a diffuse large B-cell lymphoma subgroup. In normal tissues, CD30 is expressed on some activated B and T lymphocytes. However, the physiological function of CD30 signaling and its contribution to the generation of CD30(+) lymphomas are still poorly understood. To gain a better understanding of CD30 signaling in B cells, we studied the expression of CD30 in different murine B-cell populations. We show that B1 cells expressed higher levels of CD30 than B2 cells and that CD30 was upregulated in IRF4(+) plasmablasts (PBs). Furthermore, we generated and analyzed mice expressing a constitutively active CD30 receptor in B lymphocytes. These mice displayed an increase in B1 cells in the peritoneal cavity (PerC) and secondary lymphoid organs as well as increased numbers of plasma cells (PCs). TI-2 immunization resulted in a further expansion of B1 cells and PCs. We provide evidence that the expanded B1 population in the spleen included a fraction of PBs. CD30 signals seemed to enhance PC differentiation by increasing activation of NF-kappa B and promoting higher levels of phosphorylated STAT3 and STAT6 and nuclear IRF4. In addition, chronic CD30 signaling led to B-cell lymphomagenesis in aged mice. These lymphomas were localized in the spleen and PerC and had a B1-like/plasmablastic phenotype. We conclude that our mouse model mirrors chronic B-cell activation with increased numbers of CD30(+) lymphocytes and provides experimental proof that chronic CD30 signaling increases the risk of B-cell lymphomagenesis.
Wissenschaftlicher Artikel
Scientific Article
Ratner, C. ; He, Z. ; Grunddal, K.V. ; Skov, L.J. ; Hartmann, B. ; Zhang, F. ; Feuchtinger, A. ; Bjerregaard, A. ; Christoffersen, C. ; Tschöp, M.H. ; Finan, B. ; DiMarchi, R.D. ; Leinninger, G.M. ; Williams, K.W. ; Clemmensen, C. ; Holst, B.
Diabetes 68, 1329-1340 (2019)
Neurotensin (NT), a gut hormone and neuropeptide, increases in circulation after bariatric surgery in rodents and humans and inhibits food intake in mice. However, its potential to treat obesity and the subsequent metabolic dysfunctions have been difficult to assess owing to its short half-life in vivo. Here, we demonstrate that a long-acting, pegylated analog of the NT peptide (P-NT) reduces food intake, body weight, and adiposity in diet-induced obese mice when administered once daily for 6 days. Strikingly, when P-NT was combined with the glucagon-like peptide 1 mimetic liraglutide, the two peptides syner-gized to reduce food intake and body weight relative to each monotherapy, without inducing a taste aversion. Further, P-NT and liraglutide coadministration improved glycemia and reduced steatohepatitis. Finally, we show that the melanocortin pathway is central for P-NT–induced anorexia and necessary for the full synergistic effect of P-NT and liraglutide combination therapy. Overall, our data suggest that P-NT and liraglutide combination therapy could be an enhanced treatment for obesity with improved tolerability compared with liraglutide monotherapy.
Wissenschaftlicher Artikel
Scientific Article
Gujrati, V. ; Prakash, J. ; Malekzadeh Najafabadi, J. ; Stiel, A.-C. ; Klemm, U. ; Mettenleiter, G. ; Aichler, M. ; Walch, A.K. ; Ntziachristos, V.
Nat. Commun. 10:1114 (2019)
Advances in genetic engineering have enabled the use of bacterial outer membrane vesicles (OMVs) to deliver vaccines, drugs and immunotherapy agents, as a strategy to circumvent biocompatibility and large-scale production issues associated with synthetic nanomaterials. We investigate bioengineered OMVs for contrast enhancement in optoacoustic (photoacoustic) imaging. We produce OMVs encapsulating biopolymer-melanin (OMVMel) using a bacterial strain expressing a tyrosinase transgene. Our results show that upon near-infrared light irradiation, OMVMel generates strong optoacoustic signals appropriate for imaging applications. In addition, we show that OMVMel builds up intense heat from the absorbed laser energy and mediates photothermal effects both in vitro and in vivo. Using multispectral optoacoustic tomography, we noninvasively monitor the spatio-temporal, tumour-associated OMVMel distribution in vivo. This work points to the use of bioengineered vesicles as potent alternatives to synthetic particles more commonly employed for optoacoustic imaging, with the potential to enable both image enhancement and photothermal applications.
Wissenschaftlicher Artikel
Scientific Article
Papathomas, T.G. ; Sun, N. ; Chortis, V. ; Taylor, A.E. ; Arlt, W. ; Richter, S. ; Eisenhofer, G. ; Ruiz-Babot, G. ; Guasti, L. ; Walch, A.K.
Histochem. Cell Biol. 151, 201-216 (2019)
Metabolic alterations have implications in a spectrum of tissue functions and disease. Aided by novel molecular biological and computational tools, our understanding of physiological and pathological processes underpinning endocrine and endocrine-related disease has significantly expanded over the last decade. Herein, we focus on novel metabolomics-related methodologies in adrenal research: in situ metabolomics by mass spectrometry imaging, steroid metabolomics by gas and liquid chromatography-mass spectrometry, energy pathway metabologenomics by liquid chromatography-mass spectrometry-based metabolomics of Krebs cycle intermediates, and cellular reprogramming to generate functional steroidogenic cells and hence to modulate the steroid metabolome. All four techniques to assess and/or modulate the metabolome in biological systems provide tremendous opportunities to manage neoplastic and non-neoplastic disease of the adrenal glands in the era of precision medicine. In this context, we discuss emerging clinical applications and/or promising metabolic-driven research towards diagnostic, prognostic, predictive and therapeutic biomarkers in tumours arising from the adrenal gland and extra-adrenal paraganglia as well as modern approaches to delineate and reprogram adrenal metabolism.
Review
Review
Ogrinc Wagner, A. ; Friedrich, V. ; Barthels, C. ; Marconi, P. ; Blutke, A. ; Brombacher, F. ; Brocker, T.
PLoS ONE 14:e0210998 (2019)
Intestinal integrity is maintained by balanced numbers of CD103(+) Dendritic cells (DCs), which generate peripherally induced regulatory T cells (iTregs). We have developed a mouse model where DC-specific constitutive CD40 signals caused a strong reduction of CD103(+) DCs in the lamina propria (LP) and intestinal lymph nodes (LN). As a consequence, also iTregs were strongly reduced and transgenic mice on the C57Bl/6-background (B6) developed fatal colitis. Here we describe that transgenic mice on a pure Balb/c-background (B/c) do not show any pathologies, while transgenic C57Bl/6 x Balb/c (F1) mice develop weak colon inflammation, without fatal colitis. This graded pathology correlated with the effects of CD40-signalling on DCs in each background, with striking loss of CD103(+) DCs in B6, but reduced in F1 and diminished in B/c background. We further show direct correlation of CD103(+) DC-numbers with numbers of iTregs, the frequencies of which behave correspondingly. Striking effects on B6-DCs reflected robust loss of surface MHCII, known to be crucial for iTreg induction. Furthermore, elevated levels of IL-23 together with IL-1, found only in B6 mice, support generation of intestinal IFN-gamma(+) IL-17(+) Th17 cells and IFN-gamma(+) Th1 cells, responsible for onset of disease. Together, this demonstrates a novel aspect of colitis-control, depending on genetic background. Moreover, strain-specific environmental sensing might alter the CD103(+) DC/iTreg-axis to tip intestinal homeostatic balance to pathology.
Wissenschaftlicher Artikel
Scientific Article
Norheim, F. ; Hasin-Brumshtein, Y. ; Vergnes, L. ; Chella Krishnan, K. ; Pan, C. ; Seldin, M.M. ; Hui, S.T. ; Mehrabian, M. ; Zhou, Z. ; Gupta, S. ; Parks, B.W. ; Walch, A.K. ; Reue, K. ; Hofmann, S.M. ; Arnold, A.P. ; Lusis, A.J.
Cell Metab. 29, 932-949.e4 (2019)
We studied sex differences in over 50 cardio-metabolic traits in a panel of 100 diverse inbred strains of mice. The results clearly showed that the effects of sex on both clinical phenotypes and gene expression depend on the genetic background. In support of this, genetic loci associated with the traits frequently showed sex specificity. For example, Lyplal1, a gene implicated in human obesity, was shown to underlie a sex-specific locus for diet induced obesity. Global gene expression analyses of tissues across the panel implicated adipose tissue "beiging" and mitochondrial functions in the sex differences. Isolated mitochondria showed gene-bysex interactions in oxidative functions, such that some strains (C57BL/6J) showed similar function between sexes, whereas others (DBA/2J and A/J) showed increased function in females. Reduced adipose mitochondria! function in males as compared to females was associated with increased susceptibility to obesity and insulin resistance. Gonadectomy studies indicated that gonadal hormones acting in a tissue-specific manner were responsible in part for the sex differences.
Wissenschaftlicher Artikel
Scientific Article
Balluff, B. ; Buck, A. ; Martin-Lorenzo, M. ; Dewez, F. ; Langer, R. ; McDonnell, L.A. ; Walch, A.K. ; Heeren, R.M.A.
Proteomics Clin. Appl. 13:e1800137 (2019)
Scope In biomedical research, mass spectrometry imaging (MSI) can obtain spatially-resolved molecular information from tissue sections. Especially matrix-assisted laser desorption/ionization (MALDI) MSI offers, depending on the type of matrix, the detection of a broad variety of molecules ranging from metabolites to proteins, thereby facilitating the collection of multilevel molecular data. Lately, integrative clustering techniques have been developed that make use of the complementary information of multilevel molecular data in order to better stratify patient cohorts, but which have not yet been applied in the field of MSI. Materials and Methods In this study, the potential of integrative clustering is investigated for multilevel molecular MSI data to subdivide cancer patients into different prognostic groups. Metabolomic and peptidomic data are obtained by MALDI-MSI from a tissue microarray containing material of 46 esophageal cancer patients. The integrative clustering methods Similarity Network Fusion, iCluster, and moCluster are applied and compared to non-integrated clustering. Conclusion The results show that the combination of multilevel molecular data increases the capability of integrative algorithms to detect patient subgroups with different clinical outcome, compared to the single level or concatenated data. This underlines the potential of multilevel molecular data from the same subject using MSI for subsequent integrative clustering.
Wissenschaftlicher Artikel
Scientific Article
Yang, L. ; Feuchtinger, A. ; Möller, W. ; Ding, Y. ; Kutschke, D. ; Möller, G. ; Schittny, J.C. ; Burgstaller, G. ; Hofmann, W. ; Stöger, T. ; Razansky, D. ; Walch, A.K. ; Schmid, O.
ACS Nano 13, 1029-1041 (2019)
Deciphering biodistribution, biokinetics, and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools. Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in nondissected ex viva murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure, and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 +/- 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 +/- 0.37) for instillation. After inhalation most NPs were observed in the proximal part of the acini as predicted by computational fluid dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues, facilitating both nanotoxicology studies and the development of nanomedicines.
Wissenschaftlicher Artikel
Scientific Article
Görgülü, K. ; Diakopoulos, K.N. ; Ai, J. ; Schoeps, B. ; Kabacaoglu, D. ; Karpathaki, A.F. ; Ciecielski, K.J. ; Kaya-Aksoy, E. ; Ruess, D.A. ; Berninger, A. ; Kowalska, M. ; Stevanovic, M. ; Wörmann, S.M. ; Wartmann, T. ; Zhao, Y. ; Halangk, W. ; Voronina, S. ; Tepikin, A. ; Schlitter, A.M. ; Steiger, K. ; Artati, A. ; Adamski, J. ; Aichler, M. ; Walch, A.K. ; Jastroch, M. ; Hartleben, G. ; Mantzoros, C.S. ; Weichert, W. ; Schmid, R.M. ; Herzig, S. ; Krüger, A. ; Sainz, B. ; Lesina, M. ; Algül, H.
Gastroenterology 156, 203-217.e20 (2019)
BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5; Kras) or heterozygous disruption of Atg5 (A5(+/-); Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5(+/-); Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5; Kras mice did not develop any tumors. Cultured A5(+/-); Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2thorn oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5(+/-); Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5(+/-); Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.
Wissenschaftlicher Artikel
Scientific Article
Harrison, L. ; Schriever, S.C. ; Feuchtinger, A. ; Kyriakou, E. ; Baumann, P. ; Pfuhlmann, K. ; Messias, A.C. ; Walch, A.K. ; Tschöp, M.H. ; Pfluger, P.T.
Int. J. Obes. 43, 1305-1318 (2019)
Background/objectives Individuals carrying loss-of-function gene mutations for the adipocyte hormone leptin are morbidly obese, but respond favorably to replacement therapy. Recombinant leptin is however largely ineffective for the vast majority of obese individuals due to leptin resistance. One theory underlying leptin resistance is impaired leptin transport across the blood-brain-barrier (BBB). Here, we aim to gain new insights into the mechanisms of leptin BBB transport, and its role in leptin resistance.Methods We developed a novel tool for visualizing leptin transport using infrared fluorescently labeled leptin, combined with tissue clearing and light-sheet fluorescence microscopy. We corroborated these data using western blotting.Results Using 3D whole brain imaging, we display comparable leptin accumulation in circumventricular organs of lean and obese mice, predominantly in the choroid plexus (CP). Protein quantification revealed comparable leptin levels in micro-dissected mediobasal hypothalami (MBH) of lean and obese mice (p = 0.99). We further found increased leptin receptor expression in the CP (p = 0.025, p = 0.0002) and a trend toward elevated leptin protein levels in the MBH (p = 0.17, p = 0.078) of obese mice undergoing weight loss interventions by calorie restriction or exendin-4 treatment.Conclusions Overall, our findings suggest a crucial role for the CP in controlling the transport of leptin into the cerebrospinal fluid and from there to target areas such as the MBH, potentially mediated via the leptin receptor. Similar leptin levels in circumventricular organs and the MBH of lean and obese mice further suggest intact leptin BBB transport in leptin resistant mice.
Wissenschaftlicher Artikel
Scientific Article
Hess-Rieger, J. ; Unger, K. ; Maihoefer, C. ; Schüttrumpf, L. ; Wintergerst, L. ; Heider, T. ; Weber, P. ; Marschner, S. ; Braselmann, H. ; Samaga, D. ; Kuger, S. ; Pflugradt, U. ; Baumeister, P. ; Walch, A.K. ; Woischke, C. ; Kirchner, T. ; Werner, M. ; Werner, K. ; Baumann, M. ; Budach, V. ; Combs, S.E. ; Debus, J. ; Grosu, A.-L. ; Krause, M. ; Linge, A. ; Rödel, C. ; Stuschke, M. ; Zips, D. ; Zitzelsberger, H. ; Ganswindt, U. ; Henke, M. ; Belka, C.
Clin. Cancer Res. 25, 1505-1516 (2019)
Purpose: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is associated with unfavorable prognosis, while independent prognostic markers remain to be defined.Experimental Design: We retrospectively performed miRNA expression profiling. Patients were operated for locally advanced HPV-negative HNSCC and had received radiochemotherapy in eight different hospitals (DKTK-ROG; n = 85). Selection fulfilled comparable demographic, treatment, and follow-up characteristics. Findings were validated in an independent single-center patient sample (LMU-KKG; n = 77). A prognostic miRNA signature was developed for freedom from recurrence and tested for other endpoints. Recursivepartitioning analysis was performed on the miRNA signature, tumor and nodal stage, and extracapsular nodal spread. Technical validation used qRT-PCR. An miRNA-mRNA target network was generated and analyzed.Results: For DKTK-ROG and LMU-KKG patients, the median follow-up was 5.1 and 5.3 years, and the 5-year freedom from recurrence rate was 63.5% and 75.3%, respectively. A five-miRNA signature (hsa-let-7g-3p, hsamiR- 6508-5p, hsa-miR-210-5p, hsa-miR-4306, and hsa-miR-7161-3p) predicted freedom from recurrence in DKTK-ROG [hazard ratio (HR) 4.42; 95% confidence interval (CI), 1.98-9.88, P < 0.001], which was confirmed in LMU-KKG (HR 4.24; 95% CI, 1.40-12.81, P = 0.005). The signature also predicted overall survival (HR 3.03; 95% CI, 1.50-6.12, P = 0.001), recurrence-free survival (HR 3.16; 95% CI, 1.65-6.04, P < 0.001), and disease-specific survival (HR 5.12; 95% CI, 1.88-13.92, P < 0.001), all confirmed in LMU-KKG data. Adjustment for relevant covariates maintained the miRNA signature predicting all endpoints. Recursive- partitioning analysis of both samples combined classified patients into low (n = 17), low-intermediate (n = 80), high-intermediate (n = 48), or high risk (n = 17) for recurrence (P < 0.001).Conclusions: The five-miRNA signature is a strong and independent prognostic factor for disease recurrence and survival of patients with HPV-negative HNSCC.
Wissenschaftlicher Artikel
Scientific Article
2018
Fernandez, I.E. ; Sun, N. ; Wei, M. ; Witting, M. ; Aichler, M. ; Verleden, S. ; Schmitt-Kopplin, P. ; Walch, A.K. ; Eickelberg, O.
Eur. Respir. J. 52 (2018)
Meeting abstract
Meeting abstract
Clemmensen, C. ; Jall, S. ; Kleinert, M. ; Quarta, C. ; Gruber, T. ; Reber, J. ; Sachs, S. ; Fischer, K. ; Feuchtinger, A. ; Karlas, A. ; Simonds, S.E. ; Grandl, G. ; Loher, D. ; Sanchez-Quant, E. ; Keipert, S. ; Jastroch, M. ; Hofmann, S.M. ; Nascimento, E.B.M. ; Schrauwen, P. ; Ntziachristos, V. ; Cowley, M.A. ; Finan, B. ; Müller, T.D. ; Tschöp, M.H.
Nat. Commun. 9:4975 (2018)
In the original PDF version of this article, affiliation 1, 'Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Muenchen & German Center for Diabetes Research (DZD), Neuherberg, Germany', was incorrectly given as 'Institute of Diabetes and Regeneration Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germany '. This has now been corrected in the PDF version of the article; the HTML version was correct at the time of publication.
Fernandez, I.E. ; Sun, N. ; Wei, M. ; Witting, M. ; Aichler, M. ; Verleden, S. ; Schmitt-Kopplin, P. ; Eickelberg, O. ; Walch, A.K.
Am. J. Respir. Crit. Care Med. 197 (2018)
Meeting abstract
Meeting abstract
Clemmensen, C. ; Jall, S. ; Kleinert, M. ; Quarta, C. ; Gruber, T. ; Reber, J. ; Sachs, S. ; Fischer, K. ; Feuchtinger, A. ; Karlas, A. ; Simonds, S.E. ; Grandl, G. ; Loher, D. ; Sanchez-Quant, E. ; Keipert, S. ; Jastroch, M. ; Hofmann, S.M. ; Nascimento, E.B.M. ; Schrauwen, P. ; Ntziachristos, V. ; Cowley, M.A. ; Finan, B. ; Müller, T.D. ; Tschöp, M.H.
Nat. Commun. 9:4304 (2018)
Pharmacological stimulation of brown adipose tissue (BAT) thermogenesis to increase energy expenditure is progressively being pursued as a viable anti-obesity strategy. Here, we report that pharmacological activation of the cold receptor transient receptor potential cation channel subfamily M member 8 (TRPM8) with agonist icilin mimics the metabolic benefits of cold exposure. In diet-induced obese (DIO) mice, treatment with icilin enhances energy expenditure, and decreases body weight, without affecting food intake. To further potentiate the thermogenic action profile of icilin and add complementary anorexigenic mechanisms, we set out to identify pharmacological partners next to icilin. To that end, we specifically targeted nicotinic acetylcholine receptor (nAChR) subtype alpha3beta4 (α3β4), which we had recognized as a potential regulator of energy homeostasis and glucose metabolism. Combinatorial targeting of TRPM8 and nAChR α3β4 by icilin and dimethylphenylpiperazinium (DMPP) orchestrates synergistic anorexic and thermogenic pathways to reverse diet-induced obesity, dyslipidemia, and glucose intolerance in DIO mice.
Wissenschaftlicher Artikel
Scientific Article
Wintergerst, L. ; Selmansberger, M. ; Maihoefer, C. ; Schüttrumpf, L. ; Walch, A.K. ; Wilke, C. ; Pitea, A. ; Woischke, C. ; Baumeister, P. ; Kirchner, T. ; Belka, C. ; Ganswindt, U. ; Zitzelsberger, H. ; Unger, K. ; Hess-Rieger, J.
Mol. Oncol., DOI: 10.1002/1878-0261.12388 (2018)
Previously, we have shown that copy number gain of the chromosomal band 16q24.3 is associated with impaired clinical outcome of radiotherapy-treated head and neck squamous cell carcinoma (HNSCC) patients. We set out to identify a prognostic mRNA signature from genes located on 16q24.3 in radio(chemo)therapy-treated HNSCC patients of the TCGA (The Cancer Genome Atlas, n = 99) cohort. We applied stepwise forward selection using expression data of 41 16q24.3 genes. The resulting optimal Cox-proportional hazards regression model included the genes APRT, CENPBD1, CHMP1A, and GALNS. Afterward, the prognostic value of the classifier was confirmed in an independent cohort of HNSCC patients treated by adjuvant radio(chemo)therapy (LMU-KKG cohort). The signature significantly differentiated high- and low-risk patients with regard to overall survival (HR = 2.01, 95% CI 1.10–3.70; P = 0.02125), recurrence-free survival (HR = 1.84, 95% CI 1.01–3.34; P = 0.04206), and locoregional recurrence-free survival (HR = 1.87, 95% CI 1.03–3.40; P = 0.03641). The functional impact of the four signature genes was investigated after reconstruction of a gene association network from transcriptome data of the TCGA HNSCC cohort using a partial correlation approach. Subsequent pathway enrichment analysis of the network neighborhood (first and second) of the signature genes suggests involvement of HNSCC-associated signaling pathways such as apoptosis, cell cycle, cell adhesion, EGFR, JAK-STAT, and mTOR. Furthermore, a detailed analysis of the first neighborhood revealed a cluster of co-expressed genes located on chromosome 16q, substantiating the impact of 16q24.3 alterations in poor clinical outcome of HNSCC. The reported gene expression signature represents a prognostic marker in HNSCC patients following postoperative radio(chemo)therapy.
Wissenschaftlicher Artikel
Scientific Article
Suwandhi, L. ; Hausmann, S. ; Braun, A. ; Gruber, T. ; Heinzmann, S.S. ; Gálvez, E.J.C. ; Buck, A. ; Legutko, B. ; Israel, A. ; Feuchtinger, A. ; Haythorne, E. ; Staiger, H. ; Heni, M. ; Häring, H.-U. ; Schmitt-Kopplin, P. ; Walch, A.K. ; Cáceres, C.G. ; Tschöp, M.H. ; Rutter, G.A. ; Strowig, T. ; Elsner, M. ; Ussar, S.
Mol. Metab., DOI: 10.1016/j.molmet.2018.07.002 (2018)
OBJECTIVE: The metabolic role of d-serine, a non-proteinogenic NMDA receptor co-agonist, is poorly understood. Conversely, inhibition of pancreatic NMDA receptors as well as loss of the d-serine producing enzyme serine racemase have been shown to modulate insulin secretion. Thus, we aim to study the impact of chronic and acute d-serine supplementation on insulin secretion and other parameters of glucose homeostasis. METHODS: We apply MALDI FT-ICR mass spectrometry imaging, NMR based metabolomics, 16s rRNA gene sequencing of gut microbiota in combination with a detailed physiological characterization to unravel the metabolic action of d-serine in mice acutely and chronically treated with 1% d-serine in drinking water in combination with either chow or high fat diet feeding. Moreover, we identify SNPs in SRR, the enzyme converting L-to d-serine and two subunits of the NMDA receptor to associate with insulin secretion in humans, based on the analysis of 2760 non-diabetic Caucasian individuals. RESULTS: We show that chronic elevation of d-serine results in reduced high fat diet intake. In addition, d-serine leads to diet-independent hyperglycemia due to blunted insulin secretion from pancreatic beta cells. Inhibition of alpha 2-adrenergic receptors rapidly restores glycemia and glucose tolerance in d-serine supplemented mice. Moreover, we show that single nucleotide polymorphisms (SNPs) in SRR as well as in individual NMDAR subunits are associated with insulin secretion in humans. CONCLUSION: Thus, we identify a novel role of d-serine in regulating systemic glucose metabolism through modulating insulin secretion.
Wissenschaftlicher Artikel
Scientific Article
Zoni, E. ; Buck, A. ; Feuchtinger, A. ; Spahn, M. ; Walch, A.K. ; Kruithof-de Julio, M.
Cancer Res. 78, 61-62 (2018)
Meeting abstract
Meeting abstract
Clemen, C.S. ; Winter, L. ; Strucksberg, K.H. ; Berwanger, C. ; Türk, M. ; Kornblum, C. ; Florin, A. ; Aguilar-Pimentel, J.A. ; Amarie, O.V. ; Becker, L. ; Garrett, L. ; Hans, W. ; Moreth, K. ; Neff, F. ; Pingen, L. ; Rathkolb, B. ; Rácz, I. ; Rozman, J. ; Treise, I. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Vorgerd, M. ; Eichinger, L. ; Schröder, R.
Biochem. Biophys. Res. Commun. 503, 2770-2777 (2018)
Heterozygous missense mutations in the human VCP gene cause inclusion body myopathy associated with Paget disease of bone and fronto-temporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). The exact molecular mechanisms by which VCP mutations cause disease manifestation in different tissues are incompletely understood. In the present study, we report the comprehensive analysis of a newly generated R155C VCP knock-in mouse model, which expresses the ortholog of the second most frequently occurring human pathogenic VCP mutation. Heterozygous R155C VCP knock-in mice showed decreased plasma lactate, serum albumin and total protein concentrations, platelet numbers, and liver to body weight ratios, and increased oxygen consumption and CD8+/Ly6C + T-cell fractions, but none of the typical human IBMPFD or ALS pathologies. Breeding of heterozygous mice did not yield in the generation of homozygous R155C VCP knock-in animals. Immunoblotting showed identical total VCP protein levels in human IBMPFD and murine R155C VCP knock-in tissues as compared to wild-type controls. However, while in human IBMPFD skeletal muscle tissue 70% of the total VCP mRNA was derived from the mutant allele, in R155C VCP knock-in mice only 5% and 7% mutant mRNA were detected in skeletal muscle and brain tissue, respectively. The lack of any obvious IBMPFD or ALS pathology could thus be a consequence of the very low expression of mutant VCP. We conclude that the increased and decreased fractions of the R155C mutant VCP mRNA in man and mice, respectively, are due to missense mutation-induced, divergent alterations in the biological half-life of the human and murine mutant mRNAs. Furthermore, our work suggests that therapy approaches lowering the expression of the mutant VCP mRNA below a critical threshold may ameliorate the intrinsic disease pathology.
Wissenschaftlicher Artikel
Scientific Article
Sun, N. ; Fernandez, I.E. ; Wei, M. ; Witting, M. ; Aichler, M. ; Feuchtinger, A. ; Burgstaller, G. ; Verleden, S.E. ; Schmitt-Kopplin, P. ; Eickelberg, O. ; Walch, A.K.
Eur. Respir. J. 52:1702314 (2018)
Idiopathic pulmonary fibrosis (IPF) is a fatal condition that reduces life expectancy and shows a limited response to available therapies. Pirfenidone has been approved for treatment of IPF, but little is known about the distinct metabolic changes that occur in the lung upon pirfenidone administration.Here, we performed a proof-of-concept study using high-resolution quantitative matrix-assisted laser desorption/ionisation Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI) to simultaneously detect, visualise and quantify in situ endogenous and exogenous metabolites in lungs of mice subjected to experimental fibrosis and human patients with IPF, and to assess the effect of pirfenidone treatment on metabolite levels.Metabolic pathway analysis and endogenous metabolite quantification revealed that pirfenidone treatment restores redox imbalance and glycolysis in IPF tissues, and downregulates ascorbate and aldarate metabolism, thereby likely contributing to in situ modulation of collagen processing. As such, we detected specific alterations in metabolite pathways in fibrosis and, importantly, metabolic recalibration following pirfenidone treatment.Together, these results highlight the suitability of high-resolution MALDI-FTICR-MSI for deciphering the therapeutic effects of pirfenidone and provide a preliminary analysis of the metabolic changes that occur during pirfenidone treatment in vivo These data may therefore contribute to improvement of currently available therapies for IPF.
Wissenschaftlicher Artikel
Scientific Article
Gora, T. ; Burkhardt, R. ; Umkehrer, S. ; Herzen, J. ; Schilling, D. ; Feuchtinger, A. ; Walch, A.K. ; Schmid, T.E. ; Multhoff, G. ; Noel, P.B. ; Rummeny, E.J. ; Pfeiffer, F. ; Combs, S.E. ; Wilkens, J.J.
Radiother. Oncol. 127, E581-E582 (2018)
Meeting abstract
Meeting abstract
Martin-Medina, A. ; Lehmann, M. ; Burgy, O. ; Hermann, S. ; Baarsma, H.A. ; Wagner, D.E. ; De Santis, M. ; Ciolek, F. ; Hofer, T.P. ; Frankenberger, M. ; Aichler, M. ; Lindner, M. ; Gesierich, W. ; Guenther, A. ; Walch, A.K. ; Coughlan, C. ; Wolters, P.  ; Lee, J.S. ; Behr, J. ; Königshoff, M.
Am. J. Respir. Crit. Care Med. 198, 1527-1538 (2018)
Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo) fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-beta in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.
Wissenschaftlicher Artikel
Scientific Article
Buck, A. ; Heijs, B. ; Beine, B. ; Schepers, J. ; Cassese, A. ; Heeren, R.M.A. ; McDonnell, L.A. ; Henkel, C. ; Walch, A.K. ; Balluff, B.
Anal. Bioanal. Chem. 410, 5969–5980 (2018)
Mass spectrometry imaging (MSI) has provided many results with translational character, which still have to be proven robust in large patient cohorts and across different centers. Although formalin-fixed paraffin-embedded (FFPE) specimens are most common in clinical practice, no MSI multicenter study has been reported for FFPE samples. Here, we report the results of the first round robin MSI study on FFPE tissues with the goal to investigate the consequences of inter-and intracenter technical variation on masking biological effects. A total of four centers were involved with similar MSI instrumentation and sample preparation equipment. A FFPE multi-organ tissue microarray containing eight different types of tissue was analyzed on a peptide and metabolite level, which enabled investigating different molecular and biological differences. Statistical analyses revealed that peptide intercenter variation was significantly lower and metabolite intercenter variation was significantly higher than the respective intracenter variations. When looking at relative univariate effects of mass signals with statistical discriminatory power, the metabolite data was more reproducible across centers compared to the peptide data. With respect to absolute effects (cross-center common intensity scale), multivariate classifiers were able to reach on average > 90% accuracy for peptides and > 80% for metabolites if trained with sufficient amount of cross-center data. Overall, our study showed that MSI data from FFPE samples could be reproduced to a high degree across centers. While metabolite data exhibited more reproducibility with respect to relative effects, peptide data-based classifiers were more directly transferable between centers and therefore more robust than expected.
Wissenschaftlicher Artikel
Scientific Article
Maihoefer, C. ; Schüttrumpf, L. ; Macht, C. ; Pflugradt, U. ; Hess-Rieger, J. ; Schneider, L. ; Woischke, C. ; Walch, A.K. ; Baumeister, P. ; Kirchner, T. ; Zitzelsberger, H. ; Belka, C. ; Ganswindt, U.
Radiat. Oncol. 13:123 (2018)
Background: Postoperative (chemo) radiation improves tumor control and survival in high-risk patients with head and neck squamous cell carcinoma based on established risk factors. The clinical cooperation group "Personalized Radiotherapy in Head and Neck Cancer" focuses on the identification and validation of new biomarkers, which are aimed at eventually stratifying and personalizing the therapy concept. Hence, we reviewed all patients with head and neck squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx, treated with postoperative (chemo) radiation from 06/2008 until 06/2015 at the Department of Radiation Oncology in the University Hospital, LMU Munich. Here we report the clinical results of the cohort, laying the foundation for further research within the framework of a clinical cooperation group.Methods: Patient data were retrospectively (until 2013) and prospectively (from 2013) collected and analyzed for outcome and treatment failures with regard to previously described and established risk factors.Results: We identified 302 patients (median follow-up 45 months, average age 60.7 years), having received postoperative (chemo) radiation (median 64 Gy). Chemotherapy was added in 58% of cases, mostly Cisplatin/5-Fluorouracil in concordance with the ARO 96-3 study. The 3-year overall survival, local, locoregional and distant failure estimates were 70.5, 9.7, 12.2 and 13.5%, respectively. Human papillomavirus-associated oropharyngeal cancer was associated with a significant improved overall survival, locoregional, distant and overall tumor control rates in multivariate analysis. Additionally, in multivariate analysis, for local failure, resection status and perineural invasion, for locoregional and distant failure extracapsular extension and for overall survival the presence of nodal disease were significant adverse factors. Moreover, 138 patients have been treated in concordance with the ARO 96-3 protocol, corroborating the results of this study.Conclusions: Our cohort represents a large unselected cohort of patients with head and neck squamous cell carcinoma treated with postoperative (chemo) radiation. Tumor control rates and survival rates are consistent with the results of previously reported data.
Wissenschaftlicher Artikel
Scientific Article
Bräuer, K. ; Brockers, K. ; Moneer, J. ; Feuchtinger, A. ; Wollscheid-Lengeling, E. ; Lengeling, A. ; Wolf, A.
BMC Evol. Biol. 18:96 (2018)
Background Translation of specific mRNAs can be highly regulated in different cells, tissues or under pathological conditions. Ribosome heterogeneity can originate from variable expression or post-translational modifications of ribosomal proteins. The ribosomal oxygenases RIOX1 (NO66) and RIOX2 (MINA53) modify ribosomal proteins by histidine hydroxylation. A similar mechanism is present in prokaryotes. Thus, ribosome hydroxylation may be a well-conserved regulatory mechanism with implications in disease and development. However, little is known about the evolutionary history of Riox1 and Riox2 genes and their encoded proteins across eukaryotic taxa. Results In this study, we have analysed Riox1 and Riox2 orthologous genes from 49 metazoen species and have constructed phylogenomic trees for both genes. Our genomic and phylogenetic analyses revealed that Arthropoda, Annelida, Nematoda and Mollusca lack the Riox2 gene, although in the early phylum Cnidaria both genes, Riox1 and Riox2, are present and expressed. Riox1 is an intronless single-exon-gene in several species, including humans. In contrast to Riox2, Riox1 is ubiquitously present throughout the animal kingdom suggesting that Riox1 is the phylogenetically older gene from which Riox2 has evolved. Both proteins have maintained a unique protein architecture with conservation of active sites within the JmjC domains, a dimerization domain, and a winged-helix domain. In addition, Riox1 proteins possess a unique N-terminal extension domain. Immunofluorescence analyses in Hela cells and in Hydra vulgaris identified a nucleolar localisation signal within the extended N-terminal domain of human RIOX1 and an altered subnuclear localisation for the Hydra Riox2. Conclusions Conserved active site residues and uniform protein domain architecture suggest a consistent enzymatic activity within the Riox orthologs throughout evolution. However, differences in genomic architecture, like single exon genes and alterations in subnuclear localisation, as described for Hydra, point towards adaption mechanisms that may correlate with taxa- or species-specific requirements. The diversification of Riox1/Riox2 gene structures throughout evolution suggest that functional requirements in expression of protein isoforms and/or subcellular localisation of proteins may have evolved by adaptation to lifestyle.  
Wissenschaftlicher Artikel
Scientific Article
Saller, M.M. ; Huettl, R.E. ; Mayer, J.M. ; Feuchtinger, A. ; Krug, C. ; Holzbach, T. ; Volkmer, E.
Neural Regen. Res. 13, 854-861 (2018)
Flatworms of the species Schmidtea mediterranea are immortal-adult animals contain a large pool of pluripotent stem cells that continuously differentiate into all adult cell types. Therefore, single-cell transcriptome profiling of adult animals should reveal mature and progenitor cells. By combining perturbation experiments, gene expression analysis, a computational method that predicts future cell states from transcriptional changes, and a lineage reconstruction method, we placed all major cell types onto a single lineage tree that connects all cells to a single stem cell compartment. We characterized gene expression changes during differentiation and discovered cell types important for regeneration. Our results demonstrate the importance of single-cell transcriptome analysis for mapping and reconstructing fundamental processes of developmental and regenerative biology at high resolution.
Wissenschaftlicher Artikel
Scientific Article
Lohöfer, F. ; Hoffmann, L. ; Buchholz, R. ; Huber, K. ; Glinzer, A. ; Kosanke, K. ; Feuchtinger, A. ; Aichler, M. ; Feuerecker, B. ; Kaissis, G. ; Rummeny, E.J. ; Höltke, C. ; Faber, C. ; Schilling, F. ; Botnar, R.M. ; Walch, A.K. ; Karst, U. ; Wildgruber, M.
Heliyon 4:e00606 (2018)
Background: Molecular MRI is becoming increasingly important for preclinical research. Validation of targeted gadolinium probes in tissue however has been cumbersome up to now. Novel methodology to assess gadolinium distribution in tissue after in vivo application is therefore needed. Purpose: To establish combined Magnetic Resonance Imaging (MRI) and Mass Spectrometry Imaging (MSI) for improved detection and quantification of Gadofluorine P deposition in scar formation and myocardial remodeling. Materials and methods: Animal studies were performed according to institutionally approved protocols. Myocardial infarction was induced by permanent ligation of the left ascending artery (LAD) in C57BL/6J mice. MRI was performed at 7T at 1 week and 6 weeks after myocardial infarction. Gadofluorine P was used for dynamic T1mapping of extracellular matrix synthesis during myocardial healing and compared to Gd-DTPA. After in vivo imaging contrast agent concentration as well as distribution in tissue were validated and quantified by spatially resolved Matrix-Assisted Laser Desorption Ionization (MALDI) MSI and Laser Ablation – Inductively Coupled Plasma – Mass Spectrometry (LA-ICP-MS) imaging. Results: Both Gadofluorine P enhancement as well as local tissue content in the myocardial scar were highest at 15 minutes post injection. R1values increased from 1 to 6 weeks after MI (1.62 s−1vs 2.68 s−1, p = 0.059) paralleled by an increase in Gadofluorine P concentration in the infarct from 0.019 mM at 1 week to 0.028 mM at 6 weeks (p = 0.048), whereas Gd-DTPA enhancement showed no differences (3.95 s−1vs 3.47 s−1, p = 0.701). MALDI-MSI results were corroborated by elemental LA-ICP-MS of Gadolinium in healthy and infarcted myocardium. Histology confirmed increased extracellular matrix synthesis at 6 weeks compared to 1 week. Conclusion: Adding quantitative MSI to MR imaging enables a quantitative validation of Gadofluorine P distribution in the heart after MI for molecular imaging.
Wissenschaftlicher Artikel
Scientific Article
Rodriguez Camargo, D.C. ; Garg, D. ; Buday, K. ; Frankó, A. ; Rodriguez Camargo, A. ; Schmidt, F. ; Cox, S.J. ; Suladze, S. ; Haslbeck, M. ; Mideksa, Y.G. ; Gemmecker, G. ; Aichler, M. ; Mettenleiter, G. ; Schulz, M. ; Walch, A.K. ; Hrabě de Angelis, M. ; Feige, M.J. ; Sierra, C.A. ; Conrad, M. ; Tripsianes, K. ; Ramamoorthy, A. ; Reif, B.
Chem. Commun. 54, 5426-5429 (2018)
In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in β-sheet rich oligomers with increased β-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.
Wissenschaftlicher Artikel
Scientific Article
Wilke, C. ; Braselmann, H. ; Hess-Rieger, J. ; Klymenko, S.V. ; Chumak, V.V. ; Zakhartseva, L.M. ; Bakhanova, E.V. ; Walch, A.K. ; Selmansberger, M. ; Samaga, D. ; Weber, P. ; Schneider, L. ; Fend, F. ; Bösmüller, H.C. ; Zitzelsberger, H. ; Unger, K.
Int. J. Cancer 143, 1505-1515 (2018)
Breast cancer is the second leading cause of cancer death among women worldwide and besides life style, age and genetic risk factors, exposure to ionizing radiation is known to increase the risk for breast cancer. Further, DNA copy number alterations (CNAs), which can result from radiation-induced double-strand breaks, are frequently occurring in breast cancer cells. We set out to identify a signature of CNAs discriminating breast cancers from radiation-exposed and non-exposed female patients. We analyzed resected breast cancer tissues from 68 exposed female Chernobyl clean-up workers and evacuees and 68 matched non-exposed control patients for CNAs by array comparative genomic hybridization analysis (aCGH). Using a stepwise forward-backward selection approach a non-complex CNA signature, that is, less than ten features, was identified in the training data set, which could be subsequently validated in the validation data set (p value <0.05). The signature consisted of nine copy number regions located on chromosomal bands 7q11.22-11.23, 7q21.3, 16q24.3, 17q21.31, 20p11.23-11.21, 1p21.1, 2q35, 2q35, 6p22.2. The signature was independent of any clinical characteristics of the patients. In all, we identified a CNA signature that has the potential to allow identification of radiation-associated breast cancer at the individual level.
Wissenschaftlicher Artikel
Scientific Article
Frankó, A. ; Rodriguez Camargo, D.C. ; Böddrich, A. ; Garg, D. ; Rodriguez Camargo, A. ; Rathkolb, B. ; Janik, D. ; Aichler, M. ; Feuchtinger, A. ; Neff, F. ; Fuchs, H. ; Wanker, E.E. ; Reif, B. ; Häring, H.-U. ; Peter, A. ; Hrabě de Angelis, M.
Sci. Rep. 8:1116 (2018)
The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.
Wissenschaftlicher Artikel
Scientific Article
Sun, N. ; Wu, Y. ; Nanba, K. ; Sbiera, S. ; Kircher, S. ; Kunzke, T. ; Aichler, M. ; Berezowska, S. ; Reibetanz, J. ; Rainey, W.E. ; Fassnacht, M. ; Walch, A.K. ; Kroiss, M.
Endocrinology 159, 1511-1524 (2018)
It is undeniably one of the greatest findings in biology that (with some very minor exceptions) every cell in the body possesses the whole genetic information needed to generate a complete individual. Today, this concept has been so thoroughly assimilated that we struggle to still see how surprising this finding actually was: all cellular phenotypes naturally occurring in one person are generated from genetic uniformity, and thus are per definition epigenetic. Transcriptional mechanisms are clearly critical for developing and protecting cell identities, because a mis-expression of few or even single genes can efficiently induce inappropriate cellular programmes. However, how transcriptional activities are molecularly controlled and which of the many known epigenomic features have causal roles remains unclear. Today, clarification of this issue is more pressing than ever because profiling efforts and epigenome-wide association studies (EWAS) continuously provide comprehensive datasets depicting epigenomic differences between tissues and disease states. In this commentary, we propagate the idea of a widespread follow-up use of epigenome editing technology in EWAS studies. This would enable them to address the questions of which features, where in the genome, and which circumstances are essential to shape development and trigger disease states.
Wissenschaftlicher Artikel
Scientific Article
Dalke, C. ; Neff, F. ; Bains, S.K. ; Bright, S. ; Lord, D.J. ; Reitmeir, P. ; Rößler, U. ; Samaga, D. ; Unger, K. ; Braselmann, H. ; Wagner, F. ; Greiter, M. ; Gomolka, M. ; Hornhardt, S. ; Kunze, S. ; Kempf, S.J. ; Garrett, L. ; Hölter, S.M. ; Wurst, W. ; Rosemann, M. ; Azimzadeh, O. ; Tapio, S. ; Aubele, M. ; Theis, F.J. ; Hoeschen, C. ; Slijepcevic, P. ; Kadhim, M. ; Atkinson, M.J. ; Zitzelsberger, H. ; Kulka, U. ; Graw, J.
Radiat. Environ. Biophys. 57, 99-113 (2018)
Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.
Wissenschaftlicher Artikel
Scientific Article
Absmaier, M. ; Napieralski, R. ; Schuster, T. ; Aubele, M. ; Walch, A.K. ; Magdolen, V. ; Dorn, J. ; Gross, E. ; Harbeck, N. ; Noske, A. ; Kiechle, M. ; Schmitt, M.
Int. J. Oncol. 52, 755-767 (2018)
Triple-negative breast cancer (TNBC) constitutes a heterogeneous breast cancer subgroup with poor prognosis; survival rates are likely to be lower with TNBC compared to other breast cancer subgroups. For this disease, systemic adjuvant chemotherapy regimens often yield suboptimal clinical results. To improve treatment regimens in TNBC, identification of molecular biomarkers may help to select patients for individualized adjuvant therapy. Evidence has accumulated that determination of the methylation status of the PITX2 gene provides a predictive value in various breast cancer subgroups, either treated with endocrine-based therapy or anthracycline-containing chemotherapy. To further explore the validity of this novel predictive candidate biomarker, in the present exploratory retrospective study, determination of the PITX2 DNA-methylation status was assessed for non-metastatic TNBC patients treated with adjuvant anthracycline-based chemotherapy by molecular analysis of breast cancer tissues. The PITX2 DNA-methylation status was determined in fresh-frozen tumor tissue specimens (n=56) by methylation-specific qRT-PCR (qMSP) and the data related to disease-free and overall survival, applying an optimized DNA-methylation score of 6.35%. For non-metastatic TNBC patients treated with adjuvant systemic anthracycline-based chemotherapy, a low PITX2 DNA-methylation status (<6.35) defines TNBC patients with poor disease-free and overall survival. Univariate and multivariate analyses demonstrate the statistically independent predictive value of PITX2 DNA-methylation. For non-metastatic TNBC patients, selective determination of the PITX2 DNA-methylation status may serve as a cancer biomarker for predicting response to anthracycline-based adjuvant chemotherapy. The assay based on methylation of the PIXT2 gene can be applied to frozen and routinely available formalin-fixed, paraffin-embedded (FFPE) breast cancer tumor tissues that will not only define those TNBC patients who may benefit from anthracycline-based chemotherapy but also those who should be spared the necessity of such potentially toxic treatment. Such patients should be allocated to alternative treatment options.
Wissenschaftlicher Artikel
Scientific Article
Ingold, I. ; Berndt, C. ; Schmitt, S. ; Doll, S. ; Poschmann, G. ; Buday, K. ; Roveri, A. ; Peng, X. ; Porto Freitas, F. ; Seibt, T. ; Mehr, L. ; Aichler, M. ; Walch, A.K. ; Lamp, D. ; Jastroch, M. ; Miyamoto, S. ; Wurst, W. ; Ursini, F. ; Arnér, E.S.J. ; Fradejas-Villar, N. ; Schweizer, U. ; Zischka, H. ; Friedmann Angeli, J.P.F. ; Conrad, M.
Cell 172, 409–422.e21 (2018)
Selenoproteins are rare proteins among all kingdoms of life containing the 21 st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4 cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided. The trace element selenium protects a critical population of interneurons from ferroptotic cell death.
Wissenschaftlicher Artikel
Scientific Article
Molatore, S. ; Kügler, A. ; Irmler, M. ; Wiedemann, T. ; Neff, F. ; Feuchtinger, A. ; Beckers, J. ; Robledo, M. ; Roncaroli, F. ; Pellegata, N.S.
Endocr. Relat. Cancer 25, 145-162 (2018)
Rats affected by the MENX syndrome spontaneously develop multiple neuroendocrine tumors (NETs) including adrenal, pituitary and thyroid gland neoplasms. MENX was initially reported to be inherited as a recessive trait and affected rats were found to be homozygous for the predisposing Cdkn1b mutation encoding p27. We here report that heterozygous MENX-mutant rats (p27+/mut) develop the same spectrum of NETs seen in the homozygous (p27mut/mut) animals but with slower progression. Consequently, p27+/mut rats have a significantly shorter lifespan compared with their wild-type (p27+/+) littermates. In the tumors of p27+/mut rats, the wild-type Cdkn1b allele is neither lost nor silenced, implying that p27 is haploinsufficient for tumor suppression in this model. Transcriptome profiling of rat adrenal (pheochromocytoma) and pituitary tumors having different p27 dosages revealed a tissue-specific, dose-dependent effect of p27 on gene expression. In p27+/mut rats, thyroid neoplasms progress to invasive and metastatic medullary thyroid carcinomas (MTCs) accompanied by increased calcitonin levels, as in humans. Comparison of expression signatures of late-stage vs early-stage MTCs from p27+/mut rats identified genes potentially involved in tumor aggressiveness. The expression of a subset of these genes was evaluated in human MTCs and found to be associated with aggressive RET-M918T-positive tumors. Altogether, p27 haploinsufficiency in MENX rats uncovered a novel, representative model of invasive and metastatic MTC exploitable for translational studies of this often aggressive and incurable cancer.
Wissenschaftlicher Artikel
Scientific Article
Aichler, M. ; Kunzke, T. ; Buck, A. ; Sun, N. ; Ackermann, M. ; Jonigk, D. ; Gaumann, A. ; Walch, A.K.
Lab. Invest. 98, 141-149 (2018)
© 2018 USCAP, Inc All rights reserved. Animal models can reproduce some model-specific aspects of human diseases, but some animal models translate poorly or fail to translate to the corresponding human disease. Here, we develop a strategy to systematically compare human and mouse tissues, and conduct a proof-of-concept experiment to identify molecular similarities and differences using patients with idiopathic pulmonary fibrosis and a bleomycin-induced fibrosis mouse model. Our novel approach employs high-throughput tissue microarrays (TMAs) of humans and mice, high-resolution matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance-mass spectrometry imaging (MALDI-FT-ICR-MSI) to spatially resolve mass spectra at the level of specific metabolites, and hierarchical clustering and pathway enrichment analysis to identify functionally similar/different molecular patterns and pathways in pathological lesions of humans and mice. We identified a large number of common molecules (n=1366) and fewer exclusive molecules in humans (n=83) and mice (n=54). Among the common molecules, the 'ascorbate and aldarate metabolism' pathway had the highest similarity in human and mouse lesions. This proof-of-concept study demonstrates that our novel strategy employing a reliable and easy-to-perform experimental design accurately identifies pathways and factors that can be directly compared between animal models and human diseases.
Wissenschaftlicher Artikel
Scientific Article
Wilke, C. ; Hess-Rieger, J. ; Klymenko, S.V. ; Chumak, V.V. ; Zakhartseva, L.M. ; Bakhanova, E.V. ; Feuchtinger, A. ; Walch, A.K. ; Selmansberger, M. ; Braselmann, H. ; Schneider, L. ; Pitea, A. ; Steinhilber, J. ; Fend, F. ; Bösmüller, H.C. ; Zitzelsberger, H. ; Unger, K.
Int. J. Cancer 142, 573-583 (2018)
Ionising radiation is a well-recognised risk factor for the development of breast cancer, however, it is unknown whether radiation-specific molecular oncogenic mechanisms exist. We investigated post-Chernobyl breast cancers from radiation-exposed female clean-up workers and non-exposed controls for molecular changes. Radiation-associated alterations identified in the discovery cohort (n=38) were subsequently validated in a second cohort (n=39). Increased expression of hsa-miR-26b-5p was associated with radiation exposure in both of the cohorts. Moreover, downregulation of the TRPS1 protein, which is a transcriptional target of hsa-miR-26b-5p was associated with radiation exposure. Since TRPS1 overexpression is common in sporadic breast cancer its observed downregulation in radiation-associated breast cancer warrants clarification of the specific functional role of TRPS1 in the radiation context. For this purpose, the impact of TRPS1 on the transcriptome was characterised in two radiation-transformed breast cell culture models after siRNA-knockdown. Deregulated genes upon TRPS1 knockdown were associated with DNA-repair, cell cycle, mitosis, cell migration, angiogenesis and EMT pathways. Furthermore, we identified the interaction partners of TRPS1 from the transcriptomic correlation networks derived from gene expression data on radiation-transformed breast cell culture models and sporadic breast cancer tissues provided by the TCGA database. The genes correlating with TRPS1 in the radiation-transformed breast cell lines were primarily linked to DNA damage response and chromosome segregation, whilst the transcriptional interaction partners in the sporadic breast cancers were mostly associated with apoptosis. Thus, upregulation of hsa-miR-26b-5p and downregulation of TRPS1 in radiation-associated breast cancer tissue samples suggests these molecules representing radiation markers in breast cancer.
Wissenschaftlicher Artikel
Scientific Article
2017
Ingold, I. ; Berndt, C. ; Schmitt, S. ; Doll, S. ; Poschmann, G. ; Roveri, A. ; Peng, X. ; Freitas, F.P. ; Aichler, M. ; Jastroch, M. ; Ursini, F. ; Arner, E.S.J. ; Fradejas-Villar, N. ; Schweizer, U. ; Zischka, H. ; Friedmann Angeli, J.P.F. ; Conrad, M.
Free Radical Biol. Med. 112, 24-24 (2017)
Meeting abstract
Meeting abstract
Klaeger, S. ; Heinzlmeir, S. ; Wilhelm, M. ; Polzer, H. ; Vick, B. ; Koenig, P.A. ; Reinecke, M. ; Ruprecht, B. ; Petzoldt, S. ; Meng, C. ; Zecha, J. ; Reiter, K. ; Qiao, H. ; Helm, D. ; Koch, H. ; Schoof, M. ; Canevari, G. ; Casale, E. ; Depaolini, S.R. ; Feuchtinger, A. ; Wu, Z. ; Schmidt, T. ; Rueckert, L. ; Becker, W. ; Huenges, J. ; Garz, A.K. ; Gohlke, B.O. ; Zolg, D.P. ; Kayser, G. ; Vooder, T. ; Preissner, R. ; Hahne, H. ; Tõnisson, N. ; Kramer, K. ; Götze, K. ; Bassermann, F. ; Schlegl, J. ; Ehrlich, H.C. ; Aiche, S. ; Walch, A.K. ; Greif, P.A. ; Schneider, S. ; Felder, E.R. ; Ruland, J. ; Médard, G. ; Jeremias, I. ; Spiekermann, K. ; Kuster, B.
Science 358:eaan4368 (2017)
Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.
Wissenschaftlicher Artikel
Scientific Article
Rodriguez Camargo, D.C. ; Korshavn, K.J. ; Jussupow, A. ; Raltchev, K. ; Goricanec, D. ; Fleisch, M. ; Sarkar, R. ; Xue, K. ; Aichler, M. ; Mettenleiter, G. ; Walch, A.K. ; Camilloni, C. ; Hagn, F. ; Reif, B. ; Ramamoorthy, A.
eLife 6:e31226 (2017)
Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique b-strand structure distinct from the conventional amyloid b-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.
Wissenschaftlicher Artikel
Scientific Article
Desbenoit, N. ; Walch, A.K. ; Spengler, B. ; Brunelle, A. ; Römpp, A.
Rapid Commun. Mass Spectrom. 32, 159-166 (2017)
RATIONALE: Mass spectrometry imaging (MSI) is a powerful tool for mapping the surface of a sample. Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) and Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization (AP-MALDI) offer complementary capabilities. Here, we present a workflow to apply both techniques to a single tissue section and combine the resulting data on the example of human colon cancer tissue. METHODS: Following cryo-sectioning, images were acquired using the high spatial resolution (1 μm pixel size) provided by TOF-SIMS. The same section was then coated with a para-nitroaniline matrix and images were acquired using AP-MALDI coupled to an Orbitrap mass spectrometer, offering high mass resolution, high mass accuracy and MS/MS capabilities. Datasets provided by both mass spectrometers were converted into the open and vendor-independent imzML file format and processed with the open-source software MSiReader. RESULTS: The TOF-SIMS and AP-MALDI-MS mass spectra show strong signals of fatty acids, cholesterol, phosphatidylcholine and sphingomyelin. We showed a high correlation between the fatty acid ions detected with TOF-SIMS in negative ion mode and the phosphatidylcholine ions detected with AP-MALDI in positive ion mode using a similar setting for visualization. Histological staining on the same section allowed the identification of the anatomical structures and their correlation with the ion images. CONCLUSIONS: This multimodal approach using two MSI platforms shows an excellent complementarity for the localization and identification of lipids. The spatial resolution of both systems is at or close to cellular dimensions and thus spatial correlation can only be obtained if the same tissue section is analyzed sequentitially. imzML-based data processing allows a real correlation of the imaging datasets provided by these two technologies and opens the way for a more complete molecular view of the anatomical structures of biological tissues.
Wissenschaftlicher Artikel
Scientific Article
Jacobs, L. ; Habringer, S. ; Slawska, J. ; Huber, K. ; Hauf, E. ; Li, Z. ; Refaeli, Y. ; Schwaiger, M. ; Rudelius, M. ; Walch, A.K. ; Keller, U.
Oncotarget 8, 78917-78929 (2017)
Aberrant B-cell receptor (BCR) signaling is known to contribute to malignant transformation. Two small molecule inhibitors targeting BCR pathway signaling include ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, and idelalisib, a specific Phosphatidylinositol-4,5-bisphosphate 3-kinase delta (PI3Kd) inhibitor, both of which have been approved for use in haematological malignancies. Despite the identification of various diffuse large B-cell lymphoma (DLBCL) subtypes, mutation status alone is not sufficient to predict patient response and therapeutic resistance can arise. Herein we apply early molecular imaging across alternative activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL subtypes to investigate the effects of BCR pathway inhibition. Treatment with both inhibitors adversely affected cell growth and viability. These effects were partially predictable based upon mutation status. Accordingly, very early 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ( 18 F-FDG-PET) and 3'-deoxy-3'[18F]-fluorothymidine positron emission tomography ( 18 F-FLT-PET) reported tumour regression and reductions in tumour metabolism and proliferation upon treatment. Furthermore, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) identified alterations in the proteome of a model of ABC DLBCL upon treatment with ibrutinib or idelalisib. In conclusion we demonstrate that very early molecular imaging adds predictive value in addition to mutational status of DLBCL that may be useful in directing patient therapy.
Wissenschaftlicher Artikel
Scientific Article
Urban, C. ; Buck, A. ; Siveke, J.T. ; Lordick, F. ; Luber, B. ; Walch, A.K. ; Aichler, M.
Biochim. Biophys. Acta 1862, 51-60 (2017)
An alcohol-based non-crosslinking tissue fixative, PAXgene Tissue System, has been proposed as alternative fixation method to formalin, providing superior and morphological preservation. To date, metabolites have not been assessed in PAXgene-fixed tissues. The study focuses on a comparison between PAXgene and standard formalin fixation for metabolomic analysis by MALDI mass spectrometry imaging. Therefore, fifty-six samples from seven mice organs were fixed with PAXgene (PFPE) or formalin (FFPE), embedded in paraffin, and processed to a tissue microarray. PAXgene was able to spatially preserve metabolites in organs achieving an overlap of common metabolites ranging from 34 to 78% with FFPE. Highly similar signal intensities and visualization of molecules demonstrated negligible differences for metabolite imaging on PFPE compared to FFPE tissues. In addition, we performed proteomic analysis of intact proteins and peptides derived from enzymatic digestion. An overlap of 33 to 58% was found between FFPE and PFPE tissue samples in peptide analysis with a higher number of PFPE-specific peaks. Analysis of intact proteins achieved an overlap in the range of 0 to 28% owing to the poor detectability of cross-linked proteins in formalin-fixed tissues. Furthermore, metabolite and peptide profiles obtained from PFPE tissues were able to correctly classify organs independent of the fixation method, whereas a distinction of organs by protein profiles was only achieved by PAXgene fixation. Finally, we applied MALDI MSI to human biopsies by sequentially analyzing metabolites and peptides within the same tissue section. Concerning prospective studies, PAXgene can be used as an alternative fixative for multi-omic tissue analysis.
Wissenschaftlicher Artikel
Scientific Article
Fichter, C.D. ; Przypadlo, C.M. ; Buck, A. ; Herbener, N. ; Riedel, B. ; Schäfer, L. ; Nakagawa, H. ; Walch, A.K. ; Reinheckel, T. ; Werner, M. ; Lassmann, S.
J. Pathol. 243, 481-495 (2017)
Oesophageal squamous cell carcinomas and oesophageal adenocarcinomas display distinct patterns of ErbB expression and dimers. The functional effects of specific ErbB homo- or heterodimers on oesophageal (cancer) cell behaviour, particularly invasion of early carcinogenesis remains unknown. Here, a new cellular model system for controlled activation of EGFR or HER2 and EGFR/HER2 or HER2/HER3 homo- and heterodimers was studied in non-neoplastic squamous oesophageal epithelial Het-1A cells. EGFR, HER2 and HER3 intracellular domains (ICDs) were fused to dimerization domains (DmrA / DmrA and DmrC), and transduced into Het-1A cells lacking ErbB expression. Dimerization of EGFR, HER2 or EGFR/HER2, HER2/HER3 ICDs was induced by synthetic ligands (A/A or A/C dimerizers). This was accompanied by phosphorylation of the respective EGFR, HER2 and HER3 ICDs and activation of distinct down-stream signalling pathways, such as PLCγ1, Akt, STAT and Src family kinases. Phenotypically, ErbB homo- and heterodimers caused cell rounding and non-apoptotic blebbing in EGFR/HER2 and HER2/HER3 heterodimer cells. In a Transwell assay, cell migration velocity was elevated in HER2-dimer as compared to empty vector cells. In addition, HER2-dimer cells showed in increased cell invasion, reaching significance for induced HER2/HER3 heterodimers (p=0.015). Importantly, in three-dimensional organotypic cultures, empty vector cells grew as a superficial cell layer, resembling oesophageal squamous epithelium. In contrast, induced HER2-dimer cells (HER2 homodimers) were highly invasive into the matrix and formed cell clusters. This was associated with partial loss of CK7 (when HER2 homodimers were modelled) and p63 (when EGFR/HER2 heterodimers were modelled), which suggests a change or loss of squamous cell differentiation. Controlled activation of specific EGFR, HER2 and HER3 homo- and heterodimers caused oesophageal squamous epithelial cell migration and/or invasion, especially in a three dimensional microenvironment, thereby functionally identifying ErbB homo- and heterodimers as important drivers of oesophageal carcinogenesis.
Wissenschaftlicher Artikel
Scientific Article
Koch, M. ; de Jong, J.S. ; Glatz, J. ; Symvoulidis, P. ; Lamberts, L.E. ; Adams, A.L.L. ; Kranendonk, M.E.G. ; van Scheltinga, A.T. ; Aichler, M. ; Jansen, L. ; de Vries, J. ; Lub-de Hooge, M. ; Schröder, C.P. ; Jorritsma-Smit, A. ; Linssen, M.D. ; de Boer, E. ; van der Vegt, B. ; Nagengast, W.B. ; Elias, S.G. ; Oliveira, S. ; Witkamp, A.J. ; Mali, W.P.Th.M. ; van der Wall, E. ; Garcia-Allende, P. ; van Diest, P.J. ; de Vries, E.G. ; Walch, A.K. ; van Dam, G.M. ; Ntziachristos, V.
Proc. SPIE 10411:104110F (2017)
In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.
Wissenschaftlicher Artikel
Scientific Article
Schlecht, A. ; Leimbeck, S.V. ; Jägle, H. ; Feuchtinger, A. ; Tamm, E.R. ; Braunger, B.M.
Am. J. Pathol. 187, 2570-2589 (2017)
The molecular pathogenesis of choroidal neovascularization (CNV), an angiogenic process that critically contributes to vision loss in age-related macular degeneration (AMD) is unclear. Here we analyzed the role of transforming growth factor (TGF)-β signaling for CNV formation by generating a series of mutant mouse models with induced conditional deletion of TGF-β signaling in the entire eye, the retinal pigment epithelium (RPE) or the vascular endothelium. Deletion of TGF-β signaling in the eye caused CNV, irrespectively if it was ablated in newborn or three-week-old mice. Areas of CNV showed photoreceptor degeneration, multilayered RPE, basal lamina deposits and accumulations of monocytes/macrophages. The changes progressed leading to marked structural and functional alterations of the retina. While the specific deletion of TGF-β signaling in the RPE caused no obvious changes, specific deletion in vascular endothelial cells caused CNV and a phenotype quite similar to that observed after the deletion in the entire eye. We conclude that impairment of TGF-β signaling in the vascular endothelium of the eye is sufficient to trigger CNV formation. Our findings highlight the importance of TGF-β signaling as key player in the development of ocular neovascularization and indicate a fundamental role of TGF-β signaling in the pathogenesis of AMD.
Wissenschaftlicher Artikel
Scientific Article
Yang, F. ; Aubele, M. ; Walch, A.K. ; Gross, E. ; Napieralski, R. ; Zhao, S. ; Ahmed, N. ; Kiechle, M. ; Reuning, U. ; Dorn, J. ; Sweep, F.C. ; Magdolen, V. ; Schmitt, M.
Biol. Chem. 398, 1151-1164 (2017)
Triple-negative breast cancer (TNBC), lacking the steroid hormone receptors ER and PR and the oncoprotein HER2, is characterized by its aggressive pattern and insensitivity to endocrine and HER2-directed therapy. Human kallikrein-related peptidases KLK1-15 provide a rich source of serine protease-type biomarkers associated with tumor growth and cancer progression for a variety of malignant diseases. In this study, recombinant KLK4 protein was generated and affinity-purified KLK4-directed polyclonal antibody pAb587 established to allow localization of KLK4 protein expression in tumor cell lines and archived formalin-fixed, paraffin-embedded TNBC tumor tissue specimens. For this, KLK4 protein expression was assessed by immunohistochemistry in primary tumor tissue sections (tissue microarrays) of 188 TNBC patients, mainly treated with anthracycline- or CMF-based polychemotherapy. KLK4 protein is localized in the cytoplasm of tumor and stroma cells. In this patient cohort, elevated stroma cell KLK4 expression, but not tumor cell KLK4 expression, is predictive for poor disease-free survival by univariate analysis (hazard ratio: 2.26, p=0.001) and multivariable analysis (hazard ratio: 2.12, p<0.01). Likewise, univariate analysis revealed a trend for statistical significance of elevated KLK4 stroma cell expression for overall survival of TNBC patients as well.
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Scientific Article
Kunzke, T. ; Balluff, B. ; Feuchtinger, A. ; Buck, A. ; Langer, R. ; Luber, B. ; Lordick, F. ; Zitzelsberger, H. ; Aichler, M. ; Walch, A.K.
Oncotarget 8, 68012-68025 (2017)
Glycosylation in cancer is a highly dynamic process that has a significant impact on tumor biology. Further, the attachment of aberrant glycan forms is already considered a hallmark of the disease state. Mass spectrometry has become a prominent approach to analyzing glycoconjugates. Specifically, matrix-assisted laser desorption/ionisation -mass spectrometric imaging (MALDI-MSI) is a powerful technique that combines mass spectrometry with histology and enables the spatially resolved and label-free detection of glycans. The most common approach to the analysis of glycans is the use of mass spectrometry adjunct to PNGase F digestion and other chemical reactions. In the current study, we perform the analysis of formalin-fixed, paraffin-embedded (FFPE) tissues for natively occurring bioactive glycan fragments without prior digestion or chemical reactions using MALDI-FT-ICR-MSI. We examined 106 primary resected gastric cancer patient tissues in a tissue microarray and correlated native-occurring fragments with clinical endpoints, therapeutic targets such as epidermal growth factor receptor (EGFR) and HER2/neu expressions and the proliferation marker MIB1. The detection of a glycosaminoglycan fragment in tumor stroma regions was determined to be an independent prognostic factor for gastric cancer patients. Native glycan fragments were significantly linked to the expression of EGFR, HER2/neu and MIB1. In conclusion, we are the first to report the in situ detection of native-occurring bioactive glycan fragments in FFPE tissues that influence patient outcomes. These findings highlight the significance of glycan fragments in gastric cancer tumor biology and patient outcome.
Wissenschaftlicher Artikel
Scientific Article
Altamura, S. ; Vegi, N. ; Hueltner, L. ; Schneider, M. ; Höppe, P. ; Schroeder, T. ; Canli, Ö. ; Greten, F.R. ; Aichler, M. ; Walch, A.K. ; Neff, F. ; Janik, D. ; Kuklik-Roos, C. ; Ladinig, C. ; Mysliwietz, J. ; Rathkolb, B. ; Buske, C. ; Conrad, M. ; Muckenthaler, M.U. ; Bornkamm, G.W.
Haematologica 102, 333-333 (2017)
Meeting abstract
Meeting abstract
Düwel, S. ; Hundshammer, C. ; Gersch, M. ; Feuerecker, B. ; Steiger, K. ; Buck, A. ; Walch, A.K. ; Haase, A. ; Glaser, S.J. ; Schwaiger, M. ; Schilling, F.
Nat. Commun. 8:15126 (2017)
Natural pH regulatory mechanisms can be overruled during several pathologies such as cancer, inflammation and ischaemia, leading to local pH changes in the human body. Here we demonstrate that (13)C-labelled zymonic acid (ZA) can be used as hyperpolarized magnetic resonance pH imaging sensor. ZA is synthesized from [1-(13)C]pyruvic acid and its (13)C resonance frequencies shift up to 3.0 p.p.m. per pH unit in the physiological pH range. The long lifetime of the hyperpolarized signal enhancement enables monitoring of pH, independent of concentration, temperature, ionic strength and protein concentration. We show in vivo pH maps within rat kidneys and subcutaneously inoculated tumours derived from a mammary adenocarcinoma cell line and characterize ZA as non-toxic compound predominantly present in the extracellular space. We suggest that ZA represents a reliable and non-invasive extracellular imaging sensor to localize and quantify pH, with the potential to improve understanding, diagnosis and therapy of diseases characterized by aberrant acid-base balance.
Wissenschaftlicher Artikel
Scientific Article
Hachmöller, O. ; Aichler, M. ; Schwamborn, K. ; Lutz, L. ; Werner, M. ; Sperling, M. ; Walch, A.K. ; Karst, U.
J. Trace Elem. Med. Biol. 44, 71-75 (2017)
The influence of rhodanine and haematoxylin and eosin (HE) staining on the copper distribution and concentration in liver needle biopsy samples originating from patients with Wilson's disease (WD), a rare autosomal recessive inherited disorder of the copper metabolism, is investigated. In contemporary diagnostic of WD, rhodanine staining is used for histopathology, since rhodanine and copper are forming a red to orange-red complex, which can be recognized in the liver tissue using a microscope. In this paper, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is applied for the analysis of eight different WD liver samples. Apart from a spatially resolved elemental detection as qualitative information, this LA-ICP-MS method offers also quantitative information by external calibration with matrix-matched gelatine standards. The sample set of this work included an unstained and a rhodanine stained section of each WD liver sample. While unstained sections of WD liver samples showed very distinct structures of the copper distribution with high copper concentrations, rhodanine stained sections revealed a blurred copper distribution with significant decreased concentrations in a range from 20 to more than 90%. This implies a copper removal from the liver tissue by complexation during the rhodanine staining. In contrast to this, a further HE stained sample of one WD liver sample did not show a significant decrease in the copper concentration and influence on the copper distribution in comparison to the unstained section. Therefore, HE staining can be combined with the analysis by means of LA-ICP-MS in two successive steps from one thin section of a biopsy specimen. This allows further information to be gained on the elemental distribution by LA-ICP-MS additional to results obtained by histological staining.
Wissenschaftlicher Artikel
Scientific Article
Medina, A.M. ; Vierkotten, S. ; Lehmann, M. ; Wagner, D. ; Baarsma, H. ; Höfer, T. ; Frankerberger, M. ; Behr, J. ; Aichler, M. ; Walch, A. ; Königshoff, M.
Am. J. Respir. Crit. Care Med. 195 (2017)
Meeting abstract
Meeting abstract
Aichler, M. ; Borgmann, D.M. ; Krumsiek, J. ; Buck, A. ; MacDonald, P.E. ; Fox, J.E.M. ; Lyon, J. ; Light, P.E. ; Keipert, S. ; Jastroch, M. ; Feuchtinger, A. ; Müller, N.S. ; Sun, N. ; Palmer, A. ; Alexandrov, T. ; Hrabě de Angelis, M. ; Neschen, S. ; Tschöp, M.H. ; Walch, A.K.
Cell Metab. 25, 1334-1347.e4 (2017)
The processes contributing to β cell dysfunction in type 2 diabetes (T2D) are uncertain, largely because it is difficult to access β cells in their intact immediate environment. We examined the pathophysiology of β cells under T2D progression directly in pancreatic tissues. We used MALDI imaging of Langerhans islets (LHIs) within mouse tissues or from human tissues to generate in situ-omics data, which we supported with in vitro experiments. Molecular interaction networks provided information on functional pathways and molecules. We found that stearoylcarnitine accumulated in β cells, leading to arrest of insulin synthesis and energy deficiency via excessive β-oxidation and depletion of TCA cycle and oxidative phosphorylation metabolites. Acetylcarnitine and an accumulation of N-acyl taurines, a group not previously detected in β cells, provoked insulin secretion. Thus, β cell dysfunction results from enhanced insulin secretion combined with an arrest of insulin synthesis.
Wissenschaftlicher Artikel
Scientific Article
Arlt, W. ; Lang, K. ; Sitch, A.J. ; Dietz, A.S. ; Rhayem, Y. ; Bancos, I. ; Feuchtinger, A. ; Chortis, V. ; Gilligan, L.C. ; Ludwig, P. ; Riester, A. ; Asbach, E. ; Hughes, B.A. ; O'Neil, D.M. ; Bidlingmaier, M. ; Tomlinson, J.W. ; Hassan-Smith, Z.K. ; Rees, D.A. ; Adolf, C. ; Hahner, S. ; Quinkler, M. ; Dekkers, T. ; Deinum, J. ; Biehl, M. ; Keevil, B.G. ; Shackleton, C.H.L. ; Deeks, J.J. ; Walch, A.K. ; Beuschlein, F. ; Reincke, M.
JCI insight 2:93136 (2017)
BACKGROUND: Adrenal aldosterone excess is the most common cause of secondary hypertension and is associated with increased cardiovascular morbidity. However, adverse metabolic risk in primary aldosteronism extends beyond hypertension, with increased rates of insulin resistance, type 2 diabetes, and osteoporosis, which cannot be easily explained by aldosterone excess. METHODS: We performed mass spectrometry-based analysis of a 24-hour urine steroid metabolome in 174 newly diagnosed patients with primary aldosteronism (103 unilateral adenomas, 71 bilateral adrenal hyperplasias) in comparison to 162 healthy controls, 56 patients with endocrine inactive adrenal adenoma, 104 patients with mild subclinical, and 47 with clinically overt adrenal cortisol excess. We also analyzed the expression of cortisol-producing CYP11B1 and aldosterone-producing CYP11B2 enzymes in adenoma tissue from 57 patients with aldosterone-producing adenoma, employing immunohistochemistry with digital image analysis. RESULTS: Primary aldosteronism patients had significantly increased cortisol and total glucocorticoid metabolite excretion (all P < 0.001), only exceeded by glucocorticoid output in patients with clinically overt adrenal Cushing syndrome. Several surrogate parameters of metabolic risk correlated significantly with glucocorticoid but not mineralocorticoid output. Intratumoral CYP11B1 expression was significantly associated with the corresponding in vivo glucocorticoid excretion. Unilateral adrenalectomy resolved both mineralocorticoid and glucocorticoid excess. Postoperative evidence of adrenal insufficiency was found in 13 (29%) of 45 consecutively tested patients. CONCLUSION: Our data indicate that glucocorticoid cosecretion is frequently found in primary aldosteronism and contributes to associated metabolic risk. Mineralocorticoid receptor antagonist therapy alone may not be sufficient to counteract adverse metabolic risk in medically treated patients with primary aldosteronism. FUNDING: Medical Research Council UK, Wellcome Trust, European Commission.
Wissenschaftlicher Artikel
Scientific Article
Knüppel, L. ; Ishikawa, Y. ; Aichler, M. ; Heinzelmann, K. ; Hatz, R. ; Behr, J. ; Walch, A.K. ; Bächinger, H.P. ; Eickelberg, O. ; Staab-Weijnitz, C.A.
Am. J. Respir. Cell Mol. Biol. 57, 77-90 (2017)
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. METHODS: Primary human fibroblasts from IPF patients and healthy donors were treated with nintedanib (0.01-1.0µM) or pirfenidone (0.1-1.0mM) in absence or presence of TGF-β1. Effects on collagen, fibronectin, FKBP10, HSP47 expression and collagen I and III secretion were analyzed by qPCR and Western Blot. Appearance of collagen fibrils was monitored by scanning electron microscopy (SEM) and kinetics of collagen fibril assembly was assessed in a light scattering approach. RESULTS: In IPF fibroblasts, nintedanib reduced the expression of collagen I, V, fibronectin and FKBP10 and attenuated secretion of collagen I and III. Pirfenidone also downregulated collagen V, but otherwise showed fewer and less pronounced effects. By and large, effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. CONCLUSIONS: Both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in downregulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused reduction and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.
Wissenschaftlicher Artikel
Scientific Article
Dislich, B. ; Stein, A. ; Seiler, C.A. ; Kröll, D. ; Berezowska, S. ; Zlobec, I. ; Galvan, J.A. ; Slotta-Huspenina, J. ; Walch, A.K. ; Langer, R.
Cancer Immunol. Immunother. 66, 777-786 (2017)
Expression analysis of programmed death-ligand 1 (PD-L1) may be helpful in guiding clinical decisions for immune checkpoint inhibition therapy, but testing by immunohistochemistry may be hampered by heterogeneous staining patterns within tumors and expression changes during metastatic course. PD-L1 expression (clone SP142) was investigated in esophageal adenocarcinomas using tissue microarrays (TMA) from 112 primary resected tumors, preoperative biopsies and full slide sections from a subset of these cases (n = 24), corresponding lymph node (n = 55) and distant metastases (n = 17). PD-L1 expression was scored as 0.1-1, >1, >5, >50% positive membranous staining of tumor cells and any positive staining of tumor-associated inflammatory infiltrates and/or stroma cells. There was a significant correlation with overall PD-L1 expression between the full slide sections and the TMA (p = 0.001), but not with the corresponding biopsies. PD-L1 expression in tumor cells >1% was detected in 8.0% of cases (9/112) and 51.8% of cases (58/112) in tumor-associated inflammatory infiltrates and/or stroma cells of primary tumors. Epithelial expression in metastases was found in 5.6% of cases (4/72) and immune cell expression in 18.1% of cases (13/72), but did not correlate with the expression pattern in the primary tumor. Overall PD-L1 expression in the primary tumor did not influence survival. However, PD-L1 expression was correlated with the number of CD3(+) tumor-infiltrating lymphocytes in the tumor center, and a combinational score of PD-L1 status/CD3(+) tumor-infiltrating lymphocytes was correlated with patients' overall survival.
Wissenschaftlicher Artikel
Scientific Article
Echtler, K. ; Konrad, I. ; Lorenz, M. ; Schneider, S. ; Hofmaier, S. ; Plenagl, F. ; Stark, K. ; Czermak, T. ; Tirniceriu, A. ; Eichhorn, M.E. ; Walch, A.K. ; Enders, G. ; Massberg, S. ; Schulz, C.
PLoS ONE 12:e0172788 (2017)
Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases.
Wissenschaftlicher Artikel
Scientific Article
Rodriguez Camargo, D.C. ; Tripsianes, K. ; Buday, K. ; Frankó, A. ; Göbl, C. ; Hartlmüller, C. ; Sarkar, R. ; Aichler, M. ; Mettenleiter, G. ; Schulz, M. ; Böddrich, A. ; Erck, C. ; Martens, H. ; Walch, A.K. ; Madl, T. ; Wanker, E.E. ; Conrad, M. ; Hrabě de Angelis, M. ; Reif, B.
Sci. Rep. 7:44041 (2017)
Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.
Wissenschaftlicher Artikel
Scientific Article
Frankó, A. ; Neschen, S. ; Rozman, J. ; Rathkolb, B. ; Aichler, M. ; Feuchtinger, A. ; Brachthäuser, L. ; Neff, F. ; Kovarova, M. ; Wolf, E. ; Fuchs, H. ; Häring, H.-U. ; Peter, A. ; Hrabě de Angelis, M.
Mol. Metab. 6, 256-266 (2017)
Objective Recently, we have shown that Bezafibrate (BEZ), the pan-PPAR (peroxisome proliferator-activated receptor) activator, ameliorated diabetes in insulin deficient streptozotocin treated diabetic mice. In order to study whether BEZ can also improve glucose metabolism in a mouse model for fatty liver and type 2 diabetes, the drug was applied to TallyHo mice. Methods TallyHo mice were divided into an early (ED) and late (LD) diabetes progression group and both groups were treated with 0.5% BEZ (BEZ group) or standard diet (SD group) for 8 weeks. We analyzed plasma parameters, pancreatic beta-cell morphology, and mass as well as glucose metabolism of the BEZ-treated and control mice. Furthermore, liver fat content and composition as well as hepatic gluconeogenesis and mitochondrial mass were determined. Results Plasma lipid and glucose levels were markedly reduced upon BEZ treatment, which was accompanied by elevated insulin sensitivity index as well as glucose tolerance, respectively. BEZ increased islet area in the pancreas. Furthermore, BEZ treatment improved energy expenditure and metabolic flexibility. In the liver, BEZ ameliorated steatosis, modified lipid composition and increased mitochondrial mass, which was accompanied by reduced hepatic gluconeogenesis. Conclusions Our data showed that BEZ ameliorates diabetes probably via reduced steatosis, enhanced hepatic mitochondrial mass, improved metabolic flexibility and elevated hepatic insulin sensitivity in TallyHo mice, suggesting that BEZ treatment could be beneficial for patients with NAFLD and impaired glucose metabolism.
Wissenschaftlicher Artikel
Scientific Article
Buck, A. ; Aichler, M. ; Huber, K. ; Walch, A.K.
Adv. Cancer Res. 134, 117-132 (2017)
Metabolomics is a rapidly evolving and a promising research field with the expectation to improve diagnosis, therapeutic treatment prediction, and prognosis of particular diseases. Among all techniques used to assess the metabolome in biological systems, mass spectrometry imaging is the method of choice to qualitatively and quantitatively analyze metabolite distribution in tissues with a high spatial resolution, thus providing molecular data in relation to cancer histopathology. The technique is ideally suited to study tissues molecular content and is able to provide molecular biomarkers or specific mass signatures which can be used in classification or the prognostic evaluation of tumors. Recently, it was shown that FFPE tissue samples are also suitable for metabolic analyses. This progress in methodology allows access to a highly valuable resource of tissues believed to widen and strengthen metabolic discovery-driven studies.
Wissenschaftlicher Artikel
Scientific Article
Ma, X. ; van Phi, V. ; Kimm, M.A. ; Prakash, J. ; Kessler, H. ; Kosanke, K. ; Feuchtinger, A. ; Aichler, M. ; Gupta, A. ; Rummeny, E.J. ; Eisenblätter, M. ; Siveke, J. ; Walch, A.K. ; Braren, R. ; Ntziachristos, V. ; Wildgruber, M.
Neoplasia 19, 8-16 (2017)
Integrins play an important role in tumor progression, invasion and metastasis. Therefore we aimed to evaluate a preclinical imaging approach applying ανβ3 integrin targeted hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) for monitoring tumor progression as well as early therapy response in a syngeneic murine Non-Small Cell Lung Cancer (NSCLC) model. Lewis Lung Carcinomas were grown orthotopically in C57BL/6 J mice and imaged in-vivo using a ανβ3 targeted near-infrared fluorescence (NIRF) probe. ανβ3-targeted FMT-XCT was able to track tumor progression. Cilengitide was able to substantially block the binding of the NIRF probe and suppress the imaging signal. Additionally mice were treated with an established chemotherapy regimen of Cisplatin and Bevacizumab or with a novel MEK inhibitor (Refametinib) for 2 weeks. While μCT revealed only a moderate slowdown of tumor growth, ανβ3 dependent signal decreased significantly compared to non-treated mice already at one week post treatment. ανβ3 targeted imaging might therefore become a promising tool for assessment of early therapy response in the future.
Wissenschaftlicher Artikel
Scientific Article
Zhao, S. ; Dorn, J. ; Napieralski, R. ; Walch, A.K. ; Diersch, S. ; Kotzsch, M. ; Ahmed, N. ; Hooper, J. ; Kiechle, M. ; Schmitt, M. ; Magdolen, V.
Biol. Chem. 398, 765-773 (2017)
n serous ovarian cancer, the clinical relevance of tumor cell-expressed plasmin(ogen) (PLG) has not yet been evaluated. Due to its proteolytic activity, plasmin supports tumorigenesis, however, angiostatin(-like) fragments, derived from PLG, can also function as potent antitumorigenic factors. In the present study, we assessed PLG protein expression in 103 cases of advanced high-grade serous ovarian cancer (FIGO III/IV) by immunohistochemistry. In 70/103 cases, positive staining of tumor cells was observed. In univariate Cox regression analysis, PLG staining was positively associated with prolonged overall survival (OS) (hazard ratio [HR] = 0.59, P = 0.026) of the patients. In multivariable analysis, PLG, together with residual tumor mass, remained a statistically significant independent prognostic marker (HR = 0.49, P = 0.009). In another small patient cohort (n=29), we assessed mRNA expression levels of PLG by quantitative PCR. Here, elevated PLG mRNA levels were also significantly associated with prolonged OS of patients (Kaplan-Meier analysis; P = 0.001). This finding was validated by in silico analysis of a microarray data set (n = 398) from The Cancer Genome Atlas (Kaplan-Meier analysis; P = 0.031). In summary, these data indicate that elevated PLG expression represents a favorable prognostic biomarker in advanced (FIGO III/IV) high-grade serous ovarian cancer.
Wissenschaftlicher Artikel
Scientific Article
Koch, M. ; de Jong, J.S. ; Glatz, J. ; Symvoulidis, P. ; Lamberts, L.E. ; Adams, A.L.L. ; Kranendonk, M.E.G. ; Terwisscha van Scheltinga, A.G. ; Aichler, M. ; Jansen, L. ; de Vries, J. ; Lub-de, Hoog, M.N. ; Schröder, C.P. ; Jorritsma-Smit, A. ; Linssen, M.D. ; de Boer, E. ; van der Vegt, B. ; Nagengast, W.B. ; Elisas,S.G. ; Oliveira, S. ; Witkamp, A.J. ; Mali, W.P.Th.M. ; van der Wall, E. ; Gracia-Allende, P.B. ; van Diest, P.J. ; de Vries, E.G. ; Walch, A.K. ; van Dam, G.M. ; Ntziachristos, V.
Cancer Res. 77, 623-631 (2017)
In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multi-scale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labelled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging.
Wissenschaftlicher Artikel
Scientific Article
Doll, S. ; Proneth, B. ; Tyurina, A.A. ; Panzilius, E. ; Kobayashi, S. ; Ingold, I. ; Irmler, M. ; Beckers, J. ; Aichler, M. ; Walch, A.K. ; Prokisch, H. ; Trümbach, D. ; Mao, G. ; Qu, F. ; Bayir, H. ; Füllekrug, J. ; Scheel, C. ; Wurst, W. ; Schick, J. ; Kagan, V.E. ; Friedmann Angeli, J.P.F. ; Conrad, M.
Nat. Chem. Biol. 13, 91-98 (2017)
© 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches—a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines—to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4–Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.
Wissenschaftlicher Artikel
Scientific Article
2016
Luber, B. ; Keller, S. ; Zwingenberger, G. ; Ebert, K. ; Maier, D. ; Geier, B. ; Theis, F.J. ; Hasenauer, J. ; Huge, S. ; Meyer-Hermann, M. ; Dehghany, J. ; Walch, A.K. ; Aichler, M. ; Lordick, F. ; Haffner, I.
Eur. J. Cancer 68, S135-S135 (2016)
Meeting abstract
Meeting abstract
Abdelmoula, W.M. ; Balluff, B. ; Englert, S. ; Dijkstra, J. ; Reinders, M.J.T. ; Walch, A.K. ; McDonnell, L.A. ; Lelieveldt, B.P.F.
Proc. Natl. Acad. Sci. U.S.A. 113, 12244-12249 (2016)
The identification of tumor subpopulations that adversely affect patient outcomes is essential for a more targeted investigation into how tumors develop detrimental phenotypes, as well as for personalized therapy. Mass spectrometry imaging has demonstrated the ability to uncover molecular intratumor heterogeneity. The challenge has been to conduct an objective analysis of the resulting data to identify those tumor subpopulations that affect patient outcome. Here we introduce spatially mapped t-distributed stochastic neighbor embedding (t-SNE), a nonlinear visualization of the data that is able to better resolve the biomolecular intratumor heterogeneity. In an unbiased manner, t-SNE can uncover tumor subpopulations that are statistically linked to patient survival in gastric cancer and metastasis status in primary tumors of breast cancer.
Wissenschaftlicher Artikel
Scientific Article
Schenck, T.L. ; Lin, S. ; Stewart, J.K. ; Koban, K.C. ; Aichler, M. ; Rezaeian, F. ; Giunta, R.E.
Brain Behav. 6:e00578 (2016)
Background: It remains a surgical challenge to treat high-grade nerve injuries of the upper extremity. Extra-anatomic reconstructions through the transfer of peripheral nerves have gained clinical importance over the past decades. This contribution outlines the anatomic and histomorphometric basis for the transfer of the superficial branch of the radial nerve (SBRN) to the median nerve (MN) and the superficial branch of the ulnar nerve (SBUN). Methods: The SBRN, MN, and SBUN were identified in 15 specimens and the nerve transfer performed. A favorable site for coaptation was chosen and its location described using relevant anatomical landmarks. Histomorphometric characteristics of donor and target were compared to evaluate the chances of a clinical success. Results: A suitable location for dissecting the SBRN was identified prior to its first bifurcation. Coaptations were possible near the pronator quadratus muscle, approximately 22 cm distal to the lateral epicondyle of the humerus. The MN and SBUN had to be dissected interfasciculary over 82 ± 5.7 mm and 49 ± 5.5 mm, respectively. Histomorphometric analysis revealed sufficient donor-to-recipient axon ratios for both transfers and identified the SBRN as a suitable donor with high axon density. Conclusion: Our anatomic and histomorphometric results indicate that the SBRN is a suitable donor for the MN and SBUN at wrist level. The measurements show feasibility of this procedure and shall help in planning this sensory nerve transfer. High axon density in the SBRN identifies it or its branches an ideal candidate for sensory reanimation of fingers and thumbs.
Wissenschaftlicher Artikel
Scientific Article
Wittmann, A. ; Grimm, M. ; Scherthan, H. ; Horsch, M. ; Beckers, J. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Ford, S.J. ; Burton, N.C. ; Razansky, D. ; Trümbach, D. ; Aichler, M. ; Walch, A.K. ; Calzada-Wack, J. ; Neff, F. ; Wurst, W. ; Hartmann, T. ; Floß, T.
PLoS ONE 11:e0164298 (2016)
Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.
Wissenschaftlicher Artikel
Scientific Article
Feuchtinger, A. ; Walch, A.K. ; Dobosz, M.
Histochem. Cell Biol. 146, 1-26 (2016)
This review delves into the rapidly evolving field of deep tissue imaging at cellular resolution, reviewing popular tissue clearing and staining methods in combination with light-sheet fluorescence microscopy (LSFM) including quantification and three-dimensional visualization tools, the field of applications and perspective, particularly with the focus on preclinical cancer research and drug development. The LSFM technique presented here allows an extremely fast optical sectioning for three-dimensional reconstruction of centimeter-sized tissue samples at cellular resolution. However, optical clearing methods are required to receive optical transparent tissue. Application of either tissue autofluorescence, in vivo fluorescence labeling, endogenous fluorescence or ex vivo whole-mount immunolabeling enables three-dimensional in situ visualization of morphological and functional features of unsectioned whole-mount tissue samples. This powerful and innovative imaging technique opens up a new dimension of tissue analysis providing detailed and comprehensive insights into biology. It enables the investigation of normal and pathological tissue features and disease progression and allows precise monitoring of potential therapeutic interventions in intact biological tissue on a cellular level.
Review
Review
Huber, M. ; Mall, R. ; Braselmann, H. ; Feuchtinger, A. ; Molatore, S. ; Lindner, K. ; Walch, A.K. ; Gross, E. ; Schmitt, M. ; Falkenberg, N. ; Aubele, M.
BMC Cancer 16:615 (2016)
BACKGROUND: Due to lack of a targeted therapy for the triple-negative breast cancer (TNBC) patients, it is important to explore this aggressive breast cancer type in more detail and to establish novel therapeutic approaches. TNBC is defined negative for the protein expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). One prominent feature of this cancer type is the frequent overexpression of major components of the urokinase-type plasminogen activator system (uPAS) including uPA, its receptor uPAR and the inhibitor PAI-1, which may be valuable as therapeutic targets. METHODS: Direct interactions of uPAR with interactors were demonstrated by immunoprecipitations and proximity ligation assays. For stable knockdowns of target proteins, lentiviral vectors were used and the effects were analysed by immunoblottings and using in vitro cell viability, migration and invasion assays. Immunohistochemical and statistical analyses of biomarkers and clinical parameters were conducted in a TNBC cohort (n = 174). RESULTS: Direct tumour-promoting interactions of uPAR with uPA and the insulin-like growth factor receptor 1 (IGF1R) were shown in TNBC cells and these interactions were significantly reduced (p = 0.001) when uPAR was downregulated. The combined knockdown of uPAR and uPA or IGF1R additively and significantly reduced cell viability, migration and invasion of the model cell lines. In TNBC tissue, the complexes formed by uPAR with uPA or with IGF1R significantly correlated with the histological grade (p = 0.0019) as well as with cathepsin B and D (p ≤ 0.0001) that are implicated in cell invasion and metastasis. CONCLUSIONS: Our outcomes show that not only overexpressed biomarkers promote tumourigenesis, but rather their interactions further potentiate tumour progression. This study emphasises the potential of combined approaches targeting uPAR and its interactors with regard to an improved therapy of TNBC.
Wissenschaftlicher Artikel
Scientific Article
Stark, K. ; Philippi, V. ; Stockhausen, S. ; Busse, J. ; Antonelli, A. ; Miller, M. ; Schubert, I. ; Hoseinpour, P. ; Chandraratne, S. ; von Brühl, M.L. ; Gärtner, F. ; Lorenz, M. ; Agresti, A. ; Coletti, R. ; Antoine, D.J. ; Heermann, R. ; Jung, K. ; Reese, S. ; Laitinen, I. ; Schwaiger, M. ; Walch, A.K. ; Sperandio, M. ; Nawroth, P.P. ; Reinhardt, C. ; Jäckel, S. ; Bianchi, M.E. ; Massberg, S.
Blood 128, 2435-2449 (2016)
Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. While sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior we identified blood-derived high-mobility group box 1 protein (HMGB1) - a prototypical mediator of sterile inflammation - to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1(-/-) chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through RAGE and TLR2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.
Wissenschaftlicher Artikel
Scientific Article
Chekkoury, A. ; Nunes, A. ; Gateau, J. ; Symvoulidis, P. ; Feuchtinger, A. ; Bézière, N. ; Ovsepian, S.V. ; Walch, A.K. ; Ntziachristos, V.
Neoplasia 18, 459-467 (2016)
Diversity of the design and alignment of illumination and ultrasonic transducers empower the fine scalability and versatility of optoacoustic imaging. In this study, we implement an innovative high-resolution optoacoustic mesoscopy for imaging the vasculature and tissue oxygenation within subcutaneous and orthotopic cancerous implants of mice in vivo through acquisition of tomographic projections over 180° at a central frequency of 24 MHz. High-resolution volumetric imaging was combined with multispectral functional measurements to resolve the exquisite inner structure and vascularization of the entire tumor mass using endogenous and exogenous optoacoustic contrast. Evidence is presented for constitutive hypoxemia within the carcinogenic tissue through analysis of the hemoglobin absorption spectra and distribution. Morphometric readouts obtained with optoacoustic mesoscopy have been verified with high-resolution ultramicroscopic studies. The findings described herein greatly extend the applications of optoacoustic mesoscopy toward structural and multispectral functional measurements of the vascularization and hemodynamics within solid tumors in vivo and are of major relevance to basic and preclinical oncological studies in small animal models.
Wissenschaftlicher Artikel
Scientific Article
Ly, A. ; Buck, A. ; Balluff, B. ; Sun, N. ; Gorzolka, K. ; Feuchtinger, A. ; Janssen, K.P. ; Kuppen, P.J. ; van de Velde, C.J. ; Weirich, G. ; Erlmeier, F. ; Langer, R. ; Aubele, M. ; Zitzelsberger, H. ; McDonnell, L.A. ; Aichler, M. ; Walch, A.K.
Nat. Protoc. 11, 1428-1443 (2016)
Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.
Wissenschaftlicher Artikel
Scientific Article
Mendler, C.T. ; Feuchtinger, A. ; Heid, I. ; Aichler, M. ; D'Alessandria, C. ; Pirsig, S. ; Blechert, B. ; Wester, H.J. ; Braren, R. ; Walch, A.K. ; Skerra, A. ; Schwaiger, M.
J. Nucl. Med. 57, 1971-1977 (2016)
Antibodies have become an established treatment modality in cancer therapy during the last decade. However, these treatments often suffer from insufficient and heterogeneous response despite validated antigen or target receptor expression in the tumor. In fact, therapeutic success depends both on the presence and accessibility of the tumor antigen by the antibody. In search of a suitable preclinical animal model to evaluate the mechanisms of tumor heterogeneity and hemodynamics, we characterized two exemplary non-Hodgkin lymphoma subtypes with comparable CD20 expression and metabolism, SUDHL-4 and Granta, using multimodal imaging techniques. METHODS: To investigate in vivo biodistribution, two differently modified αCD20 antigen-binding fragments (Fab), prepared (i) by PASylation and (ii) by fusion with an albumin-binding domain (ABD), were radiolabeled with (125)I and intravenously injected into immunocompromised mice bearing corresponding xenografts. RESULTS: Validation with (18)F-FDG revealed similar distribution of vital tumor tissue 1 h p.i. However, large differences in tumor uptake were observed when applying the CD20-specific radiotracers (125)I-Fab-ABD and (125)I-Fab-PAS200 with 12.3 and 2.4 % ID/g, respectively, for Granta in comparison with 3.5 and 0.75 % ID/g, respectively, for SUDHL-4 xenografts 24 h p.i. 3D light-sheet fluorescence microscopy with Cy5-Fab-PAS200 confirmed better tracer extravasation in the Granta tumors. Moreover, dynamic contrast enhanced MRI imaging revealed significantly reduced tumor perfusion in the SUHDL-4 xenografts. CONCLUSION: Tracer uptake was highly dependent on local tumor perfusion as well as Fab permeation in the SUDHL-4 and Granta tumors. Thus, the SUDHL-4 xenograft offers an excellent model system to investigate the influence of therapies affecting tumor angiogenesis.
Wissenschaftlicher Artikel
Scientific Article
Bader, E. ; Migliorini, A. ; Gegg, M. ; Moruzzi, N. ; Gerdes, J.M. ; Roscioni, S. ; Bakhti, M. ; Brandl, E. ; Irmler, M. ; Beckers, J. ; Aichler, M. ; Feuchtinger, A. ; Leitzinger, C. ; Zischka, H. ; Wang-Sattler, R. ; Jastroch, M. ; Tschöp, M.H. ; Machicao, F. ; Staiger, H. ; Häring, H.-U. ; Chmelova, H. ; Chouinard, J.A. ; Oskolkov, N. ; Korsgren, O. ; Speier, S. ; Lickert, H.
Nature 535, 430-434 (2016)
Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of β-cells. Pancreatic β-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene, acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature β-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger β-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for β-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional β-cell heterogeneity and induce β-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional β-cell mass in diabetic patients.
Wissenschaftlicher Artikel
Scientific Article
Mall, S. ; Yusufi, N. ; Wagner, R. ; Klar, R. ; Bianchi, H. ; Steiger, K. ; Straub, M. ; Audehm, S. ; Laitinen, I. ; Aichler, M. ; Peschel, C. ; Ziegler, S. ; Mustafa, M. ; Schwaiger, M. ; D'Alessandria, C. ; Krackhardt, A.M.
Cancer Res. 76, 4113-4123 (2016)
Sensitive in vivo imaging technologies applicable to the clinical setting are still lacking for adoptive T-cell-based immunotherapies, an important gap to fill if mechanisms of tumor rejection or escape are to be understood. Here, we propose a highly sensitive imaging technology to track human TCR-transgenic T cells in vivo by directly targeting the murinized constant TCR beta domain (TCRmu) with a zirconium-89 ((89)Zr)-labeled anti-TCRmu-F(ab')2 fragment. Binding of the labeled or unlabeled F(ab')2 fragment did not impair functionality of transgenic T cells in vitro and in vivo Using a murine xenograft model of human myeloid sarcoma, we monitored by Immuno-PET imaging human central memory T cells (TCM), which were transgenic for a myeloid peroxidase (MPO)-specific TCR. Diverse T-cell distribution patterns were detected by PET/CT imaging, depending on the tumor size and rejection phase. Results were confirmed by IHC and semiquantitative evaluation of T-cell infiltration within the tumor corresponding to the PET/CT images. Overall, these findings offer a preclinical proof of concept for an imaging approach that is readily tractable for clinical translation.
Wissenschaftlicher Artikel
Scientific Article
Huber, M. ; Falkenberg, N. ; Hauck, S.M. ; Priller, M. ; Braselmann, H. ; Feuchtinger, A. ; Walch, A.K. ; Schmitt, M. ; Aubele, M.
Oncotarget 7, 44062-44075 (2016)
The triple-negative breast cancer (TNBC) is a very aggressive tumor type often occurring in young women and is associated with a bad prognosis for the patients. TNBC lacks established targets for breast cancer therapy, such as the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Therefore, novel therapeutic targets and strategies are needed for an improved treatment of this breast cancer subtype. TNBC and respective cell lines often overexpress proteins of the urokinase plasminogen activator system (uPAS) including uPA, its receptor uPAR and inhibitor PAI-1, which together with co-factors contribute to the malignancy of TNBC. Here, two novel interacting partners of uPAR, the cysteine-rich angiogenic inducer 61 (Cyr61) and the Y-box-binding protein 1 (YB-1) were identified and their differential expression demonstrated in TNBC cells as well as in tumors. In the TNBC cohort, both interactors significantly correlated with expression levels of cathepsin B, c-Met and the tumor grade. In addition, expression levels of Cyr61 significantly correlated with cathepsin D (p=0.03), insulin receptor (p≤0.001), insulin-like growth factor receptor 1 (IGF1R, p=0.015) and also with YB-1 (p=0.0004) levels. The interactions of uPAR with Cyr61 significantly correlated with expression levels of tumor-promoting biomarkers including plasminogen (p=0.0014), cathepsin B (p=0.032), c-Met (p=0.0192) as well as with the tumor grade (p=0.02). In multivariate survival analysis, YB-1 showed independent prognostic value (p=0.01). As the novel interacting partners, also together with uPAR, contribute to tumor progression and metastasis, both may be potential therapeutic targets in breast cancer.
Wissenschaftlicher Artikel
Scientific Article
Frankó, A. ; Huypens, P. ; Neschen, S. ; Irmler, M. ; Rozman, J. ; Rathkolb, B. ; Neff, F. ; Prehn, C. ; Dubois, G. ; Baumann, M. ; Massinger, R. ; Gradinger, D. ; Przemeck, G.K.H. ; Repp, B. ; Aichler, M. ; Feuchtinger, A. ; Schommers, P. ; Stöhr, O. ; Sanchez-Lasheras, C. ; Adamski, J. ; Peter, A. ; Prokisch, H. ; Beckers, J. ; Walch, A.K. ; Fuchs, H. ; Wolf, E. ; Schubert, M. ; Wiesner, R.J. ; Hrabě de Angelis, M.
Diabetes 65, 2540-2552 (2016)
Bezafibrate (BEZ), a pan activator of peroxisome proliferator-activated receptors (PPARs), has been generally used to treat hyperlipidemia for decades. Clinical trials with type 2 diabetes patients indicated that BEZ also has beneficial effects on glucose metabolism, although the underlying mechanisms of these effects remain elusive. Even less is known about a potential role for BEZ in treating type 1 diabetes. Here we show that BEZ markedly improves hyperglycemia and glucose and insulin tolerance in streptozotocin (STZ)-treated mice, an insulin-deficient mouse model of type 1 diabetes. BEZ treatment of STZ mice significantly suppressed the hepatic expression of genes that are annotated in inflammatory processes, whereas the expression of PPAR and insulin target gene transcripts was increased. Furthermore, BEZ-treated mice also exhibited improved metabolic flexibility as well as an enhanced mitochondrial mass and function in the liver. Finally, we show that the number of pancreatic islets and the area of insulin positive cells tended to be higher in BEZ-treated mice. Our data suggest that BEZ may improve impaired glucose metabolism by augmenting hepatic mitochondrial performance, suppressing hepatic inflammatory pathways, and improving insulin sensitivity and metabolic flexibility. Thus, BEZ treatment might also be useful for patients with impaired glucose tolerance or diabetes.
Wissenschaftlicher Artikel
Scientific Article
Cassese, A. ; Ellis, S. ; Ogrinc Potočnik, N. ; Burgermeister, E. ; Ebert, M. ; Walch, A.K. ; van den Maagdenberg, A.M. ; McDonnell, L.A. ; Heeren, R.M. ; Balluff, B.
Anal. Chem. 88, 5871-5878 (2016)
Mass spectrometry imaging (MSI) is a powerful molecular imaging technique. In microprobe MSI, images are created through a grid-wise interrogation of individual spots by mass spectrometry across a surface. Classical statistical tests for within-sample comparisons fail as close-by measurement spots violate the assumption of independence of these tests, which can lead to an increased false-discovery rate. For spatial data this effect is referred to as spatial autocorrelation. In this study we investigated spatial autocorrelation in three different matrix-assisted laser desorption/ionization MSI datasets. These datasets cover different molecular classes (metabolites/drugs, lipids, and proteins) and different spatial resolutions ranging from 20 µm to 100 µm. Significant spatial autocorrelation was detected in all three datasets and found to increase with decreasing pixel size. To enable statistical testing for differences in mass signal intensities between regions of interest within MSI datasets, we propose the use of Conditional Autoregressive (CAR) models. We show that by accounting for spatial autocorrelation, discovery rates (i.e. the ratio between the features identified and the total number of features) could be reduced between 21% and 69%. The reliability of this approach was validated by control mass signals based on prior knowledge. In light of the advent of larger MSI datasets based on either an increased spatial resolution or 3D datasets, accounting for effects due to spatial autocorrelation becomes even more indispensable. Here we propose a generic and easily applicable workflow to enable within-sample statistical comparisons.
Wissenschaftlicher Artikel
Scientific Article
Buck, A. ; Balluff, B. ; Voss, A. ; Langer, R. ; Zitzelsberger, H. ; Aichler, M. ; Walch, A.K.
Anal. Chem. 88, 5281-5289 (2016)
In research and clinical settings formalin-fixed and paraffin-embedded (FFPE) tissue specimens are collected routinely and therefore this material constitutes a highly valuable source to gather insight in metabolic changes of diseases. Among mass spectrometry techniques to examine the molecular content of FFPE tissue mass spectrometry imaging (MSI) is the most appropriate when morphological and histological features shall be related to metabolic information. Currently, high resolution mass spectrometers are widely used for metabolomics studies. However, with regards to matrix-assisted laser desorption/ionization (MALDI) MSI no study has so far addressed the necessity of instrumental mass resolving power in terms of clinical diagnosis and prognosis using archived FFPE tissue. For this matter we performed for the first time a comprehensive comparison between a high mass resolution Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer and a time-of-flight (TOF) instrument with lower mass resolving power. Spectra analysis revealed that about one third of the detected peaks remained unresolved by MALDI-TOF which led to a three to five time lower number of m/z features compared to FT-ICR measurements. Overlaid peak information and background noise in TOF images made a precise assignment of molecular attributes to morphological features more difficult and limited classification approaches. This clearly demonstrates the need for high mass resolution capabilities for metabolite imaging. Nevertheless, MALDI-TOF allowed reproducing and verifying individual markers identified previously by MALDI-FT-ICR MSI. The systematic comparison gives rise to synergistically combine the different MSI platforms for high-throughput discovery and validation of biomarkers.
Wissenschaftlicher Artikel
Scientific Article
Dorn, J. ; Yassouridis, A. ; Walch, A.K. ; Diamandis, E.P. ; Schmitt, M. ; Kiechle, M. ; Wang, P. ; Drecoll, E. ; Schmalfeldt, B. ; Loessner, D. ; Kotzsch, M. ; Magdolen, V.
Am. J. Cancer Res. 6, 61-70 (2016)
Members of the human kallikrein-related peptidase (KLK) family, including KLK5, have been reported to play an important role in ovarian cancer progression. In the present study, we assessed KLK5 protein expression in ovarian cancer tissues by immunohistochemistry (IHC) and ELISA, and analyzed its association with clinicopathologic parameters and disease outcome in 95 patients with advanced ovarian cancer FIGO stage III/IV. KLK5 immunoexpression was evaluated in ovarian cancer tissue microarrays by IHC using a manual semiquantitative scoring system. KLK5 antigen levels were determined in ovarian cancer tumour tissue extracts by ELISA. KLK5 protein is expressed in ovarian cancer tissue by stromal and tumor cells. Mean KLK5 immunoscore values in tumor cells (KLK5-Tc; 5.7, range 0 to 12) were higher compared to stromal cells (KLK5-Sc; 1.2, range 0 to 9) but the correlation between KLK5-Tc and KLK5-Sc was rather low (rs = 0.34, P < 0.05). No significant associations of clinicopathological parameters with KLK5-Tc, KLK5-Sc, the combined overall score KLK5-Tc+Sc, or ELISA (KLK5-E) expression values were determined, except for KLK5-E protein expression with advanced age and high nuclear grade (G3). In univariate Cox regression analysis, elevated expression levels of KLK5-Sc are significantly linked with both prolonged overall survival (OS) (hazard ratio [HR] = 0.6, P = 0.046) and progression-free survival (PFS) (HR = 0.54, P = 0.032) of advanced ovarian cancer patients. KLK5-Tc and KLK5-Tc+Sc scores as well as the KLK5-E values were not associated with patients' outcome. In multivariable analysis, KLK5-Sc expression was found to be statistically significant for PFS. Patients with elevated KLK5-Sc had a two-fold lower risk of disease recurrence (HR = 0.53, P = 0.037) as compared to patients with low KLK5-Sc. For KLK5-Sc and OS, a trend towards statistical significance was observed (HR = 0.62, P = 0.077). These results indicate that KLK5 overexpression by stromal cells (KLK5-Sc) may be a positive modulator lowering aggressiveness of ovarian cancer.
Wissenschaftlicher Artikel
Scientific Article
Hachmöller, O. ; Aichler, M. ; Schwamborn, K. ; Lutz, L. ; Werner, M. ; Sperling, M. ; Walch, A.K. ; Karst, U.
J. Trace Elem. Med. Biol. 35, 97-102 (2016)
A laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is developed and applied for the analysis of paraffin-embedded liver needle biopsy specimens of patients with Wilson's disease (WD), a rare autosomal recessive disorder of the copper metabolism causing various hepatic, neurological and psychiatric symptoms due to a copper accumulation in the liver and the central nervous system. The sample set includes two WD liver samples and one negative control sample. The imaging analysis was performed with a spatial resolution of 10 μm. Besides copper, iron was monitored because an elevated iron concentration in the liver is known for WD. In addition to this, both elements were quantified using an external calibration based on matrix-matched gelatine standards. The presented method offers low limits of detection of 1 and 5 μg/g for copper and iron, respectively. The high detection power and good spatial resolution allow the analysis of small needle biopsy specimen using this method. The two analyzed WD samples can be well differentiated from the control sample due to their inhomogeneous copper distribution and high copper concentrations of up to 1200μg/g. Interestingly, the WD samples show an inverse correlation of regions with elevated copper concentrations and regions with high iron concentrations.
Wissenschaftlicher Artikel
Scientific Article
Trajkovic-Arsic, M. ; Kalideris, E. ; Sun, N. ; Feuchtinger, A. ; Gupta, A. ; Aichler, M. ; Schmid, R.M. ; Walch, A.K. ; Siveke, J.T.
Oncol. Res. Treat. 39, 76-77 (2016)
Meeting abstract
Meeting abstract
Haffner, I. ; Luber, B. ; Maier, D. ; Geier, B. ; Theis, F.J. ; Meyer-Hermann, M. ; Walch, A.K. ; Kretzschmar, A.K. ; von Weikersthal, L.F. ; Knorrenschild, J.R. ; Schierle, K. ; Wittekind, C. ; Lordick, F.
Oncol. Res. Treat. 39, 16-17 (2016)
Meeting abstract
Meeting abstract
Hachmöller, O. ; Buzanich, A.G. ; Aichler, M. ; Radtke, M. ; Dietrich, D. ; Schwamborn, K. ; Lutz, L. ; Werner, M. ; Sperling, M. ; Walch, A.K. ; Karst, U.
Metallomics 8, 648-653 (2016)
A liver biopsy specimen from a Wilson's disease (WD) patient was analyzed by means of micro-X-ray fluorescence (μXRF) spectroscopy to determine the elemental distribution. First, bench-top μXRF was utilized for a coarse scan of the sample under laboratory conditions. The resulting distribution maps of copper and iron enabled the determination of a region of interest (ROI) for further analysis. In order to obtain more detailed elemental information, this ROI was analyzed by synchrotron radiation (SR)-based μXRF with a beam size of 4 μm offering a resolution at the cellular level. Distribution maps of additional elements to copper and iron like zinc and manganese were obtained due to a higher sensitivity of SR-μXRF. In addition to this, X-ray absorption near edge structure spectroscopy (XANES) was performed to identify the oxidation states of copper in WD. This speciation analysis indicated a mixture of copper(i) and copper(ii) within the WD liver tissue.
Wissenschaftlicher Artikel
Scientific Article
Dislich, B. ; Stein, A. ; Seiler, C.A. ; Walch, A.K. ; Berezowska, S. ; Zlobec, I. ; Galvan, J.A. ; Langer, R.
Mod. Pathol. 29, 170A (2016)
Meeting abstract
Meeting abstract
Li, J. ; Dawidek, T. ; Liegsalz, V. ; Tzalis, V. ; Feuchtinger, A. ; Avril, S.
Mod. Pathol. 29, 53A (2016)
Meeting abstract
Meeting abstract
Ly, A. ; Merl-Pham, J. ; Priller, M. ; Gruhn, F. ; Senninger, N. ; Ueffing, M. ; Hauck, S.M.
J. Proteome Res. 15, 1350-1359 (2016)
The rd10 mouse is a model of retinitis pigmentosa characterized by the dysfunction of a rod-photoreceptor-specific phosphodiesterase. Compared to the rd1 mouse, retinal degeneration in the rd10 mouse begins later in age with a milder phenotype, making it ideal for investigating cell death and neuroprotective mechanisms. Alterations in the rd10 retina proteome at pre-, peak-, and post-degenerative time points were examined using a modified high-recovery filter-aided sample preparation (FASP) method in combination with label-free quantitative mass spectrometry, generating a proteomic dataset on almost 3000 proteins. Our data confirmed a period of protein expression similar to age-matched wild-type mice pre-degeneration, with decreases in proteins associated with phototransduction and increases in signaling proteins at peak- and post-degenerative stages. 57 proteins were differentially expressed in the rd10 retinae during peak-degeneration compared to wild-type mice after stringent FDR correction (q<0.05). Network analysis separated these proteins into a cluster of downregulated photoreceptor proteins, and one of upregulated signaling proteins centered around GFAP, STAT3, and STAT1. This is the first study to identify alterations in STAT1 in the rd10 mouse, which were confirmed with gene expression and immunoblotting experiments underpinning the efficacy of our approach. This unique proteomic dataset on protein dynamics during retinal degeneration could serve as an information source for vision research in the future.
Wissenschaftlicher Artikel
Scientific Article
Dislich, B. ; Stein, A. ; Seiler, C.A. ; Walch, A.K. ; Berezowska, S. ; Zlobec, I. ; Galvan, J.A. ; Langer, R.
Lab. Invest. 96, 170A (2016)
Meeting abstract
Meeting abstract
Li, J. ; Dawidek, T. ; Liegsalz, V. ; Tzalis, V. ; Feuchtinger, A. ; Avril, S.
Lab. Invest. 96, 53A (2016)
Meeting abstract
Meeting abstract
Grüner, B.M. ; Winkelmann, I. ; Feuchtinger, A. ; Sun, N. ; Balluff, B. ; Teichmann, N. ; Herner, A. ; Kalideris, E. ; Steiger, K. ; Braren, R. ; Aichler, M. ; Esposito, I. ; Schmid, R.M. ; Walch, A.K. ; Siveke, J.T.
Mol. Cancer Ther. 15, 1145-1152 (2016)
Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. Aim of this study was to investigate the emerging technology of matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery and spatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term-treated mice to measure drug tissue distribution. Histological and statistical analyses were performed to correlate morphology, drug distribution and survival. We found that erlotinib levels were significantly lower in PDAC compared to healthy tissue (p = 0.0078). Survival of long-term-treated mice did not correlate with overall levels of erlotinib or with overall histological tumor grade but both with the percentage of atypical glands in the cancer (p = 0.021, rs = 0.59) and the level of erlotinib in those atypical glands (p = 0.019, rs = 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution compounds in a preclinical setting and supports drug imaging-based translational approaches.
Wissenschaftlicher Artikel
Scientific Article
Lahiri, S. ; Sun, N. ; Buck, A. ; Imhof, A. ; Walch, A.K.
Expert Rev. Proteomics 13, 275-284 (2016)
Histone post-translational modifications (PTMs), histone variants and enzymes responsible for the incorporation or the removal of the PTMs are being increasingly associated with human disease. Combinations of histone PTMs and the specific incorporation of variants contribute to the establishment of cellular identity and hence are potential markers that could be exploited in disease diagnostics and prognostics and therapy response prediction. Due to the scarcity of suitable antibodies and the pre-requirement of tissue homogenization for more advanced analytical techniques, comprehensive information regarding the spatial distribution of these factors at the tissue level has been lacking. MALDI imaging mass spectrometry provides an ideal platform to measure histone PTMs and variants from tissues while maintaining the information about their spatial distribution. Discussed in this review are the relevance of histones in the context of human disease and the contribution of MALDI imaging mass spectrometry in measuring histones in situ.
Wissenschaftlicher Artikel
Scientific Article
Girst, S. ; Greubel, C. ; Reindl, J. ; Siebenwirth, C. ; Zlobinskaya, O. ; Walsh, D.W. ; Ilicic, K. ; Aichler, M. ; Walch, A.K. ; Wilkens, J.J. ; Multhoff, G. ; Dollinger, G. ; Schmid, T.E.
Int. J. Radiat. Oncol. Biol. Phys. 95, 234-241 (2016)
PURPOSE: Proton minibeam radiation therapy is a novel approach to minimize normal tissue damage in the entrance channel by spatial fractionation while keeping tumor control through a homogeneous tumor dose using beam widening with an increasing track length. In the present study, the dose distributions for homogeneous broad beam and minibeam irradiation sessions were simulated. Also, in an animal study, acute normal tissue side effects of proton minibeam irradiation were compared with homogeneous irradiation in a tumor-free mouse ear model to account for the complex effects on the immune system and vasculature in an in vivo normal tissue model. METHODS AND MATERIALS: At the ion microprobe SNAKE, 20-MeV protons were administered to the central part (7.2 × 7.2 mm(2)) of the ear of BALB/c mice, using either a homogeneous field with a dose of 60 Gy or 16 minibeams with a nominal 6000 Gy (4 × 4 minibeams, size 0.18 × 0.18 mm(2), with a distance of 1.8 mm). The same average dose was used over the irradiated area. RESULTS: No ear swelling or other skin reactions were observed at any point after minibeam irradiation. In contrast, significant ear swelling (up to fourfold), erythema, and desquamation developed in homogeneously irradiated ears 3 to 4 weeks after irradiation. Hair loss and the disappearance of sebaceous glands were only detected in the homogeneously irradiated fields. CONCLUSIONS: These results show that proton minibeam radiation therapy results in reduced adverse effects compared with conventional homogeneous broad-beam irradiation and, therefore, might have the potential to decrease the incidence of side effects resulting from clinical proton and/or heavy ion therapy.
Wissenschaftlicher Artikel
Scientific Article
Sun, N. ; Fernandez, I.E. ; Wei, M. ; Wu, Y. ; Aichler, M. ; Eickelberg, O. ; Walch, A.K.
Histochem. Cell Biol. 145, 201-211 (2016)
Given the importance of pirfenidone as the first worldwide-approved drug for idiopathic pulmonary fibrosis treatment, its pharmacodynamic properties and the metabolic response to pirfenidone treatment have not been fully elucidated. The aim of the present study was to get molecular insights of pirfenidone-related pharmacometabolomic response using MALDI-FTICR-MSI. Quantitative MALDI-FTICR-MSI was carried out for determining the pharmacokinetic properties of pirfenidone and its related metabolites 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone in lung, liver and kidney. To monitor the effect of pirfenidone administration on endogenous cell metabolism, additional in situ endogenous metabolite imaging was performed in lung tissue sections. While pirfenidone is highly abundant and delocalized across the whole micro-regions of lung, kidney and liver, 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone demonstrate heterogeneous distribution patterns in lung and kidney. In situ endogenous metabolite imaging study of lung tissue indicates no significant effects of pirfenidone on metabolic pathways. Remarkably, we found 129 discriminative m/z values which represent clear differences between control and treated lungs, the majority of which are currently unknown. PCA analysis and heatmap view can accurately distinguish control and treated groups. This is the first pharmacokinetic study to investigate the tissue distribution of orally administered pirfenidone and its related metabolites simultaneously in organs without labeling. The combination of pharmametabolome with histological features provides detailed mapping of drug effects on metabolism as response of heathy lung tissue to pirfenidone treatment.
Wissenschaftlicher Artikel
Scientific Article
Lahiri, S. ; Sun, N. ; Solis-Mezarino, V. ; Fedisch, A. ; Ninkovic, J. ; Feuchtinger, A. ; Götz, M. ; Walch, A.K. ; Imhof, A.
Proteomics 16, 437-447 (2016)
Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity and availability of anti-histone antibodies. Here, we describe a novel MALDI imaging mass spectrometry (MALDI-IMS) based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available.
Wissenschaftlicher Artikel
Scientific Article
2015
Aubele, M. ; Huber, M. ; Falkenberg, N. ; Gross, E. ; Braselmann, H. ; Walch, A.K. ; Schmitt, M.
J. Clin. Oncol. 33:150 (2015)
Meeting abstract
Meeting abstract
Fernandez, I.E. ; Na, S. ; Wei, M. ; Aichler, M. ; Walch, A.K. ; Eickelberg, O.
Am. J. Respir. Crit. Care Med. 191:A4409 (2015)
Meeting abstract
Meeting abstract
Habringer, S. ; Li, Z. ; Pietschmann, E. ; Slawska, J. ; Walch, A.K. ; Peschel, C. ; Keller, U.
Oncol. Res. Treat. 38, 99 (2015)
Meeting abstract
Meeting abstract
Chekkoury, A. ; Gateau, J. ; Driessen, W.H.P. ; Symvoulidis, P. ; Bézière, N. ; Feuchtinger, A. ; Walch, A.K. ; Ntziachristos, V.
Biomed. Opt. Express 6, 3134-3148 (2015)
Optical mesoscopy extends the capabilities of biological visualization beyond the limited penetration depth achieved by microscopy. However, imaging of opaque organisms or tissues larger than a few hundred micrometers requires invasive tissue sectioning or chemical treatment of the specimen for clearing photon scattering, an invasive process that is regardless limited with depth. We developed previously unreported broadband optoacoustic mesoscopy as a tomographic modality to enable imaging of optical contrast through several millimeters of tissue, without the need for chemical treatment of tissues. We show that the unique combination of three-dimensional projections over a broad 500 kHz-40 MHz frequency range combined with multi-wavelength illumination is necessary to render broadband multispectral optoacoustic mesoscopy (2B-MSOM) superior to previous optical or optoacoustic mesoscopy implementations.
Wissenschaftlicher Artikel
Scientific Article
Pfluger, P.T. ; Kabra, D.G. ; Aichler, M. ; Schriever, S.C. ; Pfuhlmann, K. ; Casquero García, V. ; Lehti, M. ; Weber, J. ; Kutschke, M. ; Rozman, J. ; Elrod, J.W. ; Hevener, A.L. ; Feuchtinger, A. ; Hrabě de Angelis, M. ; Walch, A.K. ; Rollmann, S.M. ; Aronow, B.J. ; Müller, T.D. ; Perez-Tilve, D. ; Jastroch, M. ; de Luca, M. ; Molkentin, J.D. ; Tschöp, M.H.
Cell Metab. 22, 838-850 (2015)
Canonical protein phosphatase 3/calcineurin signaling is central to numerous physiological processes. Here we provide evidence that calcineurin plays a pivotal role in controlling systemic energy and body weight homeostasis. Knockdown of calcineurin in Drosophila melanogaster led to a decrease in body weight and energy stores, and increased energy expenditure. In mice, global deficiency of catalytic subunit Ppp3cb, and tissue-specific ablation of regulatory subunit Ppp3r1 from skeletal muscle, but not adipose tissue or liver, led to protection from high-fat-diet-induced obesity and comorbid sequelæ. Ser637 hyperphosphorylation of dynamin-related protein 1 (Drp1) in skeletal muscle of calcineurin-deficient mice was associated with mitochondrial elongation into power-cable-shaped filaments and increased mitochondrial respiration, but also with attenuated exercise performance. Our data suggest that calcineurin acts as highly conserved pivot for the adaptive metabolic responses to environmental changes such as high-fat, high-sugar diets or exercise.
Wissenschaftlicher Artikel
Scientific Article
Kiermayer, C. ; Northrup, E. ; Schrewe, A. ; Walch, A.K. ; Hrabě de Angelis, M. ; Schoensiegel, F. ; Zischka, H. ; Prehn, C. ; Adamski, J. ; Bekeredjian, R. ; Ivandic, B. ; Kupatt, C. ; Brielmeier, M.
J. Am. Heart Assoc. 4:e002153 (2015)
BACKGROUND: Ubiquitous deletion of thioredoxin reductase 2 (Txnrd2) in mice is embryonically lethal and associated with abnormal heart development, while constitutive, heart-specific Txnrd2 inactivation leads to dilated cardiomyopathy and perinatal death. The significance of Txnrd2 in aging cardiomyocytes, however, has not yet been examined. METHODS AND RESULTS: The tamoxifen-inducible heart-specific αMHC-MerCreMer transgene was used to inactivate loxP-flanked Txnrd2 alleles in adult mice. Hearts and isolated mitochondria from aged knockout mice were morphologically and functionally analyzed. Echocardiography revealed a significant increase in left ventricular end-systolic diameters in knockouts. Fractional shortening and ejection fraction were decreased compared with controls. Ultrastructural analysis of cardiomyocytes of aged mice showed mitochondrial degeneration and accumulation of autophagic bodies. A dysregulated autophagic activity was supported by higher levels of lysosome-associated membrane protein 1 (LAMP1), microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), and p62 in knockout hearts. Isolated Txnrd2-deficient mitochondria used less oxygen and tended to produce more reactive oxygen species. Chronic hypoxia inducible factor 1, α subunit stabilization and altered transcriptional and metabolic signatures indicated that energy metabolism is deregulated. CONCLUSIONS: These results imply a novel role of Txnrd2 in sustaining heart function during aging and suggest that Txnrd2 may be a modifier of heart failure.
Wissenschaftlicher Artikel
Scientific Article
Groß, C. ; Steiger, K. ; Sayyed, S. ; Heid, I. ; Feuchtinger, A. ; Walch, A.K. ; Hess-Rieger, J. ; Unger, K. ; Zitzelsberger, H. ; Settles, M. ; Schlitter, A.M. ; Dworniczak, J. ; Altomonte, J. ; Ebert, O. ; Schwaiger, M. ; Rummeny, E.J. ; Steingötter, A. ; Esposito, I. ; Braren, R.
Clin. Cancer Res. 21, 4440-4450 (2015)
PURPOSE: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology non-invasively and identify differences between the models. EXPERIMENTAL DESIGN: DEN and McA tumor development was monitored by magnetic resonance imaging (MRI) and positron emission tomography (PET). A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. RESULTS: Histologically and biochemically confirmed liver damage resulted in increased 18F-fluordeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1-3 grading compared to uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared to McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. CONCLUSION: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies.
Wissenschaftlicher Artikel
Scientific Article
Velikova, V.B. ; Müller, C. ; Ghirardo, A. ; Rock, T. ; Aichler, M. ; Walch, A.K. ; Schmitt-Kopplin, P. ; Schnitzler, J.-P.
Plant Physiol. 168, 859-870 (2015)
Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses by improving membrane structure and scavenging reactive oxygen species. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene emitting (IE) and non-isoprene emitting (NE) poplars. We demonstrated that the total amount of mono- (MGDG), di-galactosyldiacylglycerols (DGDG), phospholipids (PL), and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amount of unsaturated fatty acids, particularly linolenic acid (18:3) in NE chloroplasts was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects PSII photochemical efficiency (ΦPSII). The low ΦPSII in NE plants was negatively correlated with non-photochemical quenching (NPQ) and the energy-dependent (qE) component of NPQ. Transmission electron microscopy (TEM) revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained less grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in isoprene-emitting species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.
Wissenschaftlicher Artikel
Scientific Article
Buck, A. ; Ly, A. ; Balluff, B. ; Sun, N. ; Gorzolka, K. ; Feuchtinger, A. ; Janssen, K.P. ; Kuppen, P.J. ; van de Velde, C.J. ; Weirich, G. ; Erlmeier, F. ; Langer, R. ; Aubele, M. ; Zitzelsberger, H. ; Aichler, M. ; Walch, A.K.
J. Pathol. 237, 123–132 (2015)
We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed paraffin-embedded (FFPE) human tissue samples. Using high-resolution Matrix-Assisted Laser Desorption/Ionization Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry Imaging (MALDI-FT-ICR MSI), we conducted a proof of principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients, and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.
Wissenschaftlicher Artikel
Scientific Article
Bauer, L. ; Takacs, A. ; Slotta-Huspenina, J. ; Langer, R. ; Becker, K. ; Novotny, A. ; Ott, K. ; Walch, A.K. ; Hapfelmeier, A. ; Keller, G.
Front. Oncol. 5:94 (2015)
BACKGROUND: NOTCH signaling can exert oncogenic or tumor suppressive functions and can contribute to chemotherapy resistance in cancer. In this study, we aimed to clarify the clinicopathological significance and the prognostic and predictive value of NOTCH1 and NOTCH2 expression in gastric cancer (GC). METHODS: NOTCH1 and NOTCH2 expression was determined immunohistochemically in 142 primarily resected GCs using tissue microarrays and in 84 pretherapeutic biopsies from patients treated by neoadjuvant chemotherapy. The results were correlated with survival, response to therapy, and clinico-pathological features. RESULTS: Primarily resected patients with NOTCH1-negative tumors demonstrated worse survival. High NOTCH1 expression was associated with early-stage tumors and with significantly increased survival in this subgroup. Higher NOTCH2 expression was associated with early-stage and intestinal-type tumors and with better survival in the subgroup of intestinal-type tumors. In pretherapeutic biopsies, higher NOTCH1 and NOTCH2 expression was more frequent in non-responding patients, but these differences were statistically not significant. CONCLUSION: Our findings suggested that, in particular, NOTCH1 expression indicated good prognosis in GC. The close relationship of high NOTCH1 and NOTCH2 expression with early tumor stages may indicate a tumor-suppressive role of NOTCH signaling in GC. The role of NOTCH1 and NOTCH2 in neoadjuvantly treated GC is limited.
Wissenschaftlicher Artikel
Scientific Article
Erlmeier, F. ; Feuchtinger, A. ; Borgmann, D.M. ; Rudelius, M. ; Autenrieth, M. ; Walch, A.K. ; Weirich, G.
Histochem. Cell Biol. 144, 147-156 (2015)
In the era of tumour type-specific therapies, the correct typing of renal tumours is of prime importance. As immunotyping and genotyping approaches are laborious and fall short of standardization, we used whole-scale computer-assisted morphometry instead. Three different types of renal tumours with different prognoses and therapies, notoriously prone to mistyping, were analysed . The sample of 335 tumours included clear cell renal cell carcinoma, chromophobe renal cell carcinoma and renal oncocytoma. The sample was analysed using H&E stains of tissue microarrrays in combination with an image-scanning software. Nuclear and cytoplasmic features were registered with the aid of computer-assisted morphometry. Features included shape, texture, colour and colour intensity for different cell compartments, e.g. nuclei and cytoplasm. The software passed several training steps for final validation. Using morphometry, we were able to classify the three renal tumour types correctly, with a 100 % specificity compared to the WHO typing. Nuclear features dominated the typing of chromophobe renal cell carcinoma, whereas cytoplasmic features were the leading classificators for renal oncocytoma. The grading of clear cell renal cell carcinoma attained a specificity of 80 %. In conclusion, modern morphometry may serve as a tool for typing renal epithelial tumours and additionally draws the attention to future nuclear research in chromophobe renal cell carcinoma.
Wissenschaftlicher Artikel
Scientific Article
Ingold, I. ; Aichler, M. ; Yefremova, E. ; Roveri, A. ; Buday, K. ; Doll, S. ; Tasdemir, A. ; Hoffard, N. ; Wurst, W. ; Walch, A.K. ; Ursini, F. ; Friedmann Angeli, J.P.F. ; Conrad, M.
J. Biol. Chem. 290, 14668-14678 (2015)
The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 (mGpx4) was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with targeted mutation of the active site selenocysteine (Sec) of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4-/- embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breedings and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoan midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mGpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared to Sec at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Since the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and being a structural protein, tightly controlled expression of functional Gpx4 emerges being key for full male fertility.
Wissenschaftlicher Artikel
Scientific Article
Oetjen, J. ; Veselkov, K. ; Watrous, J. ; McKenzie, J.S. ; Becker, M. ; Hauberg-Lotte, L. ; Kobarg, J.H. ; Strittmatter, N. ; Mróz, A.K. ; Hoffmann, F. ; Trede, D. ; Palmer, A. ; Schiffler, S. ; Steinhorst, K. ; Aichler, M. ; Goldin, R. ; Guntinas-Lichius, O. ; von Eggeling, F. ; Thiele, H. ; Maedler, K. ; Walch, A.K. ; Maass, P. ; Dorrestein, P.C. ; Takats, Z. ; Alexandrov, T.
GigaScience 4:20 (2015)
BACKGROUND: Three-dimensional (3D) imaging mass spectrometry (MS) is an analytical chemistry technique for the 3D molecular analysis of a tissue specimen, entire organ, or microbial colonies on an agar plate. 3D-imaging MS has unique advantages over existing 3D imaging techniques, offers novel perspectives for understanding the spatial organization of biological processes, and has growing potential to be introduced into routine use in both biology and medicine. Owing to the sheer quantity of data generated, the visualization, analysis, and interpretation of 3D imaging MS data remain a significant challenge. Bioinformatics research in this field is hampered by the lack of publicly available benchmark datasets needed to evaluate and compare algorithms. FINDINGS: High-quality 3D imaging MS datasets from different biological systems at several labs were acquired, supplied with overview images and scripts demonstrating how to read them, and deposited into MetaboLights, an open repository for metabolomics data. 3D imaging MS data were collected from five samples using two types of 3D imaging MS. 3D matrix-assisted laser desorption/ionization imaging (MALDI) MS data were collected from murine pancreas, murine kidney, human oral squamous cell carcinoma, and interacting microbial colonies cultured in Petri dishes. 3D desorption electrospray ionization (DESI) imaging MS data were collected from a human colorectal adenocarcinoma. CONCLUSIONS: With the aim to stimulate computational research in the field of computational 3D imaging MS, selected high-quality 3D imaging MS datasets are provided that could be used by algorithm developers as benchmark datasets.
Wissenschaftlicher Artikel
Scientific Article
Almstätter, I. ; Mykhaylyk, O. ; Settles, M. ; Altomonte, J. ; Aichler, M. ; Walch, A.K. ; Rummeny, E.J. ; Ebert, O. ; Plank, C. ; Braren, R.
Theranostics 5, 667-685 (2015)
Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring. In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r2 and r2 (*) relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 - 97 % reduction of r2 and 35 - 82 % increase of r2 (*) compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model. In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy.
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Scientific Article
Genitsch, V. ; Novotny, A. ; Seiler, C.A. ; Kröll, D. ; Walch, A.K. ; Langer, R.
Front. Oncol. 5:73 (2015)
Epstein-Barr virus (EBV)-associated gastric carcinomas (GC) represent a distinct and well-recognized subtype of gastric cancer with a prevalence of around 10% of all GC. In contrast, EBV has not been reported to play a major role in esophageal adenocarcinomas (EAC) and adenocarcinomas of the gastro-esophageal junction (GEJ). We report our experiences on EBV in collections of gastro-esophageal adenocarcinomas from two surgical centers and discuss the current state of research in this field. Tumor samples from 465 primary resected gastro-esophageal adenocarcinomas (118 EAC, 73 GEJ, and 274 GC) were investigated. Presence of EBV was determined by EBV-encoded small RNAs (EBER) in situ hybridization. Results were correlated with pathologic parameters (UICC pTNM category, Her2 status, tumor grading) and survival. EBER positivity was observed in 14 cases. None of the EAC were positive for EBER. In contrast, we observed EBER positivity in 2/73 adenocarcinomas of the GEJ (2.7%) and 12/274 GC (4.4%). These were of intestinal type (seven cases) or unclassifiable (six cases), while only one case was of diffuse type according to the Lauren classification. No association between EBV and pT, pN, or tumor grading was found, neither was there a correlation with clinical outcome. None of the EBER positive cases were Her2 positive. In conclusion, EBV does not seem to play a role in the carcinogenesis of EAC. Moreover, adenocarcinomas of the GEJ show lower rates of EBV positivity compared to GC. Our data only partially correlate with previous reports from the literature. This highlights the need for further research on this distinct entity. Recent reports, however, have identified specific epigenetic and genetic alterations in EBV-associated GC, which might lead to a distinct treatment approach for this specific subtype of GC in the future.
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Aichler, M. ; Walch, A.K.
Lab. Invest. 95, 422-431 (2015)
MALDI Imaging mass spectrometry has entered the field of tissue-based research by providing unique advantages for analyzing tissue specimen in an unprecedented detail. A broad spectrum of analytes ranging from proteins, peptides, protein modification over small molecules, drugs and their metabolites as well as pharmaceutical components, endogenous cell metabolites, lipids, and other analytes are made accessible by this in situ technique in tissue. Some of them were even not accessible in tissues within the histological context before. Thereby, the great advantage of MALDI Imaging is the correlation of molecular information with traditional histology by keeping the spatial localization information of the analytes after mass spectrometric measurement. This method is label-free and allows multiplex analysis of hundreds to thousands of molecules in the very same tissue section simultaneously. Imaging mass spectrometry brings a new quality of molecular data and links the expert discipline of pathology and deep molecular mass spectrometric analysis to tissue-based research. This review will focus on state-of-the-art of MALDI Imaging mass spectrometry, its recent applications by analyzing tissue specimen and the contributions in understanding the biology of disease as well as its perspectives for pathology research and practice.
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Schmitt, S. ; Eberhagen, C. ; Weber, S. ; Aichler, M. ; Zischka, H.
Methods Mol. Biol. 1295, 87-97 (2015)
We recently reported a new method to isolate functionally intact mitochondria from cell culture and small tissue samples (Schmitt et al., Anal Biochem 443(1):66-74, 2013). This method comprises a semi-automated cell rupture, termed pump controlled cell rupture system (PCC), which can be precisely adjusted to the specific cellular source of isolation and which can be tightly controlled (Schmitt et al., Anal Biochem 443(1):66-74, 2013). Here we provide a detailed hands-on protocol of this PCC method which results in an efficient cell breakage but preserving the mitochondrial integrity. Upon subsequent purification steps, the obtained mitochondrial fraction meets the quality and purity required for molecular analyses, e.g. proteomic comparisons, as well as for biochemical analyses, e.g. determination of diverse enzymatic activities.
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Schulz, S. ; Lichtmannegger, J. ; Schmitt, S. ; Leitzinger, C. ; Eberhagen, C. ; Einer, C. ; Kerth, J. ; Aichler, M. ; Zischka, H.
Methods Mol. Biol. 1295, 75-86 (2015)
Mitochondria are key organelles for cellular energy production and cell death decisions. Consequently, a plethora of conditions which are toxic to cells are known to directly attack these organelles. However, mitochondria originating from different tissues differ in their sensitivity to toxic insults. Thus, in order to predict the potential organ-specific toxicity of a given drug or pathological condition at the mitochondrial level, test settings are needed that directly compare the responses and vulnerabilities of mitochondria from different organs. As a prerequisite for such test strategies, we provide here a robust, prompt, and easy-to-follow step-by-step protocol to simultaneously isolate functional and intact mitochondria from rat liver, kidney, heart, and brain. This isolation procedure ensures mitochondrial preparations of comparable purity and reproducible quantities which can be subsequently analyzed for organ-specific mitochondrial toxicity.
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Aichler, M. ; Huber, K. ; Schilling, F. ; Lohöfer, F. ; Kosanke, K. ; Meier, R. ; Rummeny, E.J. ; Walch, A.K. ; Wildgruber, M.
Angew. Chem.-Int. Edit. 54, 4279-4283 (2015)
Gadolinium(III)-based contrast agents improve the sensitivity and specificity of magnetic resonance imaging (MRI), especially when targeted contrast agents are applied. Because of nonlinear correlation between the contrast agent concentration in tissue and the MRI signal obtained in vivo, quantification of certain biological or pathophysiological processes by MRI remains a challenge. Up to now, no technology has been able to provide a spatially resolved quantification of MRI agents directly within the tissue, which would allow a more precise verification of in vivo imaging results. MALDI imaging mass spectrometry for spatially resolved in situ quantification of gadolinium(III) agents, in correlation to in vivo MRI, were evaluated. Enhanced kinetics of Gadofluorine M were determined dynamically over time in a mouse model of myocardial infarction. MALDI imaging was able to corroborate the in vivo imaging MRI signals and enabled in situ quantification of the gadolinium probe with high spatial resolution.
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Bi, Z. ; Merl-Pham, J. ; Uehlein, N. ; Zimmer, I. ; Mühlhans, S. ; Aichler, M. ; Walch, A.K. ; Kaldenhoff, R. ; Palme, K. ; Schnitzler, J.-P. ; Block, K.
J. Proteomics 128, 321-332 (2015)
Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. Biological significance The present work is a comprehensive survey combining leaf plasma membrane proteomics and physiological methods to assess the functionality of the whole PIP subfamily in tree model system.
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Teiluf, K. ; Seidl, C. ; Blechert, B. ; Gaertner, F.C. ; Gilbertz, K.P. ; Fernandez, V. ; Bassermann, F. ; Endell, J. ; Boxhammer, R. ; Leclair, S. ; Vallon, M. ; Aichler, M. ; Feuchtinger, A. ; Bruchertseifer, F. ; Morgenstern, A. ; Essler, M.
Oncotarget 6, 4692-4703 (2015)
In spite of development of molecular therapeutics, multiple myeloma (MM) is fatal in most cases. CD38 is a promising target for selective treatment of MM. We tested radioimmunoconjugates consisting of the α-emitter 213Bi coupled to an anti-CD38 MAb in preclinical treatment of MM. Efficacy of 213Bi-anti-CD38-MAb was assayed towards different MM cell lines with regard to induction of DNA double-strand breaks, induction of apoptosis and initiation of cell cycle arrest. Moreover, mice bearing luciferase-expressing MM xenografts were treated with 213Bi-anti-CD38-MAb. Therapeutic efficacy was monitored by bioluminescence imaging, overall survival and histology. 213Bi-anti-CD38-MAb treatment induced DNA damage which did not result in activation of the G2 DNA-damage-response checkpoint, but instead in mitotic arrest and subsequent mitotic catastrophe. The anti-tumor effect of 213Bi-anti-CD38-MAb correlated with the expression level of CD38 in each MM cell line. In myeloma xenografts, treatment with 213Bi-anti-CD38-MAb suppressed tumor growth via induction of apoptosis in tumor tissue and significantly prolonged survival compared to controls. The major organ systems did not show any signs of 213Bi-induced toxicity. Preclinical treatment of MM with 213Bi-anti-CD38-MAb turned out as an effective therapeutic option.
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Helmbrecht, M.S. ; Soellner, H. ; Truckenbrodt, A.M. ; Sundermeier, J. ; Cohrs, C.M. ; Hans, W. ; Hrabě de Angelis, M. ; Feuchtinger, A. ; Aichler, M. ; Fouad, K. ; Huber, A.B.
Dev. Biol. 399, 2-14 (2015)
The correct wiring of neuronal circuits is of crucial importance for the function of the vertebrate nervous system. Guidance cues like the neuropilin receptors (Npn) and their ligands, the semaphorins (Sema) provide a tight spatiotemporal control of sensory and motor axon growth and guidance. Among this family of guidance partners the Sema3A-Npn1 interaction has been shown to be of great importance, since defective signaling leads to wiring deficits and defasciculation. For the embryonic stage these defects have been well described, however, also after birth the organism can adapt to new challenges by compensational mechanisms. Therefore, we used the mouse lines Olig2-Cre;Npn1(cond) and Npn1(Sema-) to investigate how postnatal organisms cope with the loss of Npn1 selectively from motor neurons or a systemic dysfunctional Sema3A-Npn1 signaling in the entire organism, respectively. While in Olig2-Cre(+);Npn1(cond-/-) mice clear anatomical deficits in paw posturing, bone structure, as well as muscle and nerve composition became evident, Npn1(Sema-) mutants appeared anatomically normal. Furthermore, Olig2-Cre(+);Npn1(cond) mutants revealed a dysfunctional extensor muscle innervation after single-train stimulation of the N.radial. Interestingly, these mice did not show obvious deficits in voluntary locomotion, however, skilled motor function was affected. In contrast, Npn1(Sema-) mutants were less affected in all behavioral tests and able to improve their performance over time. Our data suggest that loss of Sema3A-Npn1 signaling is not the only cause for the observed deficits in Olig2-Cre(+);Npn1(cond-/-) mice and that additional, yet unknown binding partners for Npn1 may be involved that allow Npn1(Sema-) mutants to compensate for their developmental deficits.
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Diakopoulos, K.N. ; Lesina, M. ; Wörmann, S. ; Song, L. ; Aichler, M. ; Schild, L. ; Artati, A. ; Römisch-Margl, W. ; Wartmann, T. ; Fischer, R. ; Kabiri, Y. ; Zischka, H. ; Halangk, W. ; Demir, I.E. ; Pilsak, C. ; Walch, A.K. ; Mantzoros, C.S. ; Steiner, J.M. ; Erkan, M. ; Schmid, R.M. ; Witt, H. ; Adamski, J. ; Algül, H.
Gastroenterology 148, 626-638 (2015)
BACKGROUND & AIMS: Little is known about the mechanisms of the progressive tissue destruction, inflammation, and fibrosis that occur during development of chronic pancreatitis. Autophagy is involved in multiple degenerative and inflammatory diseases, including pancreatitis, and requires the protein autophagy related 5 (ATG5). We created mice with defects in autophagy to determine its role in pancreatitis. METHODS: We created mice with pancreas-specific disruption of Atg5 (Ptf1aCre(ex1);Atg5(F/F) mice), and compared them to control mice. Pancreata were collected and histology, immunohistochemistry, transcriptome, and metabolome analyses were performed. ATG5-deficient mice were placed on diets containing 25% palm oil and compared to those on a standard diet. Another set of mice received the antioxidant N-acetylcysteine. Pancreatic tissues were collected from 8 patients with chronic pancreatitis (CP) and compared to pancreata from ATG5-deficient mice. RESULTS: Mice with pancreas-specific disruption of Atg5 developed atrophic CP, independent of β-cell function; a greater proportion of male mice developed CP than females. Pancreata from ATG5-deficient mice had signs of inflammation, necrosis, acinar-to-ductal metaplasia, and acinar-cell hypertrophy; this led to tissue atrophy and degeneration. Based on transcriptome and metabolome analyses, ATG5-deficient mice produced higher levels of reactive oxygen species than control mice, and had insufficient activation of glutamate-dependent metabolism. Pancreata from these mice had reduced autophagy, increased levels of p62, and increases in endoplasmic reticulum stress and mitochondrial damage, compared with tissues from control mice; p62 signaling to Nqo1 and p53 was also activated. Dietary antioxidants, especially in combination with palm oil-derived fatty acids, blocked progression to CP and pancreatic acinar atrophy. Tissues from patients with CP had many histologic similarities to those from ATG5-deficient mice. CONCLUSIONS: Mice with pancreas-specific disruption of Atg5 develop a form of CP similar to that of humans. CP development appears to involve defects in autophagy, glutamate-dependent metabolism, and increased production of reactive oxygen species. These mice might be used to identify therapeutic targets for CP.
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Ly, A. ; Schöne, C. ; Becker, M. ; Rattke, J. ; Meding, S. ; Aichler, M. ; Suckau, D. ; Walch, A.K. ; Hauck, S.M. ; Ueffing, M.
Histochem. Cell Biol. 143, 453-462 (2015)
Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) is emerging as a powerful tool for the analysis of molecular distributions in biological samples in situ. When compared to classical histology, the major benefit of this method is the ability to identify and localize many molecules in a single tissue sample. MALDI-MSI spatial resolution currently falls short of traditional microscopic methods as it is limited by instrumentation and sample preparation. Tissue preparation steps, such as matrix deposition, are critical when considering strategies to further enhance the spatial resolution. The mammalian retina was selected as the tissue of choice for method development; its stratified anatomy renders it an ideal tissue to test high-resolution MALDI-MSI as the different layers correspond to specific neuronal classes and cellular structures. We compared alcohol-fixed, paraffin-embedded retina to fresh-frozen samples and matrix that had been deposited by spray or by sublimation. We present a lipid imaging method based on MALDI-MSI of frozen retinal sections with sublimated 2,5-dihydroxybenzoic acid matrix, which results in a highly advanced resolution compared to previous established methods. Hierarchical clustering of the primary data allows robust detection and differentiation of molecular distributions at a spatial resolution between 10 and 20 μm, thus approaching single-cell resolution.
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McDonnell, L.A. ; Römpp, A. ; Balluff, B.D. ; Heeren, R.M.A. ; Albar, J.P. ; Andrén, P.E. ; Corthals, G.L. ; Walch, A.K. ; Stoeckli, M.
Anal. Bioanal. Chem. 407, 2035-2045 (2015)
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Ermolayev, V. ; Mohajerani, P. ; Ale, A.B.F ; Sarantopoulos, A. ; Aichler, M. ; Kayser, G. ; Walch, A.K. ; Ntziachristos, V.
Int. J. Cancer 137, 1107-1118 (2015)
Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation. We conducted ex-vivo and Fluorescence Molecular Tomography-X-ray Computed Tomography (FMT-XCT) in-vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex-vivo, the integrin-targeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions at all ages studied. The panel of immunofluorescence experiments demonstrated that pulmonary lesions, which only partially show cancer cell features were detected by αvβ3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be non-invasively detected in-vivo on the fluorescence channel of FMT-XCT. Our findings demonstrated αvβ3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.
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Berchtold, S. ; Grünwald, B. ; Krüger, A. ; Reithmeier, A. ; Hähl, T. ; Cheng, T. ; Feuchtinger, A. ; Born, D. ; Erkan, M. ; Kleeff, J. ; Esposito, I.
Cancer Lett. 356, 721-732 (2015)
Excessive matrix production by pancreatic stellate cells promotes local growth and metastasis of pancreatic ductal adenocarcinoma and provides a barrier for drug delivery. Collagen type V is a fibrillar, regulatory collagen up-regulated in the stroma of different malignant tumors. Here we show that collagen type V is expressed by pancreatic stellate cells in the stroma of pancreatic ductal adenocarcinoma and affects the malignant phenotype of various pancreatic cancer cell lines by promoting adhesion, migration and viability, also after treatment with chemotherapeutic drugs. Pharmacological and antibody-mediated inhibition of β1-integrin signaling abolishes collagen type V-induced effects on pancreatic cancer cells. Ablation of collagen type V secretion of pancreatic stellate cells by siRNA reduces invasion and proliferation of pancreatic cancer cells and tube formation of endothelial cells. Moreover, stable knock-down of collagen type V in pancreatic stellate cells reduces metastasis formation and angiogenesis in an orthotopic mouse model of ductal adenocarcinoma. In conclusion, paracrine loops involving cancer and stromal elements and mediated by collagen type V promote the malignant phenotype of pancreatic ductal adenocarcinoma and underline the relevance of epithelial-stromal interactions in the progression of this aggressive neoplasm.
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Kahle-Stephan, M. ; Schäfer, A. ; Seelig, A. ; Schultheiß, J. ; Wu, M. ; Aichler, M. ; Leonhardt, J. ; Rathkolb, B. ; Rozman, J. ; Sarioglu, H. ; Hauck, S.M. ; Ueffing, M. ; Wolf, E. ; Kastenmüller, G. ; Adamski, J. ; Walch, A.K. ; Hrabě de Angelis, M. ; Neschen, S.
Mol. Metab. 4, 39-50 (2015)
Objective Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. Methods We induced hepatosteatosis by feeding C3HeB/FeJ male mice a HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Results Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. Conclusions We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties – in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.
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Buck, A. ; Halbritter, S. ; Späth, C. ; Feuchtinger, A. ; Aichler, M. ; Zitzelsberger, H. ; Janssen, K.P. ; Walch, A.K.
Anal. Bioanal. Chem. 407, 2107-2116 (2015)
Tissue distribution and quantitative analysis of small molecules is a key to assess the mechanism of drug action and evaluate treatment efficacy. The prodrug irinotecan (CPT-11) is widely used for chemotherapeutic treatment of colorectal cancer. CPT-11 requires conversion into its active metabolite SN-38 to exert the desired pharmacological effect. MALDI-Fourier transform ion cyclotron resonance (FT-ICR) and MALDI-time-of-flight (TOF) mass spectrometry imaging (MSI) were performed for detection of CPT-11 and SN-38 in tissue sections from mice post CPT-11 injection. In-depth information was gained about the distribution and quantity of drug compounds in normal and tumor tissue. The prodrug was metabolized, as proven by the detection of SN-38 in liver, kidney and digestive tract. In tumors from genetic mouse models for colorectal cancer (Apc (1638N/wt) x pvillin-Kras (V12G) ), CPT-11 was detected but not the active metabolite. In order to correlate drug distribution relative to vascularization, MALDI data were superimposed with CD31 (PECAM-1) immunohistochemistry. This analysis indicated that intratumoral access of CPT-11 mainly occurred by extravasation from microvessels. The present study exploits the power of MALDI MSI in drug analysis, and presents a novel approach to monitor drug distribution in relation to vessel functionality in preclinical and clinical research.
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Schenck, T.L. ; Stewart, J. ; Lin, S. ; Aichler, M. ; Machens, H.G. ; Giunta, R.E.
J. Hand Surg. Eur. 40, 591-596 (2015)
This study focuses on the anatomical and histomorphometric features of the transfer of the anterior interosseous nerve to the deep motor branch of the ulnar nerve. The transfer was carried out in 15 cadaver specimens and is described using relevant anatomical landmarks. Nerve samples of donor and target nerves were histomorphometrically analysed and compared. The superficial and the deep ulnar branches had to be separated from each other for a length of 67 mm (SD 12; range 50–85) to reach the site of coaptation. We identified a suitable site for coaptation lying proximal to the pronator quadratus muscle, 202 mm (SD 15; range 185–230) distal to the medial epicondyle of the humerus. The features of the anterior interosseous nerve included a smaller nerve diameter, smaller cross-sectional area of fascicles, fewer fascicles and axons, but a similar axon density. The histomorphometric inferiority of the anterior interosseous nerve raises a question about whether it should be transferred only to selected parts of the deep motor branch of the ulnar nerve.
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Selmansberger, M. ; Feuchtinger, A. ; Zurnadzhy, L. ; Michna, A. ; Kaiser, J.C. ; Abend, M. ; Brenner, A. ; Bogdanova, T. ; Walch, A.K. ; Unger, K. ; Zitzelsberger, H. ; Hess-Rieger, J.
Oncogene 34, 3917-3925 (2015)
A substantial increase in papillary thyroid carcinoma (PTC) among children exposed to the radioiodine fallout has been one of the main consequences of the Chernobyl reactor accident. Recently, the investigation of PTCs from a cohort of young patients exposed to the post-Chernobyl radioiodine fallout at very young age and a matched nonexposed control group revealed a radiation-specific DNA copy number gain on chromosomal band 7q11.23 and the radiation-associated mRNA overexpression of CLIP2. In this study, we investigated the potential role of CLIP2 as a radiation marker to be used for the individual classification of PTCs into CLIP2-positive and -negative cases-a prerequisite for the integration of CLIP2 into epidemiological modelling of the risk of radiation-induced PTC. We were able to validate the radiation-associated CLIP2 overexpression at the protein level by immunohistochemistry (IHC) followed by relative quantification using digital image analysis software (P=0.0149). Furthermore, we developed a standardized workflow for the determination of CLIP2-positive and -negative cases that combines visual CLIP2 IHC scoring and CLIP2 genomic copy number status. In addition to the discovery cohort (n=33), two independent validation cohorts of PTCs (n=115) were investigated. High sensitivity and specificity rates for all three investigated cohorts were obtained, demonstrating robustness of the developed workflow. To analyse the function of CLIP2 in radiation-associated PTC, the CLIP2 gene regulatory network was reconstructed using global mRNA expression data from PTC patient samples. The genes comprising the first neighbourhood of CLIP2 (BAG2, CHST3, KIF3C, NEURL1, PPIL3 and RGS4) suggest the involvement of CLIP2 in the fundamental carcinogenic processes including apoptosis, mitogen-activated protein kinase signalling and genomic instability. In our study, we successfully developed and independently validated a workflow for the typing of PTC clinical samples into CLIP2-positive and CLIP2-negative and provided first insights into the CLIP2 interactome in the context of radiation-associated PTC.
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Baumgart, A. ; Mazur, P.K. ; Anton, M. ; Rudelius, M. ; Schwamborn, K. ; Feuchtinger, A. ; Behnke, K. ; Walch, A.K. ; Braren, R. ; Peschel, C. ; Duyster, J. ; Siveke, J.T. ; Dechow, T.
Oncogene 34, 578–588 (2015)
Lung cancer is the leading cause of cancer-related deaths worldwide. Recently, we have shown that Notch1 inhibition resulted in substantial cell death of non-small cell lung cancer (NSCLC) cells in vitro. New compounds targeting Notch signal transduction have been developed and are now being tested in clinical trials. However, the tumorigenic role of individual Notch receptors in vivo remains largely unclear. Using a Kras(G12D)-driven endogenous NSCLC mouse model, we analyzed the effect of conditional Notch1 and Notch2 receptor deletion on NSCLC tumorigenesis. Notch1 deficiency led to a reduced early tumor formation and lower activity of MAPK compared with the controls. Unexpectedly, Notch2 deletion resulted in a dramatically increased carcinogenesis and increased MAPK activity. These mice died significantly earlier due to rapidly growing tumor burden. We found that Notch1 regulates Ras/MAPK pathway via HES1-induced repression of the DUSP1 promoter encoding a phosphatase specifically suppressing pERK1/2. Interestingly, Notch1 but not Notch2 ablation leads to decreased HES1 and DUSP1 expression. However, Notch2-depleted tumors showed an appreciable increase in β-catenin expression, a known activator of HES1 and important lung cancer oncogene. Characteristically for β-catenin upregulation, we found that the majority of Notch2-deficient tumors revealed an undifferentiated phenotype as determined by their morphology, E-Cadherin and TTF1 expression levels. In addition, these carcinomas showed aggressive growth patterns with bronchus invasion and obstruction. Together, we show that Notch2 mediates differentiation and has tumor suppressor functions during lung carcinogenesis, whereas Notch1 promotes tumor initiation and progression. These data are further supported by immunohistochemical analysis of human NSCLC samples showing loss or downregulation of Notch2 compared with normal lung tissue. In conclusion, this is the first study characterizing the in vivo functions of Notch1 and Notch2 in Kras(G12D)-driven NSCLC tumorigenesis. These data highlight the clinical importance of a thorough understanding of Notch signaling especially with regard to Notch-targeted therapies.
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Scientific Article
2014
Kandemir, M. ; Feuchtinger, A. ; Walch, A.K. ; Hamprecht, F.A.
In: Proceedings (IEEE International Symposium on Biomedical Imaging, ISBI, 29. April - 02.May 2014, Beijing, China). Piscataway, NJ: IEEE, 2014. 1348-1351
We study diagnosis of Barrett's cancer from hematoxylin & eosin (H & E) stained histopathological biopsy images using multiple instance learning (MIL). We partition tissue cores into rectangular patches, and construct a feature vector consisting of a large set of cell-level and patch-level features for each patch. In MIL terms, we treat each tissue core as a bag (group of instances with a single group-level ground-truth label) and each patch an instance. After a benchmarking study on several MIL approaches, we find that a graph-based MIL algorithm, mi-Graph [1], gives the best performance (87% accuracy, 0.93 AUC), due to its inherent suitability to bags with spatially-correlated instances. In patch-level diagnosis, we reach 82% accuracy and 0.89 AUC using Bayesian logistic regression. We also pursue a study on feature importance, which shows that patch-level color and texture features and cell-level features all have significant contribution to prediction.
Huber, M. ; Falkenberg, N. ; Schmitt, M. ; Braselmann, H. ; Mall, R. ; Jakovac, M. ; Walch, A.K. ; Höfler, H. ; Aubele, M.
Eur. J. Cancer 50, S161-S162 (2014)
Meeting abstract
Meeting abstract
Selmansberger, M. ; Feuchtinger, A. ; Zurnadzhy, L. ; Höfig, I. ; Bogdanova, T. ; Walch, A.K. ; Unger, K. ; Zitzelsberger, H. ; Hess-Rieger, J.
Eur. J. Cancer 50, S113 (2014)
Meeting abstract
Meeting abstract
Becker, L. ; Kling, E. ; Schiller, E. ; Zeh, R. ; Schrewe, A. ; Hölter, S.M. ; Mossbrugger, I. ; Calzada-Wack, J. ; Strecker, V. ; Wittig, I. ; Dumitru, I. ; Wenz, T. ; Bender, A. ; Aichler, M. ; Janik, D. ; Neff, F. ; Walch, A.K. ; Quintanilla-Fend, L. ; Floß, T. ; Bekeredjian, R. ; Gailus-Durner, V. ; Fuchs, H. ; Wurst, W. ; Meitinger, T. ; Prokisch, H. ; Hrabě de Angelis, M. ; Klopstock, T.
PLoS ONE 9:e114918 (2014)
Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients.
Wissenschaftlicher Artikel
Scientific Article
Maier, S.K.
München, Technische Universität, Fakultät Wissenschaftszentrum Weihenstephan, Diss., 2014, 115 S.
MALDI imaging mass spectrometry is a powerful tool for the visualization of protein distributions in tissues. But as the molecular identity of masses detected by MALDI IMS often stays elusive the full potential of the technology cannot be unlocked. Based on the development of a method for the targeted isolation of the detectable proteins this thesis addresses the key issue of the field by combining bottom-up and top-down strategies for protein identification.
Friedmann Angeli, J.P.F. ; Schneider, M. ; Proneth, B. ; Tyurina, Y.Y. ; Tyurin, V.A. ; Hammond, V.J. ; Herbach, N. ; Aichler, M. ; Walch, A.K. ; Eggenhofer, E. ; Basavarajappa, D. ; Rådmark, O. ; Kobayashi, S. ; Seibt, T. ; Beck, H. ; Neff, F. ; Esposito, I. ; Wanke, R. ; Förster, H. ; Yefremova, O. ; Heinrichmeyer, M. ; Bornkamm, G.W. ; Geissler, E.K. ; Thomas, S.B. ; Stockwell, B.R. ; O'Donnell, V.B. ; Kagan, V.E. ; Schick, J. ; Conrad, M.
Nat. Cell Biol. 16, 1180-1191 (2014)
Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.
Wissenschaftlicher Artikel
Scientific Article
Aichler, M. ; Luber, B. ; Lordick, F. ; Walch, A.K.
World J. Gastroenterol. 20, 13648-13657 (2014)
Several new treatment options for gastric cancer have been introduced but the prognosis of patients diagnosed with gastric cancer is still poor. Disease prognosis could be improved for high-risk individuals by implementing earlier screenings. Because many patients are asymptomatic during the early stages of gastric cancer, the diagnosis is often delayed and patients present with unresectable locally advanced or metastatic disease. Cytotoxic treatment has been shown to prolong survival in general, but not all patients are responders. The application of targeted therapies and multimodal treatment has improved prognosis for those with advanced disease. However, these new therapeutic strategies do not uniformly benefit all patients. Predicting whether patients will respond to specific therapies would be of particular value and would allow for stratifying patients for personalized treatment strategies. Metabolic imaging by positron emission tomography was the first technique with the potential to predict the response of esophago-gastric cancer to neoadjuvant therapy. Exploring and validating tissue-based biomarkers are ongoing processes. In this review, we discuss the status of several targeted therapies for gastric cancer, as well as proteomic and metabolic methods for investigating biomarkers for therapy response prediction in gastric cancer.
Review
Review
Huber, K. ; Feuchtinger, A. ; Borgmann, D.M. ; Li, Z. ; Aichler, M. ; Hauck, S.M. ; Zitzelsberger, H. ; Schwaiger, M. ; Keller, U. ; Walch, A.K.
Anal. Chem. 86, 10568-10575 (2014)
Drug efficacy strongly depends on the presence of the drug substance at the target site. As vascularization is an important factor for the distribution of drugs in tissues, we analyzed drug distribution as a function of blood vessel localization in tumor tissue. In order to explore distribution of the anti-cancer drugs afatinib, erlotinib, and sorafenib, a combined approach of matrix-assisted laser desorption/ionization (MALDI) drug imaging and immunohistochemical vessel staining was applied and examined by digital image analysis. Two xenograft models were investigated: (1) mice carrying squamous cell carcinoma (FaDu) xenografts (ntumor=13) were treated with afatinib or erlotinib, and (2) sarcoma (A673) xenograft bearing mice (ntumor=8) received sorafenib treatment. MALDI drug imaging revealed a heterogeneous distribution of all anti-cancer drugs. The tumor regions containing high drug levels were associated with a higher degree of vascularization than the regions without drug signals (p<0.05). When correlating the impact of blood vessel size to drug abundance in the sarcoma model, a higher amount of small vessels was detected in the tumor regions with high drug levels compared to the tumor regions with low drug levels (p<0.05). With the analysis of co-registered MALDI imaging and CD31 immunohistochemical data by digital image analysis, we demonstrate for the first time the potential of correlating MALDI drug imaging and immunohistochemistry. Here we describe a specific and precise approach for correlating histological features and pharmacokinetic properties of drugs at microscopic level, that will provide information for the improvement of drug design, administration formula or treatment schemes.
Wissenschaftlicher Artikel
Scientific Article
Gegg, M. ; Böttcher, A. ; Burtscher, I. ; Hasenöder, S. ; van Campenhout, C.A. ; Aichler, M. ; Walch, A.K. ; Grant, S.G. ; Lickert, H.
eLife 3:e03842 (2014)
Planar cell polarity (PCP) regulates basal body (BB) docking and positioning during cilia formation, but the underlying mechanisms remain elusive. Here, we investigate the uncharacterized gene Flattop (Fltp) that is transcriptionally activated during PCP acquisition in ciliated tissues. Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells. Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells. In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3 and Fltp localize directly adjacent at the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton. Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear. Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease and asymmetric cell division.
Wissenschaftlicher Artikel
Scientific Article
Balluff, B. ; Frese, C.K. ; Maier, S.K. ; Schöne, C. ; Kuster, B. ; Schmitt, M. ; Aubele, M. ; Höfler, H. ; Deelder, A.M. ; Heck, A.J. ; Hogendoorn, P.C.W. ; Morreau, J. ; Altelaar, A.F. ; Walch, A.K. ; McDonnell, L.A.
J. Pathol. 235, 3-13 (2014)
An essential and so far unresolved factor influencing the evolution of cancer and the clinical management of patients is intra-tumor clonal and phenotypic heterogeneity. However, the de novo identification of tumor subpopulations is a so far challenging, if not an unresolved, task. Here we present the first systematic approach for the de novo discovery of clinically detrimental molecular tumor subpopulations. In this proof-of-principle study, spatially-resolved, tumor-specific mass spectra were acquired using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry from tissues of 63 gastric carcinoma and 32 breast carcinoma patients. The mass spectra, representing the proteomic heterogeneity within tumor areas, were grouped by a corroborated statistical clustering algorithm in order to obtain segmentation maps of molecularly distinct regions. These regions were presumed to represent different phenotypic tumor subpopulations. This was confirmed by linking the presence of these tumor subpopulations to the patients' clinical data. This revealed several of the detected tumor subpopulations to be associated with a different overall survival of the gastric cancer patients (P = 0.025) and the presence of locoregional metastases in patients with breast cancer (P = 0.036). The procedure presented is generic and opens novel options in cancer research as it reveals microscopically indistinct tumor subpopulations that have an adverse impact on clinical outcome. This enables their further molecular characterization for deeper insights into the biological processes of cancer which may finally lead to new targeted therapies.
Wissenschaftlicher Artikel
Scientific Article
Aichler, M. ; Motschmann, M. ; Jütting, U. ; Luber, B. ; Becker, K. ; Ott, K. ; Lordick, F. ; Langer, R. ; Feith, M. ; Siewert, J.R. ; Walch, A.K.
Oncotarget 5, 6620-6632 (2014)
Neoadjuvant platin-based therapy is accepted as a standard therapy for advanced esophageal adenocarcinoma (EAC). Patients who respond have a better survival prognosis, but still a significant number of responder patients die from tumor recurrence. Molecular markers for prognosis in neoadjuvantly treated EAC patients have not been identified yet. We investigated the epidermal growth factor receptor (EGFR) in prognosis and chemotherapy resistance in these patients. Two EAC patient cohorts, either treated by neoadjuvant cisplatin-based chemotherapy followed by surgery (n=86) or by surgical resection (n=46) were analyzed for EGFR protein expression and gene copy number. Data were correlated with clinical and histopathological response, disease-free and overall survival. In case of EGFR overexpression, the prognosis for neoadjuvant chemotherapy responders was poor as in non-responders. Responders had a significantly better disease-free survival than non-responders only if EGFR expression level (p=0.0152) or copy number (p=0.0050) was low. Comparing neoadjuvantly treated patients and primary resection patients, tumors of non-responder patients more frequently exhibited EGFR overexpression, providing evidence that EGFR is a factor for indicating chemotherapy resistance. EGFR overexpression and gene copy number are independent adverse prognostic factors for neoadjuvant chemotherapy-treated EAC patients, particularly for responders. Furthermore, EGFR overexpression is involved in resistance to cisplatin-based neoadjuvant chemotherapy.
Wissenschaftlicher Artikel
Scientific Article
Feuchtinger, A. ; Stiehler, T. ; Jütting, U. ; Marjanovic, G. ; Luber, B. ; Langer, R. ; Walch, A.K.
Histochem. Cell Biol. 143, 1-9 (2014)
Quantification of protein expression based on immunohistochemistry (IHC) is an important step in clinical diagnoses and translational tissue-based research. Manual scoring systems are used in order to evaluate protein expression based on staining intensities and distribution patterns. However, visual scoring remains an inherently subjective approach. The aim of our study was to explore whether digital image analysis proves to be an alternative or even superior tool to quantify expression of membrane-bound proteins. We analyzed five membrane-binding biomarkers (HER2, EGFR, pEGFR, β-catenin, and E-cadherin) and performed IHC on tumor tissue microarrays from 153 esophageal adenocarcinomas patients from a single center study. The tissue cores were scored visually applying an established routine scoring system as well as by using digital image analysis obtaining a continuous spectrum of average staining intensity. Subsequently, we compared both assessments by survival analysis as an end point. There were no significant correlations with patient survival using visual scoring of β-catenin, E-cadherin, pEGFR, or HER2. In contrast, the results for digital image analysis approach indicated that there were significant associations with disease-free survival for β-catenin, E-cadherin, pEGFR, and HER2 (P = 0.0125, P = 0.0014, P = 0.0299, and P = 0.0096, respectively). For EGFR, there was a greater association with patient survival when digital image analysis was used compared to when visual scoring was (visual: P = 0.0045, image analysis: P < 0.0001). The results of this study indicated that digital image analysis was superior to visual scoring. Digital image analysis is more sensitive and, therefore, better able to detect biological differences within the tissues with greater accuracy. This increased sensitivity improves the quality of quantification.
Wissenschaftlicher Artikel
Scientific Article
Maier, S.K. ; Bashkueva, K. ; Rösli, C. ; Skerra, A. ; Kuster, B.
Proteomics 14, 2427-2431 (2014)
Mass spectrometers equipped with matrix-assisted laser desorption/ionization (MALDI-MS) require frequent multi-point calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS-cal tailored for MALDI-MS based bottom-up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C-terminal Arg residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multi-point calibration of MS spectra using PAS-cal peptides compares well to current commercial reagents for protein identification by peptide mass finger printing. Calibration of tandem mass spectra from LC-MALDI experiments using the longest peptide, PAS-cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS-cal standard generically useful for bottom-up proteomics.
Wissenschaftlicher Artikel
Scientific Article
Slotta-Huspenina, J. ; Becker, K.F. ; Feith, M. ; Walch, A.K. ; Langer, R.
Cancers 6, 1382-1393 (2014)
Her2 overexpression and amplification can be found in a significant subset of esophageal adenocarcinomas. The activity of Her2 has been shown to be modulated by molecular chaperones such as HSP90. We analyzed expression/amplification data for HSP90 and Her2 on 127 primary resected esophageal adenocarcinomas in order to evaluate a possible relationship between these two molecules. HSP90 expression determined by immunohistochemistry was observed in various levels. Thirty nine (39) tumors (30.7%) were classified as Her2-positive according to their immunoreactivity and amplification status. There was a significant correlation between HSP90 expression and Her2-status (p = 0.008). This could also be demonstrated by quantitative protein expression analysis with reverse phase protein arrays (r = 0.9; p < 0.001). Her2-status was associated withpT-category (p = 0.041), lymph node metastases (p = 0.049) and tumor differentiation (p = 0.036) with a higher percentage of cases with negative Her2 status in lower tumor stagesA negative Her2-status was also associated with better survival in univariate and multivariate analysis (p = 0.001 and p = 0.014). For HSP90, no associations between clinical and pathological parameters were found. The observed association between HSP90 expression and Her2 suggests a co-regulation of these molecules in at least a subset of esophageal adenocarcinomas. Anti-HSP90 drugs, which recently have been introduced in cancer treatment, may also be an option for these tumors by targeting HSP90 alone or in combination with Her2.
Wissenschaftlicher Artikel
Scientific Article
Schmitt, S. ; Schulz, S. ; Schropp, E.-M. ; Eberhagen, C. ; Simmons, A. ; Beisker, W. ; Aichler, M. ; Zischka, H.
Mitochondrion 19, 113-123 (2014)
Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility towards inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility towards inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations.
Wissenschaftlicher Artikel
Scientific Article
Buck, A. ; Walch, A.K.
Bioanalysis 6, 1241-1253 (2014)
In recent years the analysis in mass spectrometry imaging has been expanded to detect a wide variety of low molecular weight compounds (LMWC), including exogenous and endogenous compounds. The high sensitivity and selectivity of MS imaging combined with visualization of molecular spatial distribution in tissue making it to a valuable platform in targeted drug and untargeted metabolomic analyzes in biological and clinical research. Here, we review the current and potential applications of MALDI MS imaging in these areas. The aim of advancing MALDI MS imaging in the field of LMWC is to support clinical applications by understanding drug and drug-metabolite distribution, investigating toxicity and discover new biomarkers.
Review
Review
Graf, N. ; Li, Z. ; Herrmann, K. ; Weh, D. ; Aichler, M. ; Slawska, J. ; Walch, A.K. ; Peschel, C. ; Schwaiger, M. ; Buck, A.K. ; Dechow, T. ; Keller, U.
OncoTargets Ther. 7, 789-798 (2014)
BACKGROUND: Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) and the thymidine analog, 3'-deoxy-3'-[(18)F] fluorothymidine (FLT). METHODS: The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects. RESULTS: SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue. CONCLUSION: Dual PI3K/mTOR inhibition using BGT226 is effective in ALK-positive anaplastic large cell lymphoma and can be monitored with both FDG-PET and FLT-PET early on in the course of therapy.
Wissenschaftlicher Artikel
Scientific Article
Gorzolka, K. ; Walch, A.K.
Histol. Histopathol. 29, 1365-1376 (2014)
The molecular investigation of archived formalin-fixed, paraffin-embedded (FFPE) tissue samples provides the chance to obtain molecular patterns as indicatives for treatment and clinical end points. MALDI mass spectrometry imaging is capable of localizing molecules like proteins and peptides in tissue sections and became a favorite platform for the targeted and non-targeted approaches, especially in clinical investigations for biomarker research. In FFPE tissues the recovery of proteomic information is constrained by fixation-induced cross-links of proteins. The promising new insights obtained from FFPE in combination with the comprehensive patients' data caused much progress in the optimization of MS imaging protocols to investigate FFPE samples. This review presents the past and current research in MALDI MS imaging of FFPE tissues, demonstrating the improvement of analyses, their actual limitations, but also the promising future perspectives for histopathological and tissue-based research.
Review
Review
Huber, K. ; Aichler, M. ; Sun, N. ; Buck, A. ; Li, Z. ; Fernandez, I.E. ; Hauck, S.M. ; Zitzelsberger, H. ; Eickelberg, O. ; Janssen, K.P. ; Keller, U. ; Walch, A.K.
Histochem. Cell Biol. 142, 361-371 (2014)
The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.
Wissenschaftlicher Artikel
Scientific Article
Dekker, T.J. ; Balluff, B.D. ; Jones, E.A. ; Schöne, C.D. ; Schmitt, M. ; Aubele, M. ; Kroep, J.R. ; Smit, V.T. ; Tollenaar, R.A. ; Mesker, W.E. ; Walch, A.K. ; McDonnell, L.A.
J. Proteome Res. 13, 4730-4738 (2014)
MALDI mass spectrometry imaging (MSI) has rapidly established itself as a powerful biomarker discovery tool. To date, no formal investigation has assessed the center-to-center comparability of MALDI MSI experiments, an essential step for it to develop into a new diagnostic method. To test such capabilities, we have performed a multicenter study focused on biomarkers of stromal activation in breast cancer. MALDI MSI experiments were performed in two centers using independent tissue banks, infrastructure, methods, and practitioners. One of the data sets was used for discovery and the other for validation. Areas of intra- and extratumoral stroma were selected, and their protein signals were compared. Four protein signals were found to be significantly associated with tumor-associated stroma in the discovery data set measured in Munich. Three of these peaks were also detected in the independent validation data set measured in Leiden, all of which were also significantly associated with intratumoral stroma. Hierarchical clustering displayed 100% accuracy in the Munich MSI data set and 80.9% accuracy in the Leiden MSI data set. The association of one of the identified mass signals (PA28) with stromal activation was confirmed with immunohistochemistry performed on 20 breast tumors. Independent and international MALDI MSI investigations could identify validated biomarkers of stromal activation.
Wissenschaftlicher Artikel
Scientific Article
Bézière, N. ; von Schacky, C. ; Kosanke, Y. ; Kimm, M. ; Nunes, A. ; Licha, K. ; Aichler, M. ; Walch, A.K. ; Rummeny, E.J. ; Ntziachristos, V. ; Meier, R.
Arthritis Rheum. 66, 2071-2078 (2014)
Objectives Rheumatoid Arthritis (RA) is one of the most frequent inflammatory diseases, causing pain and disability in the affected joints. Early diagnosis is essential for the efficiency of symptomatic treatment, and relies on careful clinical, serologic and imaging examinations, such as Magnetic Resonance Imaging (MRI), which is both expensive and time consuming. In an effort to provide the biomedical community with a more accessible way to assess arthritis advancement, we investigated the use of multispectral optoacoustic tomography (MSOT) in a murine model to visualize the extent of the inflammation in vivo through a L- and P-selectin targeting contrast agent. Methods Collagen induced arthritis mice were used as a rheumatoid arthritis model of the limb. MSOT was performed using a L- and P-selectin targeting contrast agent (dPGS-NIR provided by Mivenion, Germany) to increase contrast of the arthritic joint, and signal intensity ratios between healthy and arthritic legs were calculated. Contrast enhanced MR imaging as well as clinical observation, lymphocyte/granulocyte ratio and histology served as references. Results MSOT using an inflammation targeting contrast agent allowed for accurate diagnosis of inflammation in the mouse joints and for significant differentiation of inflamed to healthy joints (P = 0.023). The arthritis findings on the MSOT images were confirmed by clinical observation, blood analysis, contrast enhanced MRI and ex vivo histological examinations. Conclusion This study demonstrates that the combination of inflammation targeting contrast agent and optoacoustic tomographic imaging present a promising mean for diagnosis and staging of arthritic inflammation.
Wissenschaftlicher Artikel
Scientific Article
Kucharska, J. ; del Río, P. ; Arango-González, B. ; Gorza, M. ; Feuchtinger, A. ; Hauck, S.M. ; Ueffing, M.
J. Neurochem. 130, 227-240 (2014)
Subretinal injections with glial cell line-derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of Retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here we show that Cyr61/CCN1, one of the secreted proteins upregulated in primary RMG after GDNF stimulation, provides neuroprotective and pro-survival capacities: Recombinant Cyr61 significantly reduced photoreceptor cells (PR) death in organotypic cultures of Pde6brd1 retinas. In order to identify stimulated pathways in the retina, we treated Pde6brd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation-site specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3, and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro-survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.
Wissenschaftlicher Artikel
Scientific Article
Stribl, C.B. ; Samara, A. ; Trümbach, D. ; Augustin, R. ; Neumann, M. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Rathkolb, B. ; Wolf, E. ; Beckers, J. ; Horsch, M. ; Neff, F. ; Kremmer, E. ; Koob, S. ; Reichert, A.S. ; Hans, W. ; Rozman, J. ; Klingenspor, M. ; Aichler, M. ; Walch, A.K. ; Becker, L. ; Klopstock, T. ; Glasl, L. ; Hölter, S.M. ; Wurst, W. ; Floß, T.
J. Biol. Chem. 289, 10769-10784 (2014)
The majority of Amyotrophic Lateral Sklerosis (ALS) cases as well as many patients suffering from Frontotemporal Lobar Dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR-DNA binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange (RMCE) to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes while the endogenous mouse Tdp-43 was decreased to 20% of wildtype levels as a result of disturbed feedback regulation. Heterozygous TDP-43A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes, as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol and glucose in the blood. As seen in Transmission Electron Microscopy, neuronal cells in motor cortices of TDP-43A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90% but only slight motoric impairment was detected. The observed phenotype was interpreted as a pre-disease model which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.
Wissenschaftlicher Artikel
Scientific Article
Sun, N. ; Ly, A. ; Meding, S. ; Witting, M. ; Hauck, S.M. ; Ueffing, M. ; Schmitt-Kopplin, P. ; Aichler, M. ; Walch, A.K.
Proteomics 14, 913-923 (2014)
Mass spectrometry imaging (MSI) is a valuable tool for diagnostics and systems biology studies, being a highly sensitive, label-free technique capable of providing comprehensive spatial distribution of different classes of biomolecules. The application of MSI to the study of endogenous compounds has received considerable attention because metabolites are the result of the interactions of a biosystem with its environment. MSI can therefore enhance understanding of disease mechanisms and elucidate mechanisms for biological variation. Here we present the in situ comparative metabolomics imaging data for analyses of light- and dark-treated retina. A wide variety of tissue metabolites were imaged at a high spatial resolution. These include nucleotides, central carbon metabolism pathway intermediates, 2-oxocarboxylic acid metabolism, oxidative phosphorylation, glycerophospholipid metabolism, and cysteine and methionine metabolites. The high lateral resolution enabled the differentiation of retinal layers, allowing determination of the spatial distributions of different endogenous compounds. A number of metabolites demonstrated differences between light and dark conditions. These findings add to the understanding of metabolic activity in the retina. This article is protected by copyright. All rights reserved.
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Scientific Article
Li, Z. ; Herrmann, K. ; Pirsig, S. ; Philipp-Abbrederis, K. ; Henninger, M. ; Aichler, M. ; Feuchtinger, A. ; Walch, A.K. ; Beer, A.J. ; Ringshausen, I. ; Pomykala, K.L. ; Scheidhauer, K. ; Schwaiger, M. ; Keller, U. ; Buck, A.K.
Am. J. Nucl. Med. Mol. Imaging 4, 70-79 (2014)
The role of [(18)F]fluorodeoxyglucose ([(18)F]FDG) PET in staging of sarcoma is well established. The aim of this preclinical study was to compare [(18)F]fluorothymidine ([(18)F]FLT) PET to [(18)F]FDG PET regarding early metabolic changes of sarcoma in the course of targeted cancer therapy. SCID mice bearing sarcoma A673 xenotransplants were used for investigation of tumor response after treatment with the multikinase inhibitor Sorafenib. [(18)F]FLT and/or [(18)F]FDG-PET were performed prior to and early after initiation of treatment. Tumoral uptake (% Injected Dose per gram (%ID/g) of [(18)F]FLT-PET was compared to [(18)F]FDG-PET. Results were correlated with histopathology and in vitro data including cellular uptake, cell cycle-related protein expression, cell cycle distribution and apoptosis. In vitro experiments showed that A673 cells were sensitive to Sorafenib. In vivo, tumor growth was inhibited in comparison to a 4-fold increase of the tumor volume in control mice. Using [(18)F]FDG as tracer, a moderate reduction in tracer uptake (n=15, mean relative %ID/g 74%, range 35%-121%, p=0.03) was observed. The decrease in %ID/g using [(18)F]FLT-PET was significantly higher (p=0.003). The mean relative %ID/g in [(18)F]FLT uptake on day + 5 was significantly reduced to 54% compared to baseline (n=15, range 24%-125%, SD=29%). The PET analysis 24 hr after therapy showed a significant reduction of the mean [(18)F]FLT-%ID/g (p=0.04). The reduction of %ID/g on day + 1 in [(18)F]FDG-PET was not statistically significant (p=0.99). In conclusion, both [(18)F]FDG- and [(18)F]FLT-PET were able to predict response to Sorafenib treatment. In contrast to [(18)F]FDG-PET, [(18)F]FLT-PET was more predictive for very early response to treatment.
Wissenschaftlicher Artikel
Scientific Article
Wildgruber, M. ; Bielicki, I. ; Aichler, M. ; Kosanke, K. ; Feuchtinger, A. ; Settles, M. ; Onthank, D.C. ; Cesati, R.R. ; Robinson, S.P. ; Huber, A.M. ; Rummeny, E.J. ; Walch, A.K. ; Botnar, R.M.
Circ.-Cardiovasc. Imaging 7, 321-329 (2014)
BACKGROUND: -To prospectively evaluate an elastin-specific magnetic resonance contrast agent (ESMA) for in-vivo targeting of elastic fibers in myocardial infarction and post-infarction scar remodeling. METHODS AND RESULTS: -Myocardial infarction (MI) was induced in C57BL/6J mice (n=40) by permanent ligation of the left anterior descending coronary artery (LAD). Magnetic Resonance Imaging (MRI) was performed at 7 and 21 days post MI. The merits of gadolinium-based ESMA (Gd-ESMA) were compared to Gd-DTPA in terms of infarct-size determination, contrast-to-noise ratio (CNR) and enhancement kinetics. Specific binding in-vivo was evaluated by blocking the molecular target using non-paramagnetic Lanthanum-ESMA (La-ESMA). In-vivo imaging results were confirmed by post-mortem triphenyltetrazoliumcholride (TTC) staining, Elastica-Van-Gieson (EvG) staining and Western Blotting. Delayed enhancement MRI revealed prolonged enhancement of Gd-ESMA in the post-ischemic scar compared to Gd-DTPA. Infarct size measurements showed good agreement between Gd-ESMA and Gd-DTPA and were confirmed by ex-vivo TTC staining. Pre-injection of the blocking La-ESMA resulted in significantly lower CNR of Gd-ESMA at the infarct site (p=0.0019). While no significant differences in CNR were observed between delayed-enhancement imaging with Gd-DTPA between day 7 and 21 (1.8 ± vs 3.8, p=ns), Gd-ESMA showed markedly higher CNR on day 21 post MI (14.1 vs 4.9, p=0.0032), which correlated with increased synthesis of tropoelastin detected by Western Blot analysis and histology. Higher CNR values for Gd-ESMA further correlated with improved ejection fraction of the mice on day 21 after MI. CONCLUSIONS: -Gd-ESMA enables targeting of elastin within the infarct scar in a mouse model of myocardial infarction. The imaging properties of Gd-ESMA allow quantification of intra-scar elastin content in-vivo and thereby provides potential for non-invasive characterization of post-infarction scar remodeling.  
Wissenschaftlicher Artikel
Scientific Article
McDonnell, L.A. ; Walch, A.K. ; Stoeckli, M. ; Corthals, G.L.
J. Proteome Res. 13, 1138–1142 (2014)
The clinical application of mass spectrometry imaging has developed into a sizeable sub-discipline of proteomics and metabolomics because its seamless integration with pathology enables biomarkers and biomarker profiles to be determined that can aid patient and disease stratification (diagnosis, prognosis and response to therapy). Confident identification of the discriminating peaks remains a challenge owing to the presence of non-tryptic protein fragments, large mass-to-charge ratio ions that are not efficiently fragmented via tandem mass spectrometry or a high density of isobaric species. To aid the clinical development and implementation of mass spectrometry imaging a public database of identifications has been initiated. The MSiMass list database (www.maldi-msi.org/mass) enables users to assign identities to the peaks observed in their experiments and provides the methods by which the identifications were obtained. In contrast to existing protein databases, this list is designed as a community effort without a formal review panel. In this concept, authors can freely enter data, and can comment on existing entries. In such, the database itself is an experiment on sharing knowledge and its ability to rapidly provide quality data will be evaluated in the future.
Wissenschaftlicher Artikel
Scientific Article
Aichler, M. ; Walch, A.K.
J. Pathol. 232, 383-385 (2014)
Barrett's oesophagus is a metaplastic change, such that the normal squamous epithelial lining of the oesophagus is replaced by specialised columnar lined epithelium. Barrett's oesophagus is clinically significant and has a high health economic impact as it is associated with heightened risk of progression to oesophageal adenocarcinoma. This review discusses the pathogenesis of Barrett's oesophagus with an emphasis on the underlying molecular events.
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Scientific Article
2013
Aichler, M.
GIT Fachz. Lab. 7, 444-447 (2013)
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Scientific Article
Frankó, A. ; Baris, O.R. ; Bergschneider, E. ; von Toerne, C. ; Hauck, S.M. ; Aichler, M. ; Walch, A.K. ; Wurst, W. ; Wiesner, R.J. ; Johnston, I.C.D. ; Hrabě de Angelis, M.
PLoS ONE 8:e82392 (2013)
To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.
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Scientific Article
Ale, A.B.F ; Siebenhaar, F. ; Kosanke, K. ; Aichler, M. ; Radrich, K. ; Heydrich, S. ; Schiemann, M. ; Bielicki, I. ; Noel, P.B. ; Braren, R. ; Maurer, M. ; Walch, A.K. ; Rummeny, E.J. ; Ntziachristos, V. ; Wildgruber, M.
Theranostics 3, 903-913 (2013)
Cardiomyocyte loss via apoptosis plays a crucial role in ventricular remodeling following myocardial infarction (MI). Cell-based therapy approaches using bone marrow derived c-kit(+) pluripotent cells may attenuate apoptosis following ischemic injury. We therefore thought to examine the early course of apoptosis following myocardial infarction - in-vivo - and non-invasively determine the effect of c-kit(+) bone marrow cells on post-MI remodeling. We studied apoptosis in wild-type Kit(+/+), c-kit mutant Kit(W)/Kit(W-v) and Kit(W)/Kit(W-v) mice after cell therapy with bone-marrow derived c-kit(+) cells after ischemia-reperfusion injury. Mice were followed by hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) at 6h, 24h and 7 days after ischemia-reperfusion injury using an Annexin V-based fluorescent nanosensor targeting phosphatidylserine. Kit(W)/Kit(W-v) mice showed increased and prolonged apoptosis compared to control Kit(+/+) mice while c-kit cell therapy was able to attenuate the altered apoptosis rates. Increased apoptosis was accompanied by severe decline in heart function, determined by cardiac Magnetic Resonance Imaging, and cell therapy was able to rescue the animals from deleterious heart failure. Post-mortem cryoslicing and immunohistochemistry localized the fluorescence signal of the Annexin V sensor within the infarcted myocardium. Flow cytometry of digested infarct specimens identified apoptotic cardiomyocytes as the major source for the in-vivo Annexin V signal. In-vivo molecular imaging using hybrid FMT-XCT reveals increased cardiomyocyte apoptosis in Kit(W)/Kit(W-v) mice and shows that c-kit(+) cardioprotective cells are able to attenuate post-MI apoptosis and rescue mice from progressive heart failure.
Wissenschaftlicher Artikel
Scientific Article
Falkenberg, N. ; Anastasov, N. ; Rappl, K. ; Braselmann, H. ; Auer, G. ; Walch, A.K. ; Huber, M. ; Höfig, I. ; Schmitt, M. ; Höfler, H. ; Atkinson, M.J. ; Aubele, M.
Br. J. Cancer 109, 2714-2723 (2013)
Background:MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells.Methods:MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT-PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222.Results:In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed.Conclusion:This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.
Wissenschaftlicher Artikel
Scientific Article
Altomonte, J. ; Marozin, S. ; de Toni, E.N. ; Rizzani, A. ; Esposito, I. ; Steiger, K. ; Feuchtinger, A. ; Hellerbrand, C. ; Schmid, R.M. ; Ebert, O.
Mol. Ther. 21, 2032-2042 (2013)
Recombinant vesicular stomatitis virus (VSV) shows promise for the treatment of hepatocellular carcinoma (HCC), but its safety and efficacy when administered in a setting of hepatic fibrosis, which occurs in the majority of clinical cases, is unknown. We hypothesized that VSV could provide a novel benefit to the underlying fibrosis, due to its ability to replicate and cause cell death specifically in activated hepatic stellate cells. In addition to the ability of VSV to produce a significant oncolytic response in HCC-bearing rats in the background of thioacetamide-induced hepatic fibrosis without signs of hepatotoxicity, we observed a significant downgrading of fibrosis stage, a decrease in collagen content in the liver, and modulation of gene expression in favor of fibrotic regression. Together, this work suggests that VSV is not only safe and effective for the treatment of HCC with underlying fibrosis, but it could potentially be developed for clinical application as a novel antifibrotic agent.
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Scientific Article
Hahne, H. ; Pachl, F. ; Ruprecht, B. ; Maier, S.K. ; Klaeger, S. ; Helm, D. ; Médard, G. ; Wilm, M. ; Lemeer, S. ; Kuster, B.
Nat. Methods 10, 989-991 (2013)
We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.
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Scientific Article
Schmitt, S. ; Saathoff, F. ; Meissner, L. ; Schropp, E.-M. ; Lichtmannegger, J. ; Schulz, S. ; Eberhagen, C. ; Borchard, S. ; Aichler, M. ; Adamski, J. ; Plesnila, N. ; Rothenfusser, S. ; Kroemer, G. ; Zischka, H.
Anal. Biochem. 443, 66-74 (2013)
Mitochondrial dysfunctions decisively contribute to the progression of human diseases, implying that functional tests of isolated mitochondria may furnish conclusive information for diagnosis and therapy. Classical mitochondrial isolation methods, however, lack precisely adjustable settings for cell rupture, which is the most critical step in this procedure, and this complicates subsequent analyses. Here, we present an efficient method to isolate functionally active, intact mitochondria from cultured or primary cells and minute tissue samples in a rapid, highly reproducible manner.
Wissenschaftlicher Artikel
Scientific Article
Pallua, J.D. ; Schaefer, G. ; Seifarth, C. ; Becker, M. ; Meding, S. ; Rauser, S. ; Walch, A.K. ; Handler, M. ; Netzer, M. ; Popovscaia, M. ; Osl, M. ; Baumgartner, C. ; Lindner, H. ; Kremser, L. ; Sarg, B. ; Bartsch, G. ; Huck, C.W. ; Bonn, G.K. ; Klocker, H.
J. Proteomics 91, 500-514 (2013)
[object Object]: New biomarkers are needed to improve the specificity of prostate cancer detection and characterisation of individual tumors. In a proteomics profiling approach using MALDI-MS tissue imaging on frozen tissue sections, we identified discriminating masses. Imaging analysis of cancer, non-malignant benign epithelium and stromal areas of 15 prostatectomy specimens in a test and 10 in a validation set identified characteristic m/z peaks for each tissue type, e.g. m/z 10775 for benign epithelial, m/z 6284 and m/z 6657.5 for cancer and m/z 4965 for stromal tissue. A 10-fold cross-validation analysis showed highest discriminatory ability to separate tissue types for m/z 6284 and m/z 6657.5, both overexpressed in cancer, and a multicomponent mass peak cluster at m/z 10775-10797.4 overexpressed in benign epithelial tissue. ROC AUC values for these three masses ranged from 0.85 to 0.95 in the discrimination of malignant and non-malignant tissue. To identify the underlying proteins, prostate whole tissue extract was separated by nano-HPLC and subjected to MALDI TOF/TOF analysis. Proteins in fractions containing discriminatory m/z masses were identified by MS/MS analysis and candidate marker proteins subsequently validated by immunohistochemistry (IHC). Biliverdin reductase B (BLVRB) turned out to be overexpressed in PCa tissue. BIOLOGICAL SIGNIFICANCE: In this study on cryosections of radical prostatectomies of prostate cancer patients, we performed a MALDI-MS tissue imaging analysis and a consecutive protein identification of significant m/z masses by nano-HPLC, MALDI TOF/TOF and MS/MS analysis. We identified BLVRB as a potential biomarker in the discrimination of PCa and benign tissue, also suggesting BVR as a feasible therapeutic target.
Wissenschaftlicher Artikel
Scientific Article
Balluff, B.
München, Ludwig-Maximilians-Universität, Medizinische Fakultät, Diss., 2013, 114 S.
In the presented thesis, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was used for the proteomic analysis of gastric cancer tissue samples, with the aims of 1) identifying proteins that predict disease outcome of patients with intestinal-type gastric cancer after surgical resection, and 2) generating a proteomic classifier that determines HER2-status in order to aid in therapy decision with regard to trastuzumab (Herceptin) administration. In the first study, a seven-protein signature was found to be associated with an unfavorable overall survival independent of major clinical covariates after analyzing 63 intestinal-type primary resected gastric cancer samples by MALDI imaging. Of these seven proteins, three could be identified as CRIP1, HNP-1, and S100-A6, and validated immunohistochemically on tissue microarrays of an independent validation cohort (n=118). While HNP-1 and S100-A6 were found to further subdivide early (UICC-I) and late stage (UICC-II-III) patients into different prognostic groups, CRIP1, a protein previously unknown in gastric cancer, was confirmed as a novel and independent prognostic factor for all patients in the validation cohort. The protein pattern described here serves as a new independent indicator of patient survival complementing the previously known clinical parameters in terms of prognostic relevance. In the second study, we hypothesized that MALDI imaging mass spectrometry may be useful for generating a classifier that may determine HER2-status in gastric cancer. This assumption was based on previous results where HER2-status could be reliably predicted in breast cancer patients. Here, 59 gastric cryo tissue samples were analyzed by MALDI imaging and the obtained proteomic profiles were used to create HER2 prediction models using different classification algorithms. Astonishingly, the breast cancer proteomic classifier from the previous study was able to correctly predict HER2-status in gastric cancers with a sensitivity of 65% and a specificity of 92%. In order to create a universal classifier for HER2-status, breast and non-breast cancer samples were combined, which increased sensitivity to 78%; specificity was 88%. This study provides evidence that HER2-status can be identified on a proteomic level across different cancer types suggesting that HER2 overexpression may constitute a widely spread molecular event independent of the tumor entity.
Neubert, P. ; Walch, A.K.
Expert Rev. Proteomics 10, 259-273 (2013)
Imaging mass spectrometry (IMS) is still a relatively young imaging technique that allows molecular mapping of diverse biomolecules in their natural environment. Furthermore, IMS allows for the direct correlation of tissue histology and proteomic, metabolomic or lipidomic information. In recent years, increasing efforts have been made in the development and improvement of IMS, which aid its application in clinical research. In this article, current frontiers of clinical research applications of IMS are discussed in the context of recent developments of IMS technology. Critical stages in planning and realizing clinical studies are highlighted. Finally, a selection of recent prominent examples for successful clinical applications of IMS is presented.
Review
Review
Berezowska, S. ; Novotny, A. ; Bauer, K. ; Feuchtinger, A. ; Slotta-Huspenina, J. ; Becker, K. ; Langer, R. ; Walch, A.K.
PLoS ONE 8:e69098 (2013)
BACKGROUND: Her2 expression and amplification occurs in a significant subset of gastro-esophageal carcinomas. Her2 is a client protein of molecular chaperones, e.g. heat shock protein (HSP) 90, rendering targeted therapies against Her2/HSP90 an interesting approach. This study aimed to investigate the role and relationship of Her2 and HSP90 in gastric and gastro-esophageal adenocarcinomas. MATERIAL AND METHODS: Immunohistochemical determination of HSP90 and Her2 expression was performed on 347 primary resected tumors. Her2 amplification was additionally determined by fluorescence in situ hybridization for all cases. Expression and amplification results were correlated with pathologic parameters (UICC pTNM category, tumor grading) and survival. RESULTS: Elevated Her2 copy numbers were observed in 87 tumors, 21 of them showing amplification. 174 tumors showed Her2 immunoreactivity/expression. HSP 90 immunoreactivity was found in 125 tumors. There was no difference between gastric carcinomas and carcinomas of the gastroesophageal junction regarding Her2 or HSP90. Both high HSP90 and Her2 expression/amplification were associated with earlier tumor stages (p<0.01), absence of lymph node metastases (p<0.02) and Laurens intestinal type (p<0.001). HSP90 correlated with Her2 expression and amplification (p<0.001 each). Expressions of HSP90 and Her2, but not Her2 amplification were associated with better prognosis (p=0.02; p=0.004; p=0.802). Moreover, Her2 expression was an independent prognostic factor for overall survival in the subgroup of gastric carcinoma patients (p=0.014) besides pT category, pN category and distant metastases. CONCLUSION: Her2 expression and gene amplification occurred in a significant subset of cases. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity and the definition of Her2 status in gastric cancers. Moreover, the correlation of Her2 expression with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment.
Wissenschaftlicher Artikel
Scientific Article
Sun, N. ; Walch, A.K.
Histochem. Cell Biol. 140, 93-104 (2013)
Mass spectrometry imaging (MSI) is a rapidly evolving technology that yields qualitative and quantitative distribution maps of small pharmaceutical-active molecules and their metabolites in tissue sections in situ. The simplicity, high sensitivity and ability to provide comprehensive spatial distribution maps of different classes of biomolecules make MSI a valuable tool to complement histopathology for diagnostics and biomarker discovery. In this review, qualitative and quantitative MSI of drugs and metabolites in tissue at therapeutic levels are discussed and the impact of this technique in drug discovery and clinical research is highlighted.
Review
Review
Maier, S.K. ; Hahne, H. ; Moghaddas Gholami, A. ; Balluff, B. ; Meding, S. ; Schoene, C. ; Walch, A.K. ; Kuster, B.
Mol. Cell. Proteomics 12, 2901-2910 (2013)
MALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a generic method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1,400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. All protein identification data from this study as well as the IMS literature has been deposited into MaTisse, a new publically available database which we anticipate will become a valuable resource for the IMS community.
Wissenschaftlicher Artikel
Scientific Article
Wortmann, M. ; Schneider, M. ; Pircher, J. ; Hellfritsch, J. ; Aichler, M. ; Vegi, N. ; Koelle, P. ; Kuhlencordt, P. ; Walch, A.K. ; Pohl, U. ; Bornkamm, G.W. ; Conrad, M. ; Beck, H.
Circ. Res. 113, 408-417 (2013)
Rationale: Growing evidence indicates that oxidative stress contributes markedly to endothelial dysfunction. The selenoenzyme glutathione peroxidase 4 (Gpx4) is an intracellular antioxidant enzyme important for the protection of membranes by its unique activity to reduce complex hydroperoxides in membrane bilayers and lipoprotein particles. Yet a role of Gpx4 in endothelial cell function has remained enigmatic. Objective: To investigate the role of Gpx4 ablation and subsequent lipid peroxidation in the vascular compartment in vivo. Methods and Results: Endothelial-specific deletion of Gpx4 had no obvious impact on normal vascular homeostasis nor did it impair tumor-derived angiogenesis in mice maintained on a normal diet. By stark contrast, aortic explants from endothelial-specific Gpx4 knockout mice showed a markedly reduced number of endothelial branches in sprouting assays. To shed light onto this apparent discrepancy between the in vivo and ex vivo results, we depleted mice of a second antioxidant, vitamin E, which is normally absent under ex vivo conditions. Mice were therefore fed a vitamin E-depleted diet for 6 weeks before endothelial deletion of Gpx4 was induced by 4-hydroxytamoxifen. Surprisingly, about 80% of the knockout mice died. Histopathological analysis revealed detachment of endothelial cells from the basement membrane as well as endothelial cell death in multiple organs which triggered thrombus formation. Thromboembolic events were the likely cause of various clinical pathologies including heart failure, renal and splenic micro-infarctions or paraplegia. Conclusions: Here we show for the first time that in the absence of Gpx4, sufficient vitamin E supplementation is crucial for endothelial viability.
Wissenschaftlicher Artikel
Scientific Article
Schulz, S. ; Schmitt, S. ; Wimmer, R. ; Aichler, M. ; Eisenhofer, S. ; Lichtmannegger, J. ; Eberhagen, C. ; Artmann, R. ; Toókos, F. ; Walch, A.K. ; Krappmann, D. ; Brenner-Jan, C. ; Rust, C. ; Zischka, H.
Biochim. Biophys. Acta-Biomembr. 1828, 2121-2133 (2013)
The cell-toxic bile salts glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA) are responsible for hepatocyte demise in cholestatic liver diseases, while tauroursodeoxycholic acid (TUDCA) is regarded hepatoprotective. We demonstrate the direct mitochondrio-toxicity of bile salts which deplete the mitochondrial membrane potential and induce the mitochondrial permeability transition (MPT). The bile salt mediated mechanistic mode of destruction significantly differs from that of calcium, the prototype MPT inducer. Cell-toxic bile salts initially bind to the mitochondrial outer membrane. Subsequently, the structure of the inner boundary membrane disintegrates. And it is only thereafter that the MPT is induced. This progressive destruction occurs in a dose- and time-dependent way. We demonstrate that GCDCA and TCDCA, but not TUDCA, preferentially permeabilize liposomes containing the mitochondrial membrane protein ANT, a process resembling the MPT induction in whole mitochondria. This suggests that ANT is one decisive target for toxic bile salts. To our knowledge this is the first report unraveling the consecutive steps leading to mitochondrial destruction by cell-toxic bile salts.
Wissenschaftlicher Artikel
Scientific Article
Taruttis, A. ; Wildgruber, M. ; Kosanke, K. ; Bézière, N. ; Licha, K. ; Haag, R. ; Aichler, M. ; Walch, A.K. ; Rummeny, E. ; Ntziachristos, V.
Photoacoustics 1, 3-8 (2013)
Objectives To investigate the feasibility of a high resolution optical imaging strategy for myocardial infarction. Background Near-infrared approaches to imaging cardiovascular disease enable visualization of disease-associated biological processes in vivo. However, even at the scale of small animals, the strong scattering of light prevents high resolution imaging after the first 1–2 mm of tissue, leading to degraded signal localization. Methods Multispectral optoacoustic tomography (MSOT) was used to non-invasively image myocardial infarction (MI) in a murine model of coronary artery ligation at resolutions not possible with current deep-tissue optical imaging methods. Post-MI imaging was based on resolving the spectral absorption signature of a dendritic polyglycerol sulfate-based (dPGS) near-infrared imaging agent targeted to P- and L-selectin. Results In vivo imaging succeeded in detection of the agent in the injured myocardium after intravenous injection. The high anatomic resolution (<200 μm) achieved by the described method allowed signals originating in the infarcted heart to be distinguished from uptake in adjacent regions. Histological analysis found dPGS signal in infarcted areas, originating from leukocytes and endothelial cells. Conclusions MSOT imaging of myocardial infarction provides non-invasive visualization of optical contrast with a high spatial resolution that is not degraded by the scattering of light.
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Scientific Article
Krebs, K.M. ; Böttinger, N. ; Huang, L.R. ; Chmielewski, M. ; Arzberger, S. ; Gasteiger, G. ; Jager, C. ; Schmitt, E. ; Bohne, F. ; Aichler, M. ; Uckert, W. ; Abken, H. ; Heikenwälder, M. ; Knolle, P. ; Protzer, U.
Gastroenterology 145, 456-465 (2013)
BACKGROUND & AIMS: Antiviral agents suppress hepatitis B virus (HBV) replication but do not clear the infection. A strong effector T-cell response is required to eradicate HBV, but this does not occur in patients with chronic infection. T cells might be directed toward virus-infected cells by expressing HBV-specific receptors, and thereby clear HBV and help to prevent development of liver cancer. In mice, we studied whether redirected T cells can engraft following adoptive transfer, without prior T-cell depletion, and whether the large amounts of circulating viral antigens inactivate the transferred T cells or lead to uncontrolled, immune-mediated damage. METHODS: CD8(+) T cells were isolated from mice and stimulated using an optimized protocol. Chimeric antigen receptors (CARs) that bind HBV envelope proteins (S-CAR) and activate T cells were expressed on the surface of cells using retroviral vectors. S-CAR-expressing CD8(+) T cells, which carried the marker CD45.1, were injected into CD45.2(+) HBV transgenic mice. We compared these mice with mice that received CD8(+) T cells induced by vaccination, cells that express a CAR without a proper signaling domain, or cells that express a CAR that does not bind HBV proteins (controls). RESULTS: CD8(+) T cells that expressed HBV-specific CARs recognized different HBV subtypes and were able to engraft and expand in immune-competent HBV transgenic mice. Following adoptive transfer, the S-CAR-expressing T cells localized to and functioned in the liver; they rapidly and efficiently controlled HBV replication, compared with controls, causing only transient liver damage. The large amount of circulating viral antigen did not impair or over-activate the S-CAR grafted T cells. CONCLUSION: T cells with a CAR specific for HBV envelop proteins localize to the livers of mice to reduce HBV replication, causing only transient liver damage. This immune-cell therapy might be developed for patients with chronic hepatitis B, regardless of their HLA-type.
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Ludyga, N. ; Englert, S. ; Pflieger, K. ; Rauser, S. ; Braselmann, H. ; Walch, A.K. ; Auer, G. ; Höfler, H. ; Aubele, M.
Mol. Cancer 12:28 (2013)
BACKGROUND: CRIP1 (cysteine-rich intestinal protein 1) has been found in several tumor types, its prognostic impact and its role in cellular processes, particularly in breast cancer, are still unclear. METHODS: To elucidate the prognostic impact of CRIP1, we analyzed tissues from 113 primary invasive ductal breast carcinomas using immunohistochemistry. For the functional characterization of CRIP1, its endogenous expression was transiently downregulated in T47D and BT474 breast cancer cells and the effects analyzed by immunoblotting, WST-1 proliferation assay and invasion assay. RESULTS: We found a significant correlation between CRIP1 and HER2 (human epidermal growth factor receptor 2) expression levels (p = 0.016) in tumor tissues. In Kaplan Meier analyses, CRIP1 expression was significantly associated with the distant metastases-free survival of patients, revealing a better prognosis for high CRIP1 expression (p = 0.039). Moreover, in multivariate survival analyses, the expression of CRIP1 was an independent negative prognostic factor, along with the positive prognosticators nodal status and tumor size (p = 0.029). CRIP1 knockdown in the T47D and BT474 breast cancer cell lines led to the increased phosphorylation of MAPK and Akt, to the reduced phosphorylation of cdc2, and to a significantly elevated cell proliferation in vitro (p < 0.001). These results indicate that reduced CRIP1 levels may increase cell proliferation and activate cell growth. In addition, CRIP1 knockdown increased cell invasion in vitro. CONCLUSIONS: Because the lack of CRIP1 expression in breast cancer tissue is significantly associated with a worse prognosis for patients and low endogenous CRIP1 levels in vitro increased the malignant potential of breast cancer cells, we hypothesize that CRIP1 may act as a tumor suppressor in proliferation and invasion processes. Therefore, CRIP1 may be an independent prognostic marker with significant predictive power for use in breast cancer therapy.
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Azimzadeh, O. ; Sievert, W. ; Sarioglu, H. ; Yentrapalli, R. ; Barjaktarovic, Z. ; Sriharshan, A. ; Ueffing, M. ; Janik, D. ; Aichler, M. ; Atkinson, M.J. ; Multhoff, G. ; Tapio, S.
J. Proteome Res. 12, 2700-2714 (2013)
Radiation exposure of the thorax is associated with a markedly increased risk of cardiac morbidity and mortality with a latency period of decades. Although many studies have confirmed the damaging effect of ionizing radiation on the myocardium and cardiac endothelial structure and function, the molecular mechanism behind this damage is not yet elucidated. Peroxisome proliferator-activated receptor alpha (PPAR alpha), a transcriptional regulator of lipid metabolism in heart tissue, has recently received great attention in the development of cardiovascular disease. The goal of this study was to investigate radiation-induced cardiac damage in general and the role of PPAR alpha in this process in particular. C57BL/6 mice received local heart irradiation with X-ray doses of 8 and 16 gray (Gy) at the age of 8 weeks. The mice were sacrificed 16 weeks later. Radiation-induced changes in the cardiac proteome were quantified using the Isotope Coded Protein Label (ICPL) method followed by mass spectrometry and software analysis. Significant alterations were observed in proteins involved in lipid metabolism and oxidative phosphorylation. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPAR alpha. This was reflected as decreased expression of its target genes involved in energy metabolism and mitochondrial respiratory chain confirming the proteomics data. This study suggests that persistent alteration of cardiac metabolism due to impaired PPAR alpha activity contributes to the heart pathology after radiation.
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Tapfer, A. ; Braren, R. ; Bech, M. ; Willner, M. ; Zanette, I. ; Weitkamp, T. ; Trajkovic-Arsic, M. ; Siveke, J.T. ; Settles, M. ; Aichler, M. ; Walch, A.K. ; Pfeiffer, F.
PLoS ONE 8:e58439 (2013)
To explore the potential of grating-based x-ray phase-contrast computed tomography (CT) for preclinical research, a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) was investigated. One ex-vivo mouse specimen was scanned with different grating-based phase-contrast CT imaging setups covering two different settings: i) high-resolution synchrotron radiation (SR) imaging and ii) dose-reduced imaging using either synchrotron radiation or a conventional x-ray tube source. These experimental settings were chosen to assess the potential of phase-contrast imaging for two different types of application: i) high-performance imaging for virtual microscopy applications and ii) biomedical imaging with increased soft-tissue contrast for in-vivo applications. For validation and as a reference, histological slicing and magnetic resonance imaging (MRI) were performed on the same mouse specimen. For each x-ray imaging setup, attenuation and phase-contrast images were compared visually with regard to contrast in general, and specifically concerning the recognizability of lesions and cancerous tissue. To quantitatively assess contrast, the contrast-to-noise ratios (CNR) of selected regions of interest (ROI) in the attenuation images and the phase images were analyzed and compared. It was found that both for virtual microscopy and for in-vivo applications, there is great potential for phase-contrast imaging: in the SR-based benchmarking data, fine details about tissue composition are accessible in the phase images and the visibility of solid tumor tissue under dose-reduced conditions is markedly superior in the phase images. The present study hence demonstrates improved diagnostic value with phase-contrast CT in a mouse model of a complex endogenous cancer, promoting the use and further development of grating-based phase-contrast CT for biomedical imaging applications.
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Aichler, M. ; Elsner, M. ; Ludyga, N. ; Feuchtinger, A. ; Zangen, V. ; Maier, S.K. ; Balluff, B. ; Schöne, C. ; Hierber, L. ; Braselmann, H. ; Meding, S. ; Rauser, S. ; Zischka, H. ; Aubele, M. ; Schmitt, M. ; Feith, M. ; Hauck, S.M. ; Ueffing, M. ; Langer, R. ; Kuester, B. ; Zitzelsberger, H. ; Höfler, H. ; Walch, A.K.
J. Pathol. 230, 410-419 (2013)
Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pre-therapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.
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Oetjen, J. ; Aichler, M. ; Trede, D. ; Strehlow, J. ; Berger, J. ; Heldmann, S. ; Becker, M. ; Gottschalk, M. ; Kobarg, J.H. ; Wirtz, S. ; Schiffler, S. ; Thiele, H. ; Walch, A.K. ; Maass, P. ; Alexandrov, T.
J. Proteomics 90, 52-60 (2013)
MALDI imaging mass spectrometry (MALDI-imaging) has emerged as a spatially-resolved label-free bioanalytical technique for direct analysis of biological samples and was recently introduced for analysis of 3D tissue specimens. We present a new experimental and computational pipeline for molecular analysis of tissue specimens which integrates 3D MALDI-imaging, magnetic resonance imaging (MRI), and histological staining and microscopy, and evaluate the pipeline by applying it to analysis of a mouse kidney. To ensure sample integrity and reproducible sectioning, we utilized the PAXgene fixation and paraffin embedding and proved its compatibility with MRI. Altogether, 122 serial sections of the kidney were analyzed using MALDI-imaging, resulting in a 3D dataset of 200 GB comprised of 2 million spectra. We show that elastic image registration better compensates for local distortions of tissue sections. The computational analysis of 3D MALDI-imaging data was performed using our spatial segmentation pipeline which determines regions of distinct molecular composition and finds m/z-values co-localized with these regions. For facilitated interpretation of 3D distribution of ions, we evaluated isosurfaces providing simplified visualization. We present the data in a multimodal fashion combining 3D MALDI-imaging with the MRI volume rendering and with light microscopic images of histologically stained sections. BIOLOGICAL SIGNIFICANCE: Our novel experimental and computational pipeline for 3D MALDI-imaging can be applied to address clinical questions such as proteomic analysis of the tumor morphologic heterogeneity. Examining the protein distribution as well as the drug distribution throughout an entire tumor using our pipeline will facilitate understanding of the molecular mechanisms of carcinogenesis.
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Schöne, C. ; Höfler, H. ; Walch, A.K.
Clin. Biochem. 46, 539-545 (2013)
Despite the tendency to regard tumors as a simple mass of cancer cells, tumors have a high degree of complexity that is difficult to access with most analytical methods. Because the cancer tissue itself directly contains all information concerning proteomic and genetic changes, it represents the best possible sample material for any molecular research. However, an analytical method should also take advantage of morphological information contained within the cancer tissues, a feat that is not easily possible with methods based on sample homogenization such as conventional mass spectrometry. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a method that allows the combination of mass spectrometric analyses with simultaneous histological evaluation to analyze various analytes such as proteins, peptides, lipids, or exogenous and endogenous small molecules. Spatially resolved mass spectrometric measurements are directly taken from a tissue section without destroying it. This combination allows for direct analysis of tumor samples while retaining the morphological information contained within the tissues, making it a very valuable tool in cancer research by complementing other currently used approaches. In this review, we discuss the position that MALDI imaging mass spectrometry currently occupies in the field of cancer research by showing its fields of application as well as the results and new discoveries that could be obtained using this method.
Review
Review
Meding, S. ; Walch, A.K.
Methods Mol. Biol. 931, 537-546 (2013)
MALDI (Matrix-Assisted Laser Desorption/Ionization) Imaging mass spectrometry is a powerful new method for analyzing the spatial distribution of molecules in tissues. Several different classes of cellular constituents such as proteins, peptides, lipids, and small molecules can be analyzed in situ while maintaining the morphological integrity of the tissue. This allows a correlation of the morphology with the previously acquired molecular patterns. By this, specific molecules can be clearly assigned to their cellular origin. Here, we will present a protocol for the analysis of proteins in tissues which are either native or alcohol-fixed and paraffin-embedded.
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Müller, T.D. ; Lee, S.J. ; Jastroch, M. ; Kabra, D. ; Stemmer, K. ; Aichler, M. ; Abplanalp, B. ; Ananthakrishnan, G. ; Bhardwaj, N. ; Collins, S. ; Divanovic, S. ; Endele, M. ; Finan, B. ; Gao, Y. ; Habegger, K.M. ; Hembree, J. ; Heppner, K.M. ; Hofmann, S. ; Holland, J. ; Küchler, D. ; Kutschke, M. ; Krishna, R. ; Lehti, M. ; Oelkrug, R. ; Ottaway, N. ; Perez-Tilve, D. ; Raver, C. ; Walch, A.K. ; Schriever, S.C. ; Speakman, J. ; Tseng, Y.H. ; Diaz-Meco, M. ; Pfluger, P.T. ; Moscat, J. ; Tschöp, M.H.
J. Clin. Invest. 123, 469-478 (2013)
The scaffold protein p62 (sequestosome 1; SQSTM1) is an emerging key molecular link among the metabolic, immune, and proliferative processes of the cell. Here, we report that adipocyte-specific, but not CNS-, liver-, muscle-, or myeloid-specific p62-deficient mice are obese and exhibit a decreased metabolic rate caused by impaired nonshivering thermogenesis. Our results show that p62 regulates energy metabolism via control of mitochondrial function in brown adipose tissue (BAT). Accordingly, adipocyte-specific p62 deficiency led to impaired mitochondrial function, causing BAT to become unresponsive to β-adrenergic stimuli. Ablation of p62 leads to decreased activation of p38 targets, affecting signaling molecules that control mitochondrial function, such as ATF2, CREB, PGC1α, DIO2, NRF1, CYTC, COX2, ATP5β, and UCP1. p62 ablation in HIB1B and BAT primary cells demonstrated that p62 controls thermogenesis in a cell-autonomous manner, independently of brown adipocyte development or differentiation. Together, our data identify p62 as a novel regulator of mitochondrial function and brown fat thermogenesis.
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Marinoni, I. ; Lee, M.S. ; Mountford, S. ; Perren, A. ; Bravi, I. ; Jennen, L. ; Feuchtinger, A. ; Drouin, J. ; Roncaroli, F. ; Pellegata, N.S.
Neuropathol. Appl. Neurobiol. 39, 256-269 (2013)
Aims: The aim of this study is to evaluate the pathological features, serum hormone levels and ex-vivo cultures of pituitary adenomas that occur in rats affected by MENX syndrome. MENX is multiple endocrine neoplasia syndrome caused by a germline mutation in the cell cycle inhibitor p27. Characterisation of MENX adenomas is a prerequisite to exploit this animal model for molecular and translational studies of pituitary adenomas. Methods: We investigated MENX pituitary adenomas with immunohistochemistry, double immunofluorescence, electron microscopy, RT-PCR, measurement of serum hormone levels and ex-vivo cultures. Results: Adenomas in MENX rats belong to the gonadotroph lineage. They start from 4 months of age as multiple neoplastic nodules and progress to become large lesions that efface the gland. Adenomas are composed of chromophobic cells predominantly expressing the glycoprotein alpha-subunit (αGSU). They show mitotic activity and high Ki67 labelling. A few neoplastic cells co-express gonadotrophins and the transcription factor SF1, together with growth hormone or prolactin and Pit-1, suggesting that they are not fully committed to one cell lineage. Ex vivo cultures show features similar to the primary tumour. Conclusions: Our results suggest that p27 function is critical in regulating gonadotroph cells growth. The MENX syndrome represents a unique model to elucidate the physiological and molecular mechanisms mediating the pathogenesis of gonadotroph adenomas.  
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Bettstetter, M. ; Berezowska, S. ; Keller, G. ; Walch, A.K. ; Feuchtinger, A. ; Slotta-Huspenina, J. ; Feith, M. ; Drecoll, E. ; Höfler, H. ; Langer, R.
Hum. Pathol. 44, 829-836 (2013)
Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context.
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2012
Hahne, H. ; Neubert, P. ; Kuhn, K. ; Etienne, C. ; Bomgarden, R. ; Rogers, J.C. ; Kuster, B.
Anal. Chem. 84, 3716-3724 (2012)
N-Linked protein glycosylation is one of the most prevalent post-translational modifications and is involved in essential cellular functions such as cell-cell interactions and cellular recognition as well as in chronic diseases. In this study, we explored stable isotope labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel approach for the quantification of N-linked glycans. Glyco-TMTs bearing hydrazide- and aminooxy-functionalized groups were compared for glycan reducing end derivatization efficiency and quantification merits. Aminooxy TMTs outperform the hydrazide reagents in terms of labeling efficiency (>95% vs 65% at 0.1 μM) and mass spectrometry based quantification using heavy/light-TMT labeled glycans enabled accurate quantification in MS1 spectra (CV < 15%) over a broad dynamic range (up to 1:40). In contrast, isobaric TMT labeling with quantification of reporter ions in tandem mass spectra suffered from severe ratio compression already at low sample ratios. To demonstrate the practical utility of the developed approach, we characterized the global N-linked glycosylation profiles of the isogenic human colon carcinoma cell lines SW480 (primary tumor) and SW620 (metastatic tumor). The data revealed significant down-regulation of high-mannose glycans in the metastatic cell line.
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Feuerecker, B. ; Pirsig, S. ; Seidl, C. ; Aichler, M. ; Feuchtinger, A. ; Bruchelt, G. ; Senekowitsch-Schmidtke, R.
Cancer Biol. Ther. 13, 1425-1435 (2012)
Cancer cells convert glucose preferentially to lactate even in the presence of oxygen (aerobic glycolysis-Warburg effect). New concepts in cancer treatment aim at inhibition of aerobic glycolysis. Pyruvate dehydrogenase converts pyruvate to acetylCoA thus preventing lactate formation. Therefore, the aim of this study was to evaluate compounds that could activate pyruvate dehydrogenase in cancer cells. We investigated the effects of (R)-(+)-alpha-lipoic acid (LPA) and dichloroacetate (DCA), possible activators of pyruvate dehydrogenase, on suppression of aerobic glycolysis and induction of cell death. The neuroblastoma cell lines Kelly, SK-N-SH, Neuro-2a and the breast cancer cell line SkBr3 were incubated with different concentrations (0.1-0 mM) of LPA and DCA. The effects of both compounds on cell viability/proliferation (WST-1 assay), [18F]-FDG uptake, lactate production and induction of apoptosis (flow cytometric detection of caspase-3) were evaluated. Furthermore, NMRI nu/nu mice that had been inoculated s.c. with SkBr3 cells were treated daily for four weeks with LPA (i.p, 18.5 mg/kg) starting at day 7 p.i.. Tumour development was measured with a sliding calliper and monitored via [18F]-FDG-PET. Residual tumours after therapy were examined histopathologically. These data suggests that LPA can reduce (i) cell viability/proliferation, (ii) uptake of [18F]-FDG and (iii) lactate production and increase apoptosis in all investigated cell lines. In contrast, DCA was almost ineffective. In the mouse xenograft model with s.c. SkBr3 cells, daily treatment with LPA retarded tumor progression. Therefore, LPA seems to be a promising compound for cancer treatment.
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Malinowsky, K. ; Wolff, C. ; Berg, D. ; Schuster, T. ; Walch, A.K. ; Bronger, H. ; Mannsperger, H. ; Schmidt, C. ; Korf, U. ; Höfler, H. ; Becker, K.F.
Transl. Oncol. 5, 98-104 (2012)
The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues.
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Meding, S. ; Balluff, B. ; Elsner, M. ; Schöne, C. ; Rauser, S. ; Nitsche, U. ; Maak, M. ; Schäfer, A. ; Hauck, S.M. ; Ueffing, M. ; Langer, R. ; Höfler, H. ; Friess, H. ; Rosenberg, R. ; Walch, A.K.
J. Pathol. 228, 459-470 (2012)
Regional lymph node metastasis negatively affects prognosis in colon cancer patients. The molecular processes leading to regional lymph node metastasis are only partially understood and proteomic markers for metastasis are still scarce. Therefore, a tissue-based proteomic approach was undertaken for identifying proteins associated with regional lymph node metastasis. Two complementary tissue-based proteomic methods have been employed. MALDI imaging was used for identifying small proteins (≤25 kDa) in situ and label-free quantitative proteomics was used for identifying larger proteins. A tissue cohort comprising primary colon tumours without metastasis (UICC II, pN0, n = 21) and with lymph node metastasis (UICC III, pN2, n = 33) was analysed. Subsequent validation of identified proteins was done by immunohistochemical staining on an independent tissue cohort consisting of primary colon tumour specimens (n = 168). MALDI imaging yielded ten discriminating m/z species, and label-free quantitative proteomics 28 proteins. Two MALDI imaging-derived candidate proteins (FXYD3 and S100A11) and one from the label-free quantitative proteomics (GSTM3) were validated on the independent tissue cohort. All three markers correlated significantly with regional lymph node metastasis: FXYD3 (p = 0.0110), S100A11 (p = 0.0071), and GSTM3 (p = 0.0173). FXYD3 and S100A11 were more highly expressed in UICC II patient tumour tissues. GSTM3 was more highly expressed in UICC III patient tumour tissues. By our tissue-based proteomic approach, we could identify a large panel of proteins which are associated with regional lymph node metastasis and which have not been described so far. Here we show that novel markers for regional lymp.
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Wulfkuhle, J.D. ; Berg, D. ; Wolff, C. ; Langer, R. ; Tran, K. ; Illi, J. ; Espina, V. ; Pierobon, M. ; Deng, J. ; Demichele, A. ; Walch, A.K. ; Bronger, H. ; Becker, I. ; Waldhör, C. ; Höfler, H. ; Esserman, L. ; Liotta, L.A. ; Becker, K.F. ; Petricoin, E.F.
Clin. Cancer Res. 18, 6426-6435 (2012)
PURPOSE: Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.EXPERIMENTAL DESIGN: Three independent study sets, comprising a total of 415 individual patient samples from flash-frozen core biopsy samples and formalin-fixed and paraffin-embedded (FFPE) surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pretreatment core biopsy samples and whole-tissue lysates from 288 FFPE samples and these results were compared with FISH and immunohistochemistry (IHC).RESULTS: RPMA measurements of total HER2 were highly concordant (>90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC(+) population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC(-) tumors and, identical to that seen with FISH/IHC(+) tumors, the HER2 activation was concordant with EGF receptor (EGFR) and HER3 phosphorylation and downstream signaling endpoint activation.CONCLUSIONS: Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC(-)/FISH(-)/pHER2(+) tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR.
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Scientific Article
Graf, N. ; Herrmann, K. ; Numberger, B. ; Zwisler, D. ; Aichler, M. ; Feuchtinger, A. ; Schuster, T. ; Wester, H.-J. ; Senekowitsch-Schmidtke, R. ; Peschel, C. ; Schwaiger, M. ; Keller, U. ; Dechow, T. ; Buck, A.K.
Eur. J. Nucl. Med. Mol. Imaging 40, 34-43 (2012)
PURPOSE: Positron emission tomography (PET) with the thymidine analogue [(18)F]fluorothymidine ([(18)F]FLT) has been shown to detect early response to chemotherapy in high-grade lymphoma. In this preclinical in vitro and in vivo study we compared [(18)F]FLT to the glucose analogue [(18)F]fluorodeoxyglucose ([(18)F]FDG) regarding dose-dependent visualization and prediction of early therapy response. METHODS: Immunodeficient mice bearing human diffuse large B-cell lymphoma (SUDHL-4) xenotransplants were treated intraperitoneally with increasing doses of the cytotoxic agent doxorubicin. Metabolic and antiproliferative effects were assessed 2 days after therapy by [(18)F]FLT and [(18)F]FDG PET. Explanted lymphomas were analysed histologically and by immunostaining against Ki67 and caspase 3. In vitro, lymphoma cells were incubated with increasing concentrations of doxorubicin and analysed using the tetrazolium assay, fluorescence-activated cell sorting, and [(18)F]FLT and [(18)F]FDG uptake 48 h later. RESULTS: In vivo, tumour growth was inhibited by doses of doxorubicin ranging from 25 μg to 200 μg. The mean tumour-to-background ratio (TBR) of [(18)F]FLT on day +2 was significantly reduced in all dose groups compared to control and baseline values and preceded changes in tumour volume. Importantly, there was a significant inverse correlation between reduction in TBR and dose of chemotherapy (r = -0.54, p = 0.021). The mean TBR of [(18)F]FDG, however, increased after therapy and differed considerably between groups (r = -0.13, p = 0.668). Explanted tumours showed a dose-dependent decrease in the proliferation marker Ki67, but no change in the apoptotic marker caspase 3. In vitro, doxorubicin led to a dose-dependent reduction in cell viability and a decrease in S phase. Lymphoma cells showed a dose-dependent reduction in [(18)F]FLT uptake, in contrast to a variable and decelerated reduction in [(18)F]FDG uptake. Thus, the increase in [(18)F]FDG uptake in vivo presumably reflected nonspecific glucose metabolism of inflammatory cells, as confirmed by histology of explanted lymphomas. CONCLUSION: Early responses to dose-dependent antiproliferative treatment in high-grade lymphoma are more accurately visualized with [(18)F]FLT PET than with [(18)F]FDG PET.
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Scientific Article
Stross, L. ; Günther, J. ; Gasteiger, G. ; Asen, T. ; Graf, S. ; Aichler, M. ; Esposito, I. ; Busch, D.H. ; Knolle, P. ; Sparwasser, T. ; Protzer, U.
Hepatology 56, 873-883 (2012)
The strength of antiviral T cell responses correlates with clearance of hepatitis B virus (HBV) infection, but the immunological mechanisms mitigating or suppressing HBV-specific T cells are still poorly understood. In this study, we examined the role of CD4+ Foxp3+ regulatory T cells (Tregs) in a mouse model of acute HBV infection. We initiated HBV infection via an adenoviral vector transferring a 1.3-fold overlength HBV genome (AdHBV) into transgenic DEREG mice, where Tregs can be transiently but selectively depleted by injection of diphtheria toxin. The effect of Treg depletion on the outcome of HBV infection was characterized by detailed virological, immunological, and histopathological analysis. Numbers of Tregs increase in the liver rapidly after initiation of HBV replication. Initial depletion of Tregs revealed their complex regulatory function during acute infection. Tregs mitigated immunomediated liver damage by down-regulating the antiviral activity of effector T cells by limiting cytokine production and cytotoxicity, but did not influence development of HBV-specific CD8 T cells or development of memory T cells. Furthermore, Tregs controlled the recruitment of innate immune cells such as macrophages and dendritic cells to the infected liver. As a consequence, Tregs significantly delayed clearance of HBV from blood and infected hepatocytes. Conclusion: Tregs limit immunomediated liver damage early after an acute infection of the liver, thereby contributing to conservation of tissue integrity and organ function at the cost of prolonging virus clearance. (HEPATOLOGY 2012;56:873883)
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Scientific Article
Slotta-Huspenina, J. ; Berg, D. ; Bauer, K. ; Wolff, C. ; Malinowsky, K. ; Bauer, L. ; Drecoll, E. ; Bettstetter, M. ; Feith, M. ; Walch, A.K. ; Höfler, H. ; Becker, K.F. ; Langer, R.
PLoS ONE 7:e41420 (2012)
A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.
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Scientific Article
Vallon, M. ; Seidl, C. ; Blechert, B. ; Li, Z. ; Gilbertz, K.P. ; Baumgart, A. ; Aichler, M. ; Feuchtinger, A. ; Gaertner, F.C. ; Bruchertseifer, F. ; Morgenstern, A. ; Walch, A.K. ; Senekowitsch-Schmidtke, R. ; Essler, M.
Eur. J. Nucl. Med. Mol. Imaging 39, 1886-1897 (2012)
PURPOSE: Targeted therapy with α-particle emitting radionuclides is a promising new option in cancer therapy. Stable conjugates of the vascular tumour-homing peptide F3 with the α-emitter (213)Bi specifically target tumour cells. The aim of our study was to determine efficacy of combined (213)Bi-diethylenetriaminepentaacetic acid (DTPA)-F3 and paclitaxel treatment compared to treatment with either (213)Bi-DTPA-F3 or paclitaxel both in vitro and in vivo. METHODS: Cytotoxicity of treatment with (213)Bi-DTPA-F3 and paclitaxel, alone or in combination, was assayed towards OVCAR-3 cells using the alamarBlue assay, the clonogenic assay and flow cytometric analyses of the mode of cell death and cell cycle arrest. Therapeutic efficacy of the different treatment options was assayed after repeated treatment of mice bearing intraperitoneal OVCAR-3 xenograft tumours. Therapy monitoring was performed by bioluminescence imaging and histopathologic analysis. RESULTS: Treatment of OVCAR-3 cells in vitro with combined (213)Bi-DTPA-F3 and paclitaxel resulted in enhanced cytotoxicity, induction of apoptosis and G2/M phase arrest compared to treatment with either (213)Bi-DTPA-F3 or paclitaxel. Accordingly, i.p. xenograft OVCAR-3 tumours showed the best response following repeated (six times) combined therapy with (213)Bi-DTPA-F3 (1.85 MBq) and paclitaxel (120 μg) as demonstrated by bioluminescence imaging and histopathologic investigation of tumour spread on the mesentery of the small and large intestine. Moreover, mean survival of xenograft mice that received combined therapy with (213)Bi-DTPA-F3 and paclitaxel was significantly superior to mice treated with either (213)Bi-DTPA-F3 or paclitaxel alone. CONCLUSION: Combined treatment with (213)Bi-DTPA-F3 and paclitaxel significantly increased mean survival of mice with peritoneal carcinomatosis of ovarian origin, thus favouring future therapeutic application.
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Scientific Article
Li, Z. ; Graf, N.A. ; Herrmann, K. ; Jünger, A. ; Aichler, M. ; Feuchtinger, A. ; Baumgart, A. ; Walch, A.K. ; Peschel, C. ; Schwaiger, M. ; Buck, A. ; Keller, U. ; Dechow, T.
Cancer Res. 72, 5014-5024 (2012)
The prognosis of relapsed or refractory aggressive lymphoma is poor. The huge variety of currently evolving targeted treatment approaches would benefit from tools for early prediction of response or resistance. We used various ALK-positive anaplastic large cell lymphoma (ALCL) cell lines to evaluate two inhibitors, the HSP90 inhibitor NVP-AUY922, and the mTOR inhibitor everolimus, both of which have shown to interfere with ALK-dependent oncogenic signal transduction. Their therapeutic effect was determined in vitro by MTT assay, [18F]fluorodeoxyglucose- (FDG) and [18F]fluorothymidine- (FLT) uptake, and by biochemical analysis of ALK-induced signalling. Micro FDG- and FLT-PET imaging studies in immunodeficient mice bearing ALCL xenotransplants were performed with the cell lines SUDHL-1 and Karpas299 to assess early treatment response to NVP-AUY922 or everolimus in vivo. SUDHL-1 cells showed sensitivity to both inhibitors in vitro. Importantly, we detected a significant reduction of FLT-uptake in SUDHL-1 bearing animals using both inhibitors compared to baseline as early as 5 days after initiation of targeted therapy. Immunostaining showed a decrease in Ki-67 and an increase in cleaved caspase-3 staining. In contrast, FDG-uptake did not significantly decrease at early time points. Karpas299 xenotransplants, which are resistant to NVP-AUY922 and sensitive to everolimus treatment, showed an increase of mean FLT-uptake on day 2 after administration of NVP-AUY299, but a significant reduction in FLT-uptake upon everolimus treatment. In conclusion, we show that FLT- but not FDG-PET is able to predict response to treatment with specific inhibitors very early in the course of treatment and thus enables early prediction of treatment efficacy.
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Gehrmann, M. ; Stangl, S. ; Kirschner, A. ; Foulds, G.A. ; Sievert, W. ; Doß, B.T. ; Walch, A.K. ; Pockley, A.G. ; Multhoff, G.
PLoS ONE 7:e41341 (2012)
BACKGROUND: We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner. METHODOLOGY/PRINCIPAL FINDINGS: Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes. CONCLUSIONS/SIGNIFICANCE: These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.
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Balluff, B. ; Rauser, S. ; Ebert, M.P. ; Siveke, J.T. ; Höfler, H. ; Walch, A.K.
Gastroenterology 143, 544-549 (2012)
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Saraste, A. ; Laitinen, I. ; Weidl, E. ; Wildgruber, M. ; Weber, A.W. ; Nekolla, S.G. ; Hölzlwimmer, G. ; Esposito, I. ; Walch, A.K. ; Leppänen, P. ; Lisinen, I. ; Luppa, P.B. ; Yla-Herttuala, S. ; Wester, H.J. ; Knuuti, J. ; Schwaiger, M.
J. Nucl. Cardiol. 19, 775-784 (2012)
Expression of alpha(v)beta(3) integrin has been proposed as a marker for atherosclerotic lesion inflammation. We studied whether diet intervention reduces uptake of alpha(v)beta(3) integrin-targeted positron emission tomography tracer F-18-galacto-RGD in mouse atherosclerotic plaques. Hypercholesterolemic LDLR-/- ApoB(100/100) mice on high-fat diet for 4 months were randomized to further 3 months on high-fat diet (high-fat group, n = 8) or regular mouse chow (intervention group, n = 7). Intima-media ratio describing plaque burden was comparable between intervention and high-fat groups (2.0 +/- A 0.5 vs 2.3 +/- A 0.8, P = .5). Uptake of F-18-galacto-RGD in the aorta was lower in the intervention than high-fat group (%ID/g 0.16 vs 0.23, P < .01). Autoradiography showed 35% lower uptake of F-18-galacto-RGD in the atherosclerotic plaques in the intervention than high-fat group (P = .007). Uptake of F-18-galacto-RGD in plaques correlated with uptake of H-3-deoxyglucose and nuclear density, which was lower in the intervention than high-fat group (P = .01). Flow cytometry demonstrated macrophages expressing alpha(v) and beta(3) integrins in the aorta. Uptake of F-18-galacto-RGD in mouse atherosclerotic lesions was reduced by lipid-lowering diet intervention. Expression of alpha(v)beta(3) integrin is a potential target for evaluation of therapy response in atherosclerosis.
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Trede, D. ; Schiffler, S. ; Becker, M. ; Wirtz, S. ; Steinhorst, K. ; Strehlow, J. ; Aichler, M. ; Kobarg, J.H. ; Oetjen, J. ; Dyatlov, A. ; Heldmann, S. ; Walch, A.K. ; Thiele, H. ; Maass, P. ; Alexandrov, T.
Anal. Chem. 84, 6079-6087 (2012)
Three-dimensional (3D) imaging has a significant impact on many challenges of life sciences. Three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging label-free bioanalytical technique capturing the spatial distribution of hundreds of molecular compounds in 3D by providing a MALDI mass spectrum for each spatial point of a 3D sample. Currently, 3D MALDI-IMS cannot tap its full potential due to the lack efficient computational methods for constructing, processing, and visualizing large and complex 3D MALDI-IMS data. We present a new pipeline of efficient computational methods, which enables analysis and interpretation of a 3D MALDI-IMS data set. Construction of a MALDI-IMS data set was done according to the state-of-the-art protocols and involved sample preparation, spectra acquisition, spectra preprocessing, and registration of serial sections. For analysis and interpretation of 3D MALDI-IMS data, we applied the spatial segmentation approach which is well-accepted in analysis of two-dimensional (2D) MALDI-IMS data. In line with 2D data analysis, we used edge-preserving 3D image denoising prior to segmentation to reduce strong and chaotic spectrum-to-spectrum variation. For segmentation, we used an efficient clustering method, called bisecting k-means, which is optimized for hierarchical clustering of a large 3D MALDI-IMS data set. Using the proposed pipeline, we analyzed a central part of a mouse kidney using 33 serial sections of 3.5 μm thickness after the PAXgene tissue fixation and paraffin embedding. For each serial section, a 2D MALDI-IMS data set was acquired following the standard protocols with the high spatial resolution of 50 μm. Altogether, 512 495 mass spectra were acquired that corresponds to approximately 50 gigabytes of data. After registration of serial sections into a 3D data set, our computational pipeline allowed us to reveal the 3D kidney anatomical structure based on mass spectrometry data only. Finally, automated analysis discovered molecular masses colocalized with major anatomical regions. In the same way, the proposed pipeline can be used for analysis and interpretation of any 3D MALDI-IMS data set in particular of pathological cases.
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Baumhoer, D. ; Smida, J. ; Zillmer, S. ; Rosemann, M. ; Atkinson, M.J. ; Nelson, P.J. ; Jundt, G. ; von Luettichau, I. ; Nathrath, M.
Mod. Pathol. 25, 522-528 (2012)
Hematogenous spread determines the outcome of osteosarcoma (OS) patients, but the pathogenesis of developing metastatic disease is still unclear. Chemokines are critical regulators of cell trafficking and adhesion, and have been reported to be aberrantly expressed and to correlate with an unfavorable prognosis and metastatic spread in several malignant tumors. The chemokine receptors CXCR4 and CXCR7 together with their common ligand CXCL12 form one of the most important chemokine axes in this context. To investigate a potential role of these chemokines in OSs, we analyzed their expression in a series of 223 well-characterized and pretherapeutic OS samples. Interestingly, we found the expression of CXCL12 and CXCR4 to correlate with a better long-term outcome and with a lower prevalence of metastases. These findings suggest a distinct role of CXCR4/CXCR7/CXCL12 signaling in the tumors of bone, as has also been previously described in acute leukemia. As many malignant tumors metastasize to bone, and tumor cells are thought to be directed to bone in response to CXCL12, OS cells expressing both CXCL12 and the corresponding receptors might be detained at their site of origin. The disruption of CXCR4/CXCR7/CXCL12 signaling could therefore be crucial in OSs for the migration of tumor cells from bone into circulation and for developing systemic disease.
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Prade, E. ; Tobiasch, M. ; Hitkova, I. ; Schaffer, I. ; Lian, F. ; Xing, X.B. ; Tänzer, M. ; Rauser, S. ; Walch, A.K. ; Feith, M. ; Post, S. ; Röcken, C. ; Schmid, R.M. ; Ebert, M.P.A. ; Burgermeister, E.
Mol. Endocrinol. 26, 819-832 (2012)
Bile acids are synthesized from cholesterol and are major risk factors for Barrett adenocarcinoma (BAC) of the esophagus. Caveolin-1 (Cav1), a scaffold protein of membrane caveolae, is transcriptionally regulated by cholesterol via sterol-responsive element-binding protein-1 (SREBP1). Cav1 protects squamous epithelia by controlling cell growth and stabilizing cell junctions and matrix adhesion. Cav1 is frequently down-regulated in human cancers; however, the molecular mechanisms that lead to this event are unknown. We show that the basal layer of the nonneoplastic human esophageal squamous epithelium expressed Cav1 mainly at intercellular junctions. In contrast, Cav1 was lost in 95% of tissue specimens from BAC patients (n = 100). A strong cytoplasmic expression of Cav1 correlated with poor survival in a small subgroup (n = 5) of BAC patients, and stable expression of an oncogenic Cav1 variant (Cav1-P132L) in the human BAC cell line OE19 promoted proliferation. Cav1 was also detectable in immortalized human squamous epithelial, Barrett esophagus (CPC), and squamous cell carcinoma cells (OE21), but was low in BAC cell lines (OE19, OE33). Mechanistically, bile acids down-regulated Cav1 expression by inhibition of the proteolytic cleavage of 125-kDa pre-SREBP1 from the endoplasmic reticulum/Golgi apparatus and nuclear translocation of active 68-kDa SREBP1. This block in SREBP1's posttranslational processing impaired transcriptional activation of SREBP1 response elements in the proximal human Cav1 promoter. Cav1 was also down-regulated in esophagi from C57BL/6 mice on a diet enriched with 1% (wt/wt) chenodeoxycholic acid. Mice deficient for Cav1 or the nuclear bile acid receptor farnesoid X receptor showed hyperplasia and hyperkeratosis of the basal cell layer of esophageal epithelia, respectively. These data indicate that bile acid-mediated down-regulation of Cav1 marks early changes in the squamous epithelium, which may contribute to onset of Barrett esophagus metaplasia and progression to BAC. (Molecular Endocrinology 26: 819-832, 2012)
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Grüner, B.M. ; Hahne, H. ; Mazur, P.K. ; Trajkovic-Arsic, M. ; Maier, S.K. ; Esposito, I. ; Kalideris, E. ; Michalski, C.W. ; Kleeff, J. ; Rauser, S. ; Schmid, R.M. ; Kuster, B. ; Walch, A.K. ; Siveke, J.T.
PLoS ONE 7:e39424 (2012)
The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosinbeta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.
Wissenschaftlicher Artikel
Scientific Article
Heindl, S. ; Eggenstein, E. ; Keller, S. ; Kneissl, J. ; Keller, G. ; Mutze, K. ; Rauser, S. ; Gasteiger, G. ; Drexler, I. ; Hapfelmeier, A. ; Höfler, H. ; Luber, B.
J. Cancer Res. Clin. Oncol. 138, 843-858 (2012)
PURPOSE: The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line. METHODS: EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods. RESULTS: We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance. CONCLUSION: These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.
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Seiz, L. ; Dorn, J. ; Kotzsch, M. ; Walch, A.K. ; Grebenchtchikov, N.I. ; Gkazepis, A. ; Schmalfeldt, B. ; Kiechle, M. ; Bayani, J. ; Diamandis, E.P. ; Langer, R. ; Sweep, F.C. ; Schmitt, M. ; Magdolen, V.
Biol. Chem. 393, 391-401 (2012)
Several members of the human kallikrein-related peptidase family, including KLK6, are up-regulated in ovarian cancer. High KLK6 mRNA or protein expression, measured by quantitative polymerase chain reaction and enzyme-linked immunoassay, respectively, was previously found to be associated with a shortened overall and progression-free survival (OS and PFS, respectively). In the present study, we aimed at analyzing KLK6 protein expression in ovarian cancer tissue by immunohistochemistry. Using a newly developed mono-specific polyclonal antibody, KLK6 immunoexpression was initially evaluated in normal tissues. We observed strong staining in the brain and moderate staining in the kidney, liver, and ovary, whereas the pancreas and the skeletal muscle were unreactive, which is in line with previously published results. Next, both tumor cell- and stromal cell-associated KLK6 immunoexpression were analyzed in tumor tissue specimens of 118 ovarian cancer patients. In multivariate Cox regression analysis, only stromal cell-associated expression, besides the established clinical parameters FIGO stage and residual tumor mass, was found to be statistically significant for OS and PFS [high vs. low KLK6 expression; hazard ratio (HR), 1.92; p=0.017; HR, 1.80; p=0.042, respectively]. These results indicate that KLK6 expressed by stromal cells may considerably contribute to the aggressiveness of ovarian cancer.
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von Brühl, M.L. ; Stark, K. ; Steinhart, A. ; Chandraratne, S. ; Konrad, I. ; Lorenz, M. ; Khandoga, A. ; Tirniceriu, A. ; Coletti, R. ; Köllnberger, M. ; Byrne, R.A. ; Laitinen, I. ; Walch, A.K. ; Brill, A. ; Pfeiler, S. ; Manukyan, D. ; Braun, S. ; Lange, P. ; Riegger, J. ; Ware, J. ; Eckart, A. ; Haidari, S. ; Rudelius, M. ; Schulz, C. ; Echtler, K. ; Brinkmann, V. ; Schwaiger, M. ; Preissner, K.T. ; Wagner, D.D. ; Mackman, N. ; Engelmann, B. ; Massberg, S.
J. Exp. Med. 209, 819-835 (2012)
Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.
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Manook, A. ; Yousefi, B.H. ; Willuweit, A. ; Platzer, S. ; Reder, S. ; Voss, A. ; Huisman, M. ; Settles, M. ; Neff, F. ; Velden, J. ; Schoor, M. ; von der Kammer, H. ; Wester, H.J. ; Schwaiger, M. ; Henriksen, G. ; Drzezga, A..
PLoS ONE 7:e31310 (2012)
In vivo imaging and quantification of amyloid-β plaque (Aβ) burden in small-animal models of Alzheimer's disease (AD) is a valuable tool for translational research such as developing specific imaging markers and monitoring new therapy approaches. Methodological constraints such as image resolution of positron emission tomography (PET) and lack of suitable AD models have limited the feasibility of PET in mice. In this study, we evaluated a feasible protocol for PET imaging of Aβ in mouse brain with [(11)C]PiB and specific activities commonly used in human studies. In vivo mouse brain MRI for anatomical reference was acquired with a clinical 1.5 T system. A recently characterized APP/PS1 mouse was employed to measure Aβ at different disease stages in homozygous and hemizygous animals. We performed multi-modal cross-validations for the PET results with ex vivo and in vitro methodologies, including regional brain biodistribution, multi-label digital autoradiography, protein quantification with ELISA, fluorescence microscopy, semi-automated histological quantification and radioligand binding assays. Specific [(11)C]PiB uptake in individual brain regions with Aβ deposition was demonstrated and validated in all animals of the study cohort including homozygous AD animals as young as nine months. Corresponding to the extent of Aβ pathology, old homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous (23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was shown to be suitable as an intracerebral reference region. PET results were cross-validated and consistent with all applied ex vivo and in vitro methodologies. The results confirm that the experimental setup for non-invasive [(11)C]PiB imaging of Aβ in the APP/PS1 mice provides a feasible, reproducible and robust protocol for small-animal Aβ imaging. It allows longitudinal imaging studies with follow-up periods of approximately one and a half years and provides a foundation for translational Alzheimer neuroimaging in transgenic mice.
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Scientific Article
Grunewald, T.G. ; Ranft, A. ; Esposito, I. ; Da Silva-Buttkus, P. ; Aichler, M. ; Baumhoer, D. ; Schaefer, K.L. ; Ottaviano, L. ; Poremba, C. ; Jundt, G. ; Jürgens, H. ; Dirksen, U. ; Richter, G.H. ; Burdach, S.
Ann. Oncol. 23, 2185-2190 (2012)
BACKGROUND: Ewing's sarcoma (ES) is the second most common bone or soft-tissue sarcoma in childhood and adolescence and features a high propensity to metastasize. The six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is a membrane-bound mesenchymal stem cell marker highly expressed in ES. Here, we investigated the role of STEAP1 as an immunohistological marker for outcome prediction in patients with ES. PATIENTS AND METHODS: Membranous STEAP1 immunoreactivity was analyzed using immunohistochemistry in 114 primary pre-chemotherapy ES of patients diagnosed from 1983 to 2010 and compared with clinical parameters and patient outcome. Median follow-up was 3.85 years (range 0.43-17.51). RESULTS: A total of 62.3% of the ES samples displayed detectable STEAP1 expression with predominant localization of the protein at the plasma membrane. High membranous STEAP1 immunoreactivity was found in 53.5%, which correlated with better overall survival (P = 0.021). Accordingly, no or low membranous STEAP1 expression was identified as an independent risk factor in multivariate analysis (hazard ratio 2.65, P = 0.036). CONCLUSIONS: High membranous STEAP1 expression predicts improved outcome and may help to define a specific subgroup of ES patients, who might benefit from adapted therapy regimens.
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Scientific Article
Elsner, M. ; Rauser, S. ; Maier, S.K. ; Schöne, C. ; Balluff, B. ; Meding, S. ; Jung, G. ; Nipp, M. ; Sarioglu, H. ; Maccarrone, G. ; Aichler, M. ; Feuchtinger, A. ; Langer, R. ; Jütting, U. ; Feith, M. ; Kuster, B. ; Ueffing, M. ; Zitzelsberger, H. ; Höfler, H. ; Walch, A.K.
J. Proteomics 75, 4693-4704 (2012)
To characterize proteomic changes found in Barrett's adenocarcinoma and its premalignant stages, the proteomic profiles of histologically defined precursor and invasive carcinoma lesions were analyzed by MALDI imaging MS. For a primary proteomic screening, a discovery cohort of 38 fresh frozen Barrett's adenocarcinoma patient tissue samples was used. The goal was to find proteins that might be used as markers for monitoring cancer development as well as for predicting regional lymph node metastasis and disease outcome. Using mass spectrometry for protein identification and validating the results by immunohistochemistry on an independent validation set, we could identify two of 60 differentially expressed m/z species between Barrett's adenocarcinoma and the precursor lesion: COX7A2 and S100-A10. Furthermore, among 22m/z species that are differentially expressed in Barrett's adenocarcinoma cases with and without regional lymph node metastasis, one was identified as TAGLN2. In the validation set, we found a correlation of the expression levels of COX7A2 and TAGLN2 with a poor prognosis while S100-A10 was confirmed by multivariate analysis as a novel independent prognostic factor in Barrett's adenocarcinoma. Our results underscore the high potential of MALDI imaging for revealing new biologically significant molecular details from cancer tissues which might have potential for clinical application. This article is part of a Special Issue entitled: Translational Proteomics.
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Scientific Article
Meding, S. ; Nitsche, U. ; Balluff, B. ; Elsner, M. ; Rauser, S. ; Schöne, C. ; Nipp, M. ; Maak, M. ; Feith, M. ; Ebert, M.P. ; Friess, H. ; Langer, R. ; Höfler, H. ; Zitzelsberger, H. ; Rosenberg, R. ; Walch, A.K.
J. Proteome Res. 11, 1996-2003 (2012)
In clinical diagnostics, it is of outmost importance to correctly identify the source of a metastatic tumor, especially if no apparent primary tumor is present. Tissue-based proteomics might allow correct tumor classification. As a result, we performed MALDI imaging to generate proteomic signatures for different tumors. These signatures were used to classify common cancer types. At first, a cohort comprised of tissue samples from six adenocarcinoma entities located at different organ sites (esophagus, breast, colon, liver, stomach, thyroid gland, n = 171) was classified using two algorithms for a training and test set. For the test set, Support Vector Machine and Random Forest yielded overall accuracies of 82.74 and 81.18%, respectively. Then, colon cancer liver metastasis samples (n = 19) were introduced into the classification. The liver metastasis samples could be discriminated with high accuracy from primary tumors of colon cancer and hepatocellular carcinoma. Additionally, colon cancer liver metastasis samples could be successfully classified by using colon cancer primary tumor samples for the training of the classifier. These findings demonstrate that MALDI imaging-derived proteomic classifiers can discriminate between different tumor types at different organ sites and in the same site.
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Scientific Article
Takenaka, S. ; Möller, W. ; Semmler-Behnke, M. ; Karg, E.W. ; Wenk, A. ; Schmid, O. ; Stöger, T. ; Jennen, L. ; Aichler, M. ; Walch, A.K. ; Pokhrel, S. ; Mädler, L. ; Eickelberg, O. ; Kreyling, W.G.
Nanomed. 7, 855-865 (2012)
Aim: To investigate the relationship of alveolar macrophages and inhaled nanoparticles in the lung. Materials & methods: Rats were exposed by inhalation to 14 nm gold nanoparticles for 6 hours, and ultramicroscopic observation on the frequency and localization of gold nanoparticles within lavaged macrophages was performed up to 7 days. Results & discussion: The majority of macrophages examined on day 0 (94%) contained internalized gold nanoparticles, and the percentage decreased to 59% on day 7. Gold nanoparticles were exclusively found within cytoplasmic vesicles. On day 0, most gold nanoparticles appeared to be individual or slightly agglomerated, and they were frequently agglomerated on day 7. Conclusion: Alveolar macrophages efficiently internalized nanoparticles by endocytosis, and re-arrangements of vesicles and of nanoparticles in the vesicles of macrophages occurred.
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Scientific Article
Berg, D. ; Wolff, C. ; Malinowsky, K. ; Tran, K. ; Walch, A.K. ; Bronger, H. ; Schuster, T. ; Höfler, H. ; Becker, K.-F.
J. Cell. Physiol. 227, 204-212 (2012)
In the last few years, new approaches and developments in patient-tailored cancer therapies have raised the need to select, more precisely, those patients who will respond to personalized treatments. Therefore, the most efficient way for optimal therapy and patient selection is to provide a tumour-specific protein network portrait prior to treatment. The aim of our study was to monitor protein networks in formalin-fixed and paraffin-embedded (FFPE) breast cancer tissues, with special emphasis on epidermal growth factor receptor 2 (HER2)-mediated signalling pathways, to identify and validate new disease markers. For this purpose we used a recently developed technology to extract full-length proteins from FFPE tissues and analysed 23 molecules involved in HER2-related signalling by reverse phase protein microarray (RPPA) in a series of 106 FFPE breast cancer tissue samples. We found a significant correlation of HER2 with human epidermal growth factor receptor 3 (HER3/erbB3), epidermal growth factor receptor 1 (EGFR/HER1/erbB1) and urokinase plasminogen receptor (uPAR) in routinely used FFPE breast cancer tissues. Thus, targeting HER2, EGFR, HER3 and uPAR together may offer a more efficient treatment option for patients with breast cancer.
Wissenschaftlicher Artikel
Scientific Article
Ebert, M.P. ; Tänzer, M. ; Balluff, B. ; Burgermeister, E. ; Kretzschmar, A.K. ; Hughes, D.J. ; Tetzner, R. ; Lofton-Day, C. ; Rosenberg, R. ; Reinacher-Schick, A.C. ; Schulmann, K. ; Tannapfel, A. ; Hofheinz, R. ; Röcken, C. ; Keller, G. ; Langer, R. ; Specht, K. ; Porschen, R. ; Stöhlmacher-Williams, J. ; Schuster, T. ; Ströbel, P. ; Schmid, R.M.
N. Engl. J. Med. 366, 44-53 (2012)
BACKGROUND: Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy. METHODS: We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation. RESULTS: TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism. CONCLUSIONS: TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).
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Scientific Article
Aichler, M. ; Seiler, C. ; Tost, M. ; Siveke, J. ; Mazur, P.K. ; Da Silva-Buttkus, P. ; Bartsch, D.K. ; Langer, P. ; Chiblak, S. ; Dürr, A. ; Höfler, H. ; Klöppel, G. ; Müller-Decker, K. ; Brielmeier, M. ; Esposito, I.
J. Pathol. 226, 723-734 (2012)
Pancreatic ductal adenocarcinoma (PDAC) and its precursor lesions, pancreatic intraepithelial neoplasia, (PanIN), display a ductal phenotype. However, there is evidence in genetically defined mouse models for PDAC harbouring a mutated kras under the control of a pancreas-specific promoter that ductal cancer might arise in the centroacinar-acinar region, possibly through a process of acinar-ductal metaplasia (ADM). In order to further elucidate this model of PDAC development, an extensive expression analysis and molecular characterization of the putative and already established (PanIN) precursor lesions was performed in the Kras(G12D/+) ;Ptf1a-Cre(ex1/+) mouse model and in human tissues, focusing on lineage markers, developmental pathways, cell cycle regulators, apomucins and stromal activation markers. The results of this study show that areas of ADM are very frequent in the murine and human pancreas and represent regions of increased proliferation of cells with precursor potential. Moreover, atypical flat lesions originating in areas of ADM are the most probable precursors of PDAC in the Kras(G12D/+) ;Ptf1a-cre(ex1/+) mice and similar lesions were also found in the pancreas of three patients with a strong family history of PDAC. In conclusion, PDAC development in Kras(G12D/+) ;Ptf1a-Cre(ex1/+) mice starts from ADM and a similar process might also take place in patients with a strong family history of PDAC.
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Scientific Article
Kernt, M. ; Walch, A.K. ; Neubauer, A.S. ; Hirneiss, C. ; Haritoglou, C. ; Ulbig, M.W. ; Kampik, A.
Clin. Experiment. Ophthalmol. 40, 87-97 (2012)
Purpose:  Cumulative light exposure is significantly associated with aging and the progression of age-related macular degeneration. To prevent the retina from blue-light damage in pseudophakia, blue-light-absorbing intraocular lenses (IOLs) have been developed. This study compares the possible protective effects of a blue-light-absorbing IOL to an untinted UV-absorbing IOL with regard to light-induced oxidative stress and senescence of human retinal pigment epithelium (RPE). Methods:  As primary human RPE cells were exposed to white light, either a UV- and blue-light-absorbing IOL or UV-absorbing IOL was placed in the light beam. After 60 min of irradiation, cells were investigated by electron microscopy for viability, induction of intracellular reactive oxygen species, and senescence-associated β-galactosidase activity. Expression and secretion of matrix metalloproteinases 1 and 3 and their mRNA were determined by real-time PCR and enzyme-linked immunosorbent assay. Results:  Light exposure induced structural damage, decreased RPE cell viability, and increased reactive oxygen species, senescence-associated β-galactosidase activity, and matrix metalloproteinases 1 and 3 expression and secretion. Although both types of IOL significantly reduced these effects, the protective effects of the UV- and blue-light-absorbing IOL were significantly stronger than those of the UV-absorbing IOL. Conclusions:  The UV- and blue-light-absorbing IOL demonstrated significantly better protection against light-induced oxidative stress, senescence, and structural damage than the UV-absorbing IOL. These in vitro findings support the hypothesis that the UV- and blue-light-absorbing IOL may prevent retinal damage in clinical use.
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Scientific Article
Nipp, M. ; Elsner, M. ; Balluff, B. ; Meding, S. ; Sarioglu, H. ; Ueffing, M. ; Rauser, S. ; Unger, K. ; Höfler, H. ; Walch, A.K. ; Zitzelsberger, H.
J. Mol. Med. 90, 163-174 (2012)
In papillary thyroid carcinoma (PTC), metastasis is a feature of an aggressive tumor phenotype. To identify protein biomarkers that distinguish patients with an aggressive tumor behavior, proteomic signatures in metastatic and non-metastatic tumors were investigated comparatively. In particular, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to analyze primary tumor samples. We investigated a tumor cohort of PTC (n = 118) that were matched for age, tumor stage, and gender. Proteomic screening by MALDI-IMS was performed for a discovery set (n = 29). Proteins related to the discriminating mass peaks were identified by 1D-gel electrophoresis followed by mass spectrometry. The candidate proteins were subsequently validated by immunohistochemistry (IHC) using a tissue microarray for an independent PTC validation set (n = 89). In this study, we found 36 mass-to-charge-ratio (m/z) species that specifically distinguished metastatic from non-metastatic tumors, among which m/z 11,608 was identified as thioredoxin, m/z 11,184 as S100-A10, and m/z 10,094 as S100-A6. Furthermore, using IHC on the validation set, we showed that the overexpression of these three proteins was highly associated with lymph node metastasis in PTC (p < 0.005). For functional analysis of the metastasis-specific proteins, we performed an Ingenuity Pathway Analysis and discovered a strong relationship of all candidates with the TGF-β-dependent EMT pathway. Our results demonstrated the potential application of the MALDI-IMS proteomic approach in identifying protein markers of metastasis in PTC. The novel protein markers identified in this study may be used for risk stratification regarding metastatic potential in PTC.
Wissenschaftlicher Artikel
Scientific Article
2011
Schmidl, S.
München, Technische Universität München, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Diss., 2011, 109 S.
Peripheral B cells that encounter a cognate antigen and become activated via CD40 binding start to form germinal centres where they undergo affinity maturation. The affinity maturation process somatic hypermutation (SHM) depends on the activation-induced cytidine deaminase protein (AID) that inserts mutations into the immunoglobulin (Ig) locus, enhancing the genomic variety of antibodies. AID recruitment to the Ig locus and AID mutations must be strictly regulated to ensure genomic stability. However, the specific targeting of AID has not been clarified. In the scope of this project, cis- elements included in the Ig locus and potential trans- factors involved in the specific AID recruitment were identified. To this end a chicken DT40 cell line was used that solely undergoes somatic hypermutation. The assay for mutation was a GFP2 reporter system in the Ig locus that accumulates AID-induced mutations. The compact size of the DT40 Igl locus of 10 kb facilitates a successive deletion analysis. A deletion of the complete Igl locus demonstrated that elements essential for SHM are located in the Ig sequence. Via several staggered deletion analyses, the Igl enhancer was identified as the core sequence essential for AID recruitment and in particular a 200 bp sequence located at the 5’ end of the enhancer. This hypermutation core element (‘HyCorE’) was sufficient for AID recruitment. Multimerization of the element enhanced the mutation frequency. An identification of ‘HyCorE’ homologes in closely-related species and their efficient recruitment of AID confirmed the exclusive role of this sequence. An in silico analysis of the 200 bp core element identified binding sites for E2A, NF-kB, MEF-2, SP1 and Pax5. This is the first time demonstration of a sequence of 200 bp that is sufficient to induce hypermutation and carries binding sites for trans-acting factors for a putative AID recruitment complex. CD40 signalling, that is essential for germinal centre formation and initiation of SHM, was known to be deregulated in several lymphomas. The LMP1/CD40 mouse model, established in our lab, allowed a detailed analysis of constitutive CD40 signalling. The LMP1/CD40 mouse expresses a chimeric protein consisting of the self-activated LMP1 transmembrane domain and the CD40 intracellular domain. It has previously been shown that a constitutive CD40 signal in vivo initiates B cell expansion and promotes B cell tumourigenesis. Analysis of signalling pathways in LMP1/CD40 expressing B cells revealed that the MAPK ERK and JNK and the non-canonical NF-kB pathway are activated. To analyze, whether the canonical NF- kB pathway influences B cell expansion and lymphomagenesis, mice with a NEMO or IKK2 null deletion were crossed with LMP1/CD40 mice. The disruption of the canonical NF-kB pathway blocked the LMP1/CD40 induced B cell expansion, reduced the LMP1/CD40 mediated survival and inhibited proliferation. Interestingly, the depletion of NEMO or IKK2 in LMP1/CD40 mice did not influence the translocation of the NF-kB components, but led to diminished pERK and pJNK levels, indicating a cross-talk between the NF-kB and MAPK pathways. Previous publications demonstrated a connection between NEMO/IKK2 and ERK via Tpl2/MEK1, resulting in a specific activation of p65 (Phospho-Ser276). Indeed p65(Ser276) was specifically phosphorylated in LMP1/CD40 mice, but not in NEMO or IKK2 depleted LMP1/CD40 or in wild type mice. These data imply that the canonical NF-kB pathway contributes to LMP1/CD40 mediated B cell expansion via the Tpl2/ERK/pp65(Ser276) regulation mechanism. This result elucidates a completely new role of the canonical NF-kB pathway for CD40 signalling in B cells and creates a foundation for detailed signalling analyses.
Luber, B. ; Deplazes, J. ; Keller, G. ; Walch, A.K. ; Rauser, S. ; Eichmann, M. ; Langer, R. ; Höfler, H. ; Hegewisch-Becker, S. ; Folprecht, G. ; Wöll, E. ; Decker, T.-M. ; Endlicher, E. ; Lorenzen, S. ; Fend, F. ; Peschel, C. ; Lordick, F.
BMC Cancer 11:509 (2011)
BACKGROUND: The activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab combined with oxaliplatin/leucovorin/5-fluorouracil (FUFOX) was assessed in first-line metastatic gastric and oesophago-gastric junction (OGJ) cancer in a prospective phase II study showing a promising objective tumour response rate of 65% and a low mutation frequency of KRAS (3%). The aim of the correlative tumour tissue studies was to investigate the relationship between EGFR gene copy numbers, activation of the EGFR pathway, expression and mutation of E-cadherin, V600E BRAF mutation and clinical outcome of patients with gastric and OGJ cancer treated with cetuximab combined with FUFOX. METHODS: Patients included in this correlative study (n = 39) were a subset of patients from the clinical phase II study. The association between EGFR gene copy number, activation of the EGFR pathway, abundance and mutation of E-cadherin which plays an important role in these disorders, BRAF mutation and clinical outcome of patients was studied. EGFR gene copy number was assessed by FISH. Expression of the phosphorylated forms of EGFR and its downstream effectors Akt and MAPK, in addition to E-cadherin was analysed by immunohistochemistry. The frequency of mutant V600E BRAF was evaluated by allele-specific PCR and the mutation profile of the E-cadherin gene CDH1 was examined by DHPLC followed by direct sequence analysis. Correlations with overall survival (OS), time to progression (TTP) and overall response rate (ORR) were assessed. RESULTS: Our study showed a significant association between increased EGFR gene copy number (≥ 4.0) and OS in gastric and OGJ cancer, indicating the possibility that patients may be selected for treatment on a genetic basis. Furthermore, a significant correlation was shown between activated EGFR and shorter TTP and ORR, but not between activated EGFR and OS. No V600E BRAF mutations were identified. On the other hand, an interesting trend between high E-cadherin expression levels and better OS was observed and two CDH1 exon 9 missense mutations (A408V and D402H) were detected. CONCLUSION: Our finding that increased EGFR gene copy numbers, activated EGFR and the E-cadherin status are potentially interesting biomarkers needs to be confirmed in larger randomized clinical trials. TRIAL REGISTRATION: Multicentre clinical study with the European Clinical Trials Database number 2004-004024-12.
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Scientific Article
Baumhoer, D. ; Elsner, M. ; Smida, J. ; Zillmer, S. ; Rauser, S. ; Schoene, C. ; Balluff, B. ; Bielack, S. ; Jundt, G. ; Walch, A.K. ; Nathrath, M.
Oncotarget 2, 970-975 (2011)
Predicting the clinical course of osteosarcoma patients is a crucial prerequisite for a better treatment stratification in these highly aggressive neoplasms of bone. In search of new and reliable biomarkers we recently identified cysteine-rich intestinal protein 1 (CRIP1) to have significant prognostic impact in gastric cancer and therefore decided to investigate its role also in osteosarcoma. For this purpose we analyzed 223 pretherapeutic and well characterized osteosarcoma samples for their immunohistochemical expression of CRIP1 and correlated our findings with clinico-pathological parameters including follow‑up, systemic spread and response to chemotherapy. Interestingly and contrarily to gastric cancer, we found CRIP1 expression more frequently in patients with long‑term survival (10-year survival 73% in positive vs. 54% in negative cases, p = 0.0433) and without metastases (p = 0.0108) indicating a favorable prognostic effect. CRIP1 therefore seems to represent a promising new biomarker in osteosarcoma patients which should be considered for a prospective validation.
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Scientific Article
Pallua, J.D. ; Schaefer, G. ; Bittner, L.K. ; Pezzei, C. ; Huck-Pezzei, V. ; Schoenbichler, S.A. ; Meding, S. ; Rauser, S. ; Walch, A.K. ; Handler, M. ; Netzer, M. ; Osl, M. ; Seger, M. ; Pfeifer, B. ; Baumgartner, C. ; Lindner, H. ; Kremser, L. ; Sarg, B. ; Klocker, H. ; Bartsch, G. ; Bonn, G.K. ; Huck, C.W.
LC GC N. Am. Suppl. S, Suppl., 21-28 (2011)
Matrix-assisted laser desorption-ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool in histopathological characterization and represents a modern analytical technique, enabling two-dimensional detection of molecular components of biological samples. Using this method, it is possible to investigate the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids, and small molecules in biological systems by in-situ analysis of cell cultures and tissue sections. Recently, MALDI-IMS has become an essential tool for tissue analyses in life science applications, offering global analysis of tissue samples. An advantage of this imaging technique is the acquisition of local molecular expression profiles up to the microscopic level, while maintaining the topographic integrity of the tissue by avoiding time-consuming extraction, purification, or separation steps, respectively. With MALDI-IMS it is possible to determine the distribution of hundreds of unknown compounds in a single measurement, allowing rapid probing of the tissues' biochemistry. Moreover, MALDI-IMS results include qualitative and semiquantitative information, providing unique chemi-morphological information about the tissue status, which represents an important benefit for future analytical interpretation of pathological changes of a tissue. This article summarizes the most recent advances in sample preparation, instrumentation, and data-processing techniques for MALDI-IMS.
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Scientific Article
Marjanovic, G. ; Schricker, M. ; Walch, A.K. ; zur Hausen, A. ; Hopt, U.T. ; Imdahl, A. ; Makowiec, F.
J. Gastrointest. Surg. 15, 29-37 (2011)
BACKGROUND: Involved lymph nodes (LN) are a negative prognostic factor in esophageal cancers. To assess the role of nodal micrometastases, we performed immunohistochemical analyses of LN after resection of node-negative esophageal cancers and correlated the results with survival. METHODS: Seventy patients with esophageal cancer after curative resection and conventionally negative nodes were included. The LN were examined with six consecutive sections (three hematoxylin and eosin (HE) stained and three stained immunohistochemically with the cytokeratin (CK) antibodies AE1/AE3). Survival was evaluated uni- and multivariately. Median follow-up was 4.1 years. RESULTS: Immunohistochemical analysis showed CK-positive LN in 16 (23%) patients. Of those 16 cases with CK-positive LN, nine had aviable macrometastases, ten had CK-positive scars/fibrosis and five had viable micrometastases. All patients with aviable macrometastases or CK-positive scars/fibrosis had undergone neoadjuvant chemoradiation. Five-year survival was 48% in all patients. In univariate analysis, survival was worse in patients with CK-positive LN (5-year survival of 30% vs. 54% in CK-negative LN; p < 0.02) and in patients with squamous cell carcinoma (5-year survival of 38% vs. 75% in adenocarcinoma; p = 0.05). Multivariate analysis revealed CK-positive LN (p = 0.02) and (borderline) squamous cell carcinoma (p = 0.06) as negative prognostic factors. CONCLUSIONS: The immunohistochemical analysis of LN may detect (viable or non-viable) tumor cells in lymph nodes after resection of conventionally node-negative esophageal cancers. Conventional pathological analysis by HE, therefore, understages esophageal cancer in these cases. The detection of CK-positive cells in resected LN is an independent prognostic factor in otherwise LN-negative esophageal cancer.
Wissenschaftlicher Artikel
Scientific Article
Walch, A.K. ; Rauser, S. ; Höfler, H.
In: Stanta, G.* [Eds.]: Guidelines for Molecular Analysis in Archive Tissues. Berlin [u.a.]: Springer, 2011. 293-295
Tissue samples have been routinely used for decades to distinguish healthy from diseased tissue in histopathological characterization. While nucleic acid-based methodologies have been successfully in use for many years, protein-based techniques, in contrast, are at a very early stage (with the exception of immunohistochemistry). In this chapter, a method for the analysis of FFPE tissues by MALDI imaging mass spectrometry is described.
Schumann, R.G. ; Eibl, K.H. ; Zhao, F. ; Scheerbaum, M. ; Scheler, R. ; Schaumberger, M.M. ; Wehnes, H. ; Walch, A.K. ; Haritoglou, C. ; Kampik, A. ; Gandorfer, A.
Invest. Ophthalmol. Vis. Sci. 52, 7822-7834 (2011)
PURPOSE: To provide new information on epiretinal cell proliferation and the cells' origin in idiopathic macular holes and to overcome the effects of embedding and sectioning preparation procedures on cell-distribution patterns. METHODS: Interference and phase-contrast microscopy, immunocytochemistry, and scanning and transmission electron microscopy were performed on surgically excised whole-mounted internal limiting membrane (ILM) specimens removed from 60 eyes with idiopathic macular holes. Cell distribution and cell morphology were correlated with immunocytochemical staining characteristics. Twelve cell type-specific antibodies were used to detect glial cells, hyalocytes, retinal pigment epithelial cells, retinal ganglion cells, and immune cells. Cell viability was analyzed. RESULTS: Epiretinal cell proliferation was found in all ILM specimens, irrespective of the stage of the macular hole. Cell density showed a broad variety. Immunocytochemistry frequently revealed simultaneous expression of GFAP/CD45, GFAP/CD64, GFAP/CD68, GFAP/CRALBP, and GFAP/CD90. Some cells presented with intracellular contractile filaments (anti-αSMA); others were not immunoreactive to any antibody examined. The percentage of viable cells showed a broad variety with a mean of 73% (SD 29%). Electron microscopy demonstrated glial cells, hyalocytes, and myofibroblast-like cells. CONCLUSIONS: The presence of epiretinal cells at the ILM in all macular hole stages strongly suggests a substantial involvement of cell migration and proliferation in the course of macular hole development. Glial cells and hyalocytes play the predominant role in epiretinal cell proliferation. Given the co-expression of glial cell and hyalocyte markers, transdifferentiation of epiretinal cells needs further elucidation, especially with respect to αSMA-positive cells leading to traction at the vitreoretinal interface.
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Scientific Article
Loos, M. ; Langer, R. ; Schuster, T. ; Gertler, R. ; Walch, A.K. ; Rauser, S. ; Friess, H. ; Feith, M.
Ann. Thorac. Surg. 91, 1025-1031 (2011)
BACKGROUND: The costimulatory molecule B7-H1 (programmed death-1 ligand-1, PD-L1) has been implicated as a potential regulator of antitumor immunity in various human cancers. To date, no data are available on the role of B7-H1 in Barrett carcinoma. Therefore, we investigated the expression pattern and clinical significance of B7-H1 in a large cohort of patients with Barrett carcinoma. METHODS: Expression of B7-H1 was evaluated by immunohistochemistry in 101 patients with Barrett carcinoma. Expression data were correlated with clinicopathologic features, including TNM stage, UICC (Union Internationale Contre le Cancer) tumor stage, tumor grade, resection status, and survival, and with the number of tumor-infiltrating CD3(+), CD8(+), and CD45RO(+) T lymphocytes.RESULTS: Aberrant B7-H1 expression was found in Barrett carcinoma cells. High tumor B7-H1 expression was significantly associated with advanced T stage (p = 0.002), advanced UICC tumor stage (p = 0.022), and incomplete resection status (p = 0.009). The median survival of patients with high tumor B7-H1 expression was 38 months compared with 136 months for patients with no or low tumor B7-H1 expression. In the multivariable analysis, high tumor B7-H1 expression was significantly associated with an increased risk of death from Barrett carcinoma (hazard ratio, 3.50; 95% confidence interval, 1.66 to 7.38; p < 0.001). CONCLUSIONS: Our data suggest that B7-H1 may represent a new prognostic marker for patients with Barrett carcinoma. Furthermore, given its immune-inhibitory function, B7-H1 may represent a potential target in the treatment of Barrett carcinoma.
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Scientific Article
Zischka, H. ; Lichtmannegger, J. ; Schmitt, S. ; Jägemann, N. ; Schulz, S. ; Wartini, D. ; Jennen, L. ; Rust, C. ; Larochette, N. ; Galluzzi, L. ; Chajes, V. ; Bandow, N. ; Gilles, V.S. ; DiSpirito, A.A. ; Esposito, I. ; Göttlicher, M. ; Summer, K.H. ; Kroemer, G.
J. Clin. Invest. 121, 1508-1518 (2011)
Wilson disease (WD) is a rare hereditary condition that is caused by a genetic defect in the copper-transporting ATPase ATP7B that results in hepatic copper accumulation and lethal liver failure. The present study focuses on the structural mitochondrial alterations that precede clinical symptoms in the livers of rats lacking Atp7b, an animal model for WD. Liver mitochondria from these Atp7b–/– rats contained enlarged cristae and widened intermembrane spaces, which coincided with a massive mitochondrial accumulation of copper. These changes, however, preceded detectable deficits in oxidative phosphorylation and biochemical signs of oxidative damage, suggesting that the ultrastructural modifications were not the result of oxidative stress imposed by copper- dependent Fenton chemistry. In a cell-free system containing a reducing dithiol agent, isolated mitochondria exposed to copper underwent modifications that were closely related to those observed in vivo. In this cell-free system, copper induced thiol modifications of three abundant mitochondrial membrane proteins, and this correlated with reversible intramitochondrial membrane crosslinking, which was also observed in liver mitochondria from Atp7b–/– rats. In vivo, copper-chelating agents reversed mitochondrial accumulation of copper, as well as signs of intra-mitochondrial membrane crosslinking, thereby preserving the functional and structural integrity of mitochondria. Together, these findings suggest that the mitochondrion constitutes a pivotal target of copper in WD.
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Balluff, B. ; Schöne, C. ; Höfler, H. ; Walch, A.K.
Histochem. Cell Biol. 136, 227-244 (2011)
Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a method that allows the investigation of the molecular content of tissues within its morphological context. Since it is able to measure the distribution of hundreds of analytes at once, while being label free, this method has great potential which has been increasingly recognized in the field of tissue-based research. In the last few years, MALDI-IMS has been successfully used for the molecular assessment of tissue samples mainly in biomedical research and also in other scientific fields. The present article will give an update on the application of MALDI-IMS in clinical and preclinical research. It will also give an overview of the multitude of technical advancements of this method in recent years. This includes developments in instrumentation, sample preparation, computational data analysis and protein identification. It will also highlight a number of emerging fields for application of MALDI-IMS like drug imaging where MALDI-IMS is used for studying the spatial distribution of drugs in tissues.
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Lian, F. ; Xing, X. ; Yuan, G. ; Schäfer, C. ; Rauser, S. ; Walch, A.K. ; Röcken, C. ; Ebeling, M. ; Wright, M.B. ; Schmid, R.M. ; Ebert, M.P. ; Burgermeister, E.
Biochem. J. 438, 315-323 (2011)
Bile acids from duodenogastric reflux promote inflammation and increase the risk for gastro-oesophageal cancers. FXR (farnesoid X receptor/NR1H4) is a transcription factor regulated by bile acids such as CDCA (chenodeoxycholic acid). FXR protects the liver and the intestinal tract against bile acid overload; however, a functional role for FXR in the stomach has not been described. We detected FXR expression in the normal human stomach and in GC (gastric cancer). FXR mRNA and protein were also present in the human GC cell lines MKN45 and SNU5, but not in the AGS cell line. Transfection of FXR into AGS cells protected against TNFα (tumour necrosis factor α)-induced cell damage. We identified K13 (keratin 13), an anti-apoptotic protein of desmosomes, as a novel CDCA-regulated FXR-target gene. FXR bound to a conserved regulatory element in the proximal human K13 promoter. Gastric expression of K13 mRNA was increased in an FXR-dependent manner by a chow diet enriched with 1% (w/w) CDCA and by indomethacin (35 mg/kg of body weight intraperitoneal) in C57BL/6 mice. FXR-deficient mice were more susceptible to indomethacin-induced gastric ulceration than their WT (wild-type) littermates. These results suggest that FXR increases the resistance of human and murine gastric epithelial cells to inflammation-mediated damage and may thus participate in the development of GC.
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Alexandrov, T. ; Meding, S. ; Trede, D. ; Kobarg, J.H. ; Balluff, B. ; Walch, A.K. ; Thiele, H. ; Maass, P.
J. Proteomics 75, 237-245 (2011)
In the last decade, imaging mass spectrometry has seen incredible technological advances in its applications to biological samples. One computational method of data mining in this field is the spatial segmentation of a sample, which produces a segmentation map highlighting chemically similar regions. An important issue for any imaging mass spectrometry technology is its relatively low spatial or lateral resolution (i.e. a large size of pixel) as compared with microscopy. Thus, the spatial resolution of a segmentation map is also relatively low, that complicates its visual examination and interpretation when compared with microscopy data, as well as reduces the accuracy of any automated comparison. We address this issue by proposing an approach to improve the spatial resolution of a segmentation map. Given a segmentation map, our method magnifies it up to some factor, producing a super-resolution segmentation map. The super-resolution map can be overlaid and compared with a high-res microscopy image. The proposed method is based on recent advances in image processing and smoothes the "pixilated" region boundaries while preserving fine details. Moreover, it neither eliminates nor splits any region. We evaluated the proposed super-resolution segmentation approach on three MALDI-imaging datasets of human tissue sections and demonstrated the superiority of the super-segmentation maps over standard segmentation maps.
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Wu, Z. ; Doondeea, J.B. ; Moghaddas Gholami, A. ; Janning, M.C. ; Lemeer, S. ; Kramer, K. ; Eccles, S.A. ; Gollin, S.M. ; Grenman, R. ; Walch, A.K. ; Feller, S.M. ; Kuster, B.
Mol. Cell. Proteomics 10:M111.011635 (2011)
Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. In order to identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant inter-cell line differences for 42 kinases (p<0.05), and loss of function experiments using siRNA in high- and low- expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1 and EPHA2involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib and erlotinib as well as siRNA led to strong reduction of viability in high- but not low- expressing lines confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor, dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.
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Balluff, B. ; Rauser, S. ; Meding, S. ; Elsner, M. ; Schöne, C. ; Feuchtinger, A. ; Schuhmacher, C. ; Novotny, A. ; Jütting, U. ; Maccarrone, G. ; Sarioglu, H. ; Ueffing, M. ; Braselmann, H. ; Zitzelsberger, H. ; Schmid, R.M. ; Höfler, H. ; Ebert, M.P. ; Walch, A.K.
Am. J. Pathol. 179, 2720-2729 (2011)
Proteomics-based approaches allow us to investigate the biology of cancer beyond genomic initiatives. We used histology-based matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that predict disease outcome in gastric cancer after surgical resection. A total of 181 intestinal-type primary resected gastric cancer tissues from two independent patient cohorts were analyzed. Protein profiles of the discovery cohort (n = 63) were directly obtained from tumor tissue sections by MALDI imaging. A seven-protein signature was associated with an unfavorable overall survival independent of major clinical covariates. The prognostic significance of three individual proteins identified (CRIP1, HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of an independent validation cohort (n = 118). Whereas HNP-1 and S100-A6 were found to further subdivide early-stage (Union Internationale Contre le Cancer [UICC]-I) and late-stage (UICC II and III) cancer patients into different prognostic groups, CRIP1, a protein previously unknown in gastric cancer, was confirmed as a novel and independent prognostic factor for all patients in the validation cohort. The protein pattern described here serves as a new independent indicator of patient survival complementing the previously known clinical parameters in terms of prognostic relevance. These results show that this tissue-based proteomic approach may provide clinically relevant information that might be beneficial in improving risk stratification for gastric cancer patients.
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Langer, R. ; Rauser, S. ; Feith, M. ; Nährig, J.M. ; Feuchtinger, A. ; Friess, H. ; Höfler, H. ; Walch, A.K.
Mod. Pathol. 24, 908-916 (2011)
Amplification and overexpression of ErbB2 (Her2) is a frequent event in oesophageal adenocarcinomas. Assessment of ErbB2 status is crucial for identifying patients who are likely to benefit from treatment with trastuzumab. In this study, we performed a comprehensive analysis of ErbB2 amplification and expression in 142 oesophageal adenocarcinomas by comparing the most commonly used methods for ErbB2 assessment: ErbB2 expression was determined by immunohistochemistry and was scored (0, 1+, 2+ and 3+) according to a recently described modified scoring system for gastric cancer. ErbB2 amplification was evaluated by bright field double in situ hybridisation. The results were compared with pathologic features, patients' survival and previously published data from fluorescence in situ hybridisation analysis. On the basis of immunohistochemistry, which was applicable in 110 cores of the cases, 83 tumours (75%) had a score of 0 or 1+ (immunohistochemistry negative), 13 tumours (12%) were scored as 2+ and 14 tumours (13%) were scored as 3+. In situ hybridisation data were obtained from 142 cases. There was a highly significant correlation of immunohistochemistry, bright field in situ hybridisation and fluorescent in situ hybridisation (P<0.001 each). In total, 41 tumours (29%) were categorised as ErbB2 positive, which was defined as immunohistochemistry 3+ and/or an ErbB2/Chr17 quotient of ≥2 as assessed by either bright field double in situ hybridisation or fluorescence in situ hybridisation. ErbB2 positivity was observed more frequently in tumours with lower differentiation grades (P=0.029). Patients with ErbB2-positive tumours had a significantly worse prognosis, both in univariate analysis (P=0.004) and in multivariate analysis (P=0.03). In conclusion, we demonstrate that a significant number of oesophageal adenocarcinomas are positive for ErbB2. Assessment of ErbB2 amplification can be equivalently performed by conventional fluorescence in situ hybridisation or other light-microscopy-based methods, such as the novel bright field double in situ hybridisation technique.
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Timme, S. ; Schmitt, E. ; Stein, S. ; Schwarz-Finsterle, J. ; Wagner, J. ; Walch, A.K. ; Werner, M. ; Hausmann, M. ; Wiech, T.
Anal. Cell. Pathol. 34, 21-33 (2011)
Cell type specific radial positioning of chromosome territories (CTs) and their sub-domains in the interphase seem to have functional relevance in non-neoplastic human nuclei, while much less is known about nuclear architecture in carcinoma cells and its development during tumor progression. We analyzed the 3D-architecture of the chromosome 8 territory (CT8) in carcinoma and corresponding non-neoplastic ductal pancreatic epithelium. Fluorescence-in-situ-hybridization (FISH) with whole chromosome painting (WCP) probes on sections from formalin-fixed, paraffin wax-embedded tissues from six patients with ductal adenocarcinoma of the pancreas was used. Radial positions and shape parameters of CT8 were analyzed by 3D-microscopy. None of the parameters showed significant inter-individual changes. CT8 was localized in the nuclear periphery in carcinoma cells and normal ductal epithelial cells. Normalized volume and surface of CT8 did not differ significantly. In contrast, the normalized roundness was significantly lower in carcinoma cells, implying an elongation of neoplastic cell nuclei. Unexpectedly, radial positioning of CT8, a dominant parameter of nuclear architecture, did not change significantly when comparing neoplastic with non-neoplastic cells. A significant deformation of CT8, however, accompanies nuclear atypia of carcinoma cells. This decreased roundness of CTs may reflect the genomic and transcriptional alterations in carcinoma.
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Deininger, S.O. ; Cornett, D.S. ; Paape, R. ; Becker, M. ; Pineau, C. ; Rauser, S. ; Walch, A.K. ; Wolski, E.
Anal. Bioanal. Chem. 401, 167-181 (2011)
Normalization is critically important for the proper interpretation of matrix-assisted laser desorption/ionization (MALDI) imaging datasets. The effects of the commonly used normalization techniques based on total ion count (TIC) or vector norm normalization are significant, and they are frequently beneficial. In certain cases, however, these normalization algorithms may produce misleading results and possibly lead to wrong conclusions, e.g. regarding to potential biomarker distributions. This is typical for tissues in which signals of prominent abundance are present in confined areas, such as insulin in the pancreas or β-amyloid peptides in the brain. In this work, we investigated whether normalization can be improved if dominant signals are excluded from the calculation. Because manual interaction with the data (e.g., defining the abundant signals) is not desired for routine analysis, we investigated two alternatives: normalization on the spectra noise level or on the median of signal intensities in the spectrum. Normalization on the median and the noise level was found to be significantly more robust against artifact generation compared to normalization on the TIC. Therefore, we propose to include these normalization methods in the standard "toolbox" of MALDI imaging for reliable results under conditions of automation.
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Vela-Chávez, T. ; Adam, P. ; Kremer, M. ; Bink, K. ; Bacon, C.M. ; Menon, G. ; Ferry, J.A. ; Fend, F. ; Jaffe, E.S. ; Quintanilla-Martinez, L.
Leuk. Lymphoma 52, 458-466 (2011)
The aims of this study were to analyze the incidence and morphology of cyclin D1+ DLBCL and cases of Richter transformation (RT), and to elucidate possible molecular mechanisms of cyclin D1 overexpression. Seventy-two cases of de novo DLBCL and 12 cases of RT were included in this study. Cyclin D1 positivity was found in 10/66 (15%) cases of unselected de novo DLBCL and in 2/11 (18%) cases of RT. Seven independently identified cases of cyclin D1+ DLBCL, including one RT, were added to the study. Centroblastic morphology was found in 17/19 (89%) cases of cyclin D1+, most with a post-germinal center phenotype (CD10-, BCL6+, MUM1+). No alterations in the CCND1 gene indicative for a translocation t(11;14) were identified by FISH. Analysis of the MYC locus yielded gene copy alterations in five cases and no disruption of the gene locus in any case, suggesting an alternative mechanism of cyclin D1 deregulation.
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Berg, D. ; Langer, R. ; Tran, K. ; Walch, A.K. ; Schuster, T. ; Bronger, H. ; Becker, K.F.
Appl. Immunohistochem. 19, 300-305 (2011)
Currently, core biopsies are routinely used for diagnosis of breast cancer and they are often the only sample for providing prognostic and predictive markers before treatment. However, biopsies may not accurately reflect protein expression profiles from the whole tumor. In the last few years, reverse phase protein arrays (RPPA) have become a very promising tool for biomarker profiling allowing quick, precise, and simultaneous analysis of many components of a protein network. After extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) tissues, we compared human epidermal growth factor receptor 2 (HER2), estrogen receptor (ERα), and progesterone receptor (PGR) expression levels in a series of 35 FFPE breast cancer surgical specimens and their corresponding core biopsies using RPPA. We found a high concordance between protein expression in core biopsies and surgical specimens with concordance and κ-values of 91.4% and κ=0.677 for HER2; 80% and κ=0.587 for ERα; and 82.8% and κ=0.656 for PGR. In this study, we could show that HER2, ERα, and PGR expression can be assessed reliably on core biopsies of FFPE breast cancer tissues using RRPA. These results might facilitate the implementation of RPPA technology in routine clinical settings.
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Eberhardt, C. ; Amann, B. ; Feuchtinger, A. ; Hauck, S.M. ; Deeg, C.A.
Glia 59, 697-707 (2011)
Reactive gliosis is a well-established response to virtually every retinal disease. Autoimmune uveitis, a sight threatening disease, is characterized by recurrent relapses through autoaggressive T-cells. The purpose of this study was to assess retinal Müller glial cell function in equine recurrent uveitis (ERU), a spontaneous disease model resembling the human disease, by investigating membrane proteins implicated in ion and water homeostasis. We found that Kir2.1 was highly expressed in diseased retinas, whereas Kir4.1 was downregulated in comparison to controls. Distribution of Kir2.1 appeared Müller cell associated in controls, whereas staining of cell somata in the inner nuclear layer was observed in uveitis. In contrast to other subunits, Kir4.1 was evenly expressed along equine Müller cells, whereas in ERU, Kir4.1 almost disappeared from Müller cells. Hence, we suggest a different mechanism for potassium buffering in the avascular equine retina and, moreover, an impairment in uveitis. Uveitic retinas showed significantly increased expression of AQP4 as well as a displaced expression from Müller cells in healthy specimens to an intense circular expression pattern in the outer nuclear layer in ERU cases. Most interestingly, we detected the aquaporin family member protein AQP5 to be expressed in Müller cells with strong enrichments in Müller cell secondary processes. This finding indicates that fluid regulation within the equine retina may be achieved by an additional aquaporin. Furthermore, AQP5 was significantly decreased in uveitis. We conclude that the Müller cell response in autoimmune uveitis implies considerable changes in its potassium and water physiology.
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Sibbing, D. ; Pfeufer, A. ; Perisic, T. ; Mannes, A.M. ; Fritz-Wolf, K. ; Unwin, S. ; Sinner, M.F. ; Gieger, C. ; Gloeckner, C.J. ; Wichmann, H.-E. ; Kremmer, E. ; Schäfer, Z. ; Walch, A.K. ; Hinterseer, M. ; Näbauer, M. ; Kääb, S. ; Kastrati, A. ; Schömig, A. ; Meitinger, T. ; Bornkamm, G.W. ; Conrad, M. ; von Beckerath, N.
Eur. Heart J. 32, 1121-1133 (2011)
Aims Cardiac energy requirement is met to a large extent by oxidative phosphorylation in mitochondria that are highly abundant in cardiac myocytes. Human mitochondrial thioredoxin reductase (TXNRD2) is a selenocysteine-containing enzyme essential for mitochondrial oxygen radical scavenging. Cardiac-specific deletion of Txnrd2 in mice results in dilated cardiomyopathy (DCM). The aim of this study was to investigate whether TXNRD2 mutations explain a fraction of monogenic DCM cases. Methods and results Sequencing and subsequent genotyping of TXNRD2 in patients diagnosed with DCM (n = 227) and in DCM-free (n = 683) individuals from the general population sample KORA S4 was performed. The functional impact of observed mutations on Txnrd2 function was tested in mouse fibroblasts. We identified two novel amino acid residue-altering TXNRD2 mutations [175G > A (Ala59Thr) and 1124G > A (Gly375Arg)] in three heterozygous carriers among 227 patients that were not observed in the 683 DCM-free individuals. Both DCM-associated mutations result in amino acid substitutions of highly conserved residues in helices contributing to the flavin-adenine dinucleotide (FAD)-binding domain of TXNRD2. Functional analysis of both mutations in Txnrd2(-/-) mouse fibroblasts revealed that contrasting to wild-type (wt) Txnrd2, neither mutant did restore Txnrd2 function. Mutants even impaired the survival of Txnrd2 wt cells under oxidative stress by a dominant-negative mechanism. Conclusion For the first time, we describe mutations in DCM patients in a gene involved in the regulation of cellular redox state. TXNRD2 mutations may explain a fraction of human DCM disease burden.
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Saarikangas, J. ; Mattila, PK. ; Varjosalo, M. ; Bovellan, M. ; Hakanen, J. ; Calzada-Wack, J. ; Tost, M. ; Jennen, L. ; Rathkolb, B. ; Hans, W. ; Horsch, M. ; Hyvönen, M.E. ; Perälä, N. ; Fuchs, H. ; Gailus-Durner, V. ; Esposito, I. ; Wolf, E. ; Hrabě de Angelis, M. ; Frilander, MJ. ; Savilahti, H. ; Sariola, H. ; Sainio, K. ; Lehtonen, S. ; Taipale, J. ; Salminen, M. ; Lappalainen, P.
J. Cell Sci. 124, 1245-1255 (2011)
MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.
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Lagarrigue, M. ; Becker, M. ; Lavigne, R. ; Deininger, S.O. ; Walch, A.K. ; Aubry, F. ; Suckau, D. ; Pineau, C.
Mol. Cell. Proteomics 10:M110.005991 (2011)
Matrix-assisted laser desorption/ionization (MALDI) molecular imaging technology attracts increasing attention in the field of biomarker discovery. The unambiguous correlation between histopathology and MALDI images is a key feature for success. MALDI imaging mass spectrometry (IMS) at high definition thus calls for technological developments that were established by a number of small steps. This included tissue and matrix preparation steps, dedicated lasers for MALDI imaging, increase of the robustness against cell debris and matrix sublimation, software for precision matching of molecular and microscopic images, and the analysis of MALDI imaging data using multivariate statistical methods. The goal of these developments is to approach single cell resolution with IMS. Currently a performance level of 20 µm image resolution was achieved with an unmodified and commercially available instrument for proteins detected in the 2-16 kDa range. The rat testis was used as a relevant model for validating and optimizing our technological developments. Indeed, testicular anatomy is amongst the most complex found in mammalian bodies. In the present study, we were able to visualize, at 20 µm image resolution level, different stages of germ cell development in testicular seminiferous tubules, to provide a molecular correlate for its well-established stage-specific classification, to identify proteins of interest using a top-down approach and superimpose molecular and immunohistochemistry images.
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Wolff, C. ; Malinowsky, K. ; Berg, D. ; Schragner, K. ; Schuster, T: ; Walch, A.K. ; Bronger, H. ; Höfler, H. ; Becker, K.F.
J. Pathol. 223, 54-63 (2011)
The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.
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Stangl, S. ; Gehrmann, M. ; Dressel, R. ; Alves, F. ; Dullin, C. ; Themelis, G. ; Ntziachristos, V. ; Staeblein, E. ; Walch, A.K. ; Winkelmann, I. ; Multhoff, G.
J. Cell. Mol. Med. 15, 874-887 (2011)
The major stress-inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumors, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumor mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4 degrees C. After a temperature shift to 37 degrees C, the cmHsp70.1-FITC mAb translocates into early endosomes and lysosomes Intraoperative and near-infrared fluorescence (NIRF) imaging revealed an enrichment of Cy5.5-conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype-matched control, in i.p. and s.c. located CT26 tumors, as soon as 30min after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane-bound Hsp70, the fluorescence-labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumor, reaching a maximum after 24h and remained detectable at least up to 96h after a single i.v. injection. The tumor-selective internalization of mAb cmHsp70.1 at the physiological temperature of 37 degrees C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane-positive tumors. The anti-tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody dependent cytotoxicity (ADCC).
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2010
Rauser, S. ; Deininger, S.O. ; Suckau, D. ; Höfler, H. ; Walch, A.K.
Expert Rev. Proteomics 7, 927-941 (2010)
MALDI imaging mass spectrometry ('MALDI imaging') is an increasingly recognized technique for biomarker research. After years of method development in the scientific community, the technique is now increasingly applied in clinical research. In this article, we discuss the use of MALDI imaging in clinical proteomics and put it in context with classical proteomics techniques. We also highlight a number of upcoming challenges for personalized medicine, development of targeted therapies and diagnostic molecular pathology where MALDI imaging could help.
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Balluf, B. ; Herrmann, K. ; Höfler, H. ; Walch, A.K. ; Ebert, M.
Vortrag: Molekulare Bildgebung 2010, 4-6 November 2010, Seeon. (2010)
Ergin, B. ; Meding, S. ; Langer, R. ; Kap, M. ; Viertler, C. ; Schott, C. ; Ferch, U. ; Riegman, P. ; Zatloukal, K. ; Walch, A.K. ; Becker, K.F.
J. Proteome Res. 9, 5188-5196 (2010)
Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.
Wissenschaftlicher Artikel
Scientific Article
Rauser, S. ; Langer, R. ; Tschernitz, S. ; Gais, P. ; Jütting, U. ; Feith, M. ; Höfler, H. ; Walch, A.K.
BMC Cancer 10:608 (2010)
BACKGROUND: The validation of novel prognostic indicators is of greatest interest for the management of esophageal adenocarcinoma (Barrett's cancer), particularly for non-metastasized (stage I-IIA) disease. The prognostic role of tumor infiltrating T-lymphocytes (TILs) in Barrett's cancer has not been reported so far. Here we evaluated the impact of TILs on survival, recurrence, and metastasis in Barrett's cancer, particularly in stage I-IIA patients. METHODS: The levels of the adaptive immune markers CD3, CD8, and CD45RO were analyzed by immunohistochemistry and image analysis in tissue microarrays consisting of tumor tissues of 118 patients with primary resected Barrett's cancer. The findings were correlated with clinicopathological parameters including patient outcome. RESULTS: In multivariate analysis, a low density of intratumoral CD45RO+ immune cells was an independent unfavorable factor for disease-free survival in stages I-IIA patients (P = 0.004, RR = 4.7, 95% CI = 1.6-13.5) as well in the entire cohort (P = 0.048, RR = 2.0, 95% CI = 1.0-4.0). High CD3+ and CD45RO+ levels were associated with prolonged disease-free survival and overall survival as well with low recurrence rates of disease (P = 0.005 and P = 0.018, respectively). In addition, low CD3+ levels were correlated with a higher frequency of lymph node metastasis (P = 0.025). CONCLUSIONS: This study demonstrates that the density of CD45RO+ TILs is an independent prognostic factor in non-metastasized (stage I-IIA) Barrett's cancer patients and indicates an important role for the adaptive immunologic microenvironment. The inclusion of CD45RO+ density may help to improve the management of stage I-IIA Barrett's cancer.
Wissenschaftlicher Artikel
Scientific Article
Hipp, S. ; Berg, D. ; Ergin, B. ; Schuster, T. ; Hapfelmeier, A. ; Walch, A.K. ; Avril, S. ; Schmalfeldt, B. ; Höfler, H. ; Becker, K.F.
Virchows Arch. 457, 705-713 (2010)
Epithelial ovarian cancer is a highly metastatic disease and the leading cause of death among cancer of the female genital tract. Abnormal epidermal growth factor receptor (EGFR) signalling has been shown to be involved in epithelial-mesenchymal transition (EMT), an early step during metastasis. Additionally, over-expression of the E-cadherin repressor Snail, a key regulator of EMT, has previously been found to be associated with unfavourable prognostic features. Thus, the aim of our study was to elucidate the role of EGFR-dependent signalling pathways for Snail expression in ovarian cancer. For this purpose, we analysed 25 formalin-fixed and paraffin-embedded (FFPE) primary tumours and their corresponding metastases for the expression of 25 signalling pathway molecules by reverse phase protein arrays. We found a significant correlation of Snail with EGFR((Tyr1086)) and p38 MAPK((Thr180/Tyr182)) in primary ovarian carcinoma and with EGFR((Tyr1086)) in their corresponding metastasis. Additionally, we showed that high expression levels of Snail in primary tumours combined with high expression levels of the phosphorylated p38 MAPK((Thr180/Tyr182)) in metastasis lead to an increased risk for death in ovarian carcinoma patients. Thus, for future combinatorial cancer therapy, drug combinations that best target the deregulated protein network in each individual patient should be selected.
Wissenschaftlicher Artikel
Scientific Article
Herbert, Z. ; Rauser, S. ; Williams, L. ; Kapan, N. ; Güntner, M: ; Walch, A.K. ; Boyan, G.
J. Morphol. 271, 1509-1526 (2010)
The central complex is a major integrative region within the insect brain with demonstrated roles in spatial orientation, the regulation of locomotor behavior, and sound production. In the hemimetabolous grasshopper, the central complex comprises the protocerebral bridge, central body (CB), ellipsoid body, noduli, and accessory lobes, and this modular organization develops entirely during embryogenesis. From a biochemical perspective, a range of neuroactive substances has been demonstrated in these modules of the adult central complex, but little is known about their developmental expression. In this study, we use matrix-assisted laser desorption/ionization-imaging mass spectrometry on single brain slices to confirm the presence of several peptide families (tachykinin, allatostatin, periviscerokinin/pyrokinin, FLRFamide, and neuropeptide F) in the adult central complex and then use immunohistochemistry and histology to examine their developmental expression, together with that of the indolamin serotonin, and the endogenous messenger nitric oxide (NO; via its synthesizing enzyme). We find that each neuromodulator is expressed according to a unique, stereotypic, pattern within the various modules making up the central complex. Neuropeptides such as tachykinin (55%) and allatostatin (65%), and the NO-synthesizing enzyme diaphorase (70%), are expressed earlier during embryonic development than the biogenic amine serotonin (80%), whereas periviscerokinin-like peptides and FLRFamide-like peptides begin to be expressed only postembryonically. Within the CB, these neuroactive substances are present in tangential projection neurons before they appear in columnar neurons. There is also no colocalization of serotonin-positive and peptide-positive projections up to the third larval instar during development, consistent with the clear dorsoventral layering of the neuropil we observe. Our results provide the first neurochemical fingerprint of the developing central complex in an hemimetabolous insect.
Wissenschaftlicher Artikel
Scientific Article
Becker, K.-F. ; Berg, D. ; Malinowsky, K. ; Wolff, C. ; Ergin, B. ; Meding, S. ; Walch, A.K. ; Höfler, H.
Pathologe 31, (Suppl.2), 263-267 (2010)
Gewebeproben werden seit Jahrzehnten weltweit routinemäßig zur histopathologischen Charakterisierung verwendet, um erkranktes von gesundem Gewebe zu unterscheiden. Während Nukleinsäure-basierte Analysen formalinfixierter und paraffineingebetteter (FFPE-) Gewebeproben schon länger erfolgreich angewendet werden, stehen Untersuchungen auf Proteinebene erst am Anfang (abgesehen von der Immunhistochemie). Es zeichnet sich jedoch ab, dass viele Proteinuntersuchungsmethoden, die an frischen oder gefrorenen Gewebeproben eingesetzt werden, auch an FFPE-Proben angewendet werden können. Hierzu gehören z. B. Western-blot, Protein-Mikroarray, bildgebende Massenspektrometrie (MALDI Imaging) und 2-D-Gelelektrophorese. Diese Ergebnisse sind überraschend, da die Wissenschaftsgemeinde lange der Überzeugung war, dass FFPE-Proben für Proteinanalysen - außer Immunhistochemie - nicht geeignet sind. In diesem Übersichtsbeitrag berichten wir über neueste Entwicklungen auf diesem Gebiet und gehen dabei besonders auf quantitative Proteinbestimmungen und Hochdurchsatztechniken ein, die in Zukunft in den Routineablauf zur Proteinbiomarkerbestimmung integriert werden können.
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Scientific Article
Hauck, S.M. ; Dietter, J. ; Kramer, R.L. ; Hofmaier, F. ; Zipplies, J.K. ; Amann, B. ; Feuchtinger, A. ; Deeg, C.A. ; Ueffing, M.
Mol. Cell. Proteomics 9, 2292-2305 (2010)
Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis.
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Scientific Article
Rüschoff, J. ; Dietel, M. ; Baretton, G. ; Arbogast, S. ; Walch, A.K. ; Monges, G. ; Chenard, M.-P. ; Penault-Llorca, F. ; Nagelmeier, I. ; Schlake, W. ; Höfler, H. ; Kreipe, H.H.
Virchows Arch. 457, 299-307 (2010)
Trastuzumab-based therapy has been shown to confer overall survival benefit in HER2-positive patients with advanced gastric cancer in a large multicentric trial (ToGA study). Subgroup analysis identified adenocarcinomas of the stomach and gastroesophageal (GE) junction with overexpression of HER2 according to immunohistochemistry (IHC) as potential responders. Due to recent approval of trastuzumab for HER2 positive metastatic gastric and GE-junction cancer in Europe (EMEA) HER2 diagnostics is now mandatory with IHC being the primary test followed by fluorescence in situ hybridization (FISH) in IHC2+ cases. However, in order to not miss patients potentially responding to targeted therapy determination of a HER2-positive status for gastric cancer required modification of scoring as had been proposed in a pre-ToGA study. To validate this new HER2 status testing procedure in terms of inter-laboratory and inter-observer consensus for IHC scoring a series of 547 gastric cancer tissue samples on a tissue microarray (TMA) was used. In the first step, 30 representative cores were used to identify specific IHC HER2 scoring issues among eight French and German laboratories, while in the second step the full set of 547 cores was used to determine IHC HER2 intensity and area score concordance between six German pathologists. Specific issues relating to discordance were identified and recommendations formulated which proved to be effective to reliably determine HER2 status in a prospective test series of 447 diagnostic gastric cancer specimens.
Wissenschaftlicher Artikel
Scientific Article
Smida, J. ; Baumhoer, D. ; Rosemann, M. ; Walch, A.K. ; Bielack, S. ; Poremba, C. ; Remberger, K. ; Korsching, E. ; Scheurlen, W. ; Dierkes, C. ; Burdach, S. ; Jundt, G. ; Atkinson, M.J. ; Nathrath, M.
Clin. Cancer Res. 16, 4256-4267 (2010)
PURPOSE: Osteosarcoma, the most common primary malignant tumor of the bone, is characterized by complex karyotypes with numerous structural and numerical alterations. Despite attempts to establish molecular prognostic markers at the time of diagnosis, the most accepted predictive factor remains the histologic evaluation of necrosis after neoadjuvant chemotherapy. The present approach was carried out to search for genome-wide recurrent loss of heterozygosity and copy number variations that could have prognostic and therapeutic impact for osteosarcoma patients. EXPERIMENTAL DESIGN: Pretherapeutic biopsy samples of 45 osteosarcoma patients were analyzed using Affymetrix 10K2 high-density single nucleotide polymorphism arrays. Numerical aberrations and allelic imbalances were correlated with the histologically assessed response to therapy and clinical follow-up. RESULTS: The most frequent genomic alterations included amplifications of chromosome 6p21 (15.6%), 8q24 (15.6%, harboring MYC), and 12q14 (11.1%, harboring CDK4), as well as loss of heterozygosity of 10q21.1 (44.4%). All these aberrations and the total degree of heterozygosity of each tumor were significantly associated with an adverse outcome of patients and were used to define a chromosomal alteration staging system with a superior predictive potential compared with the histologic regression grading. CONCLUSIONS: Structural chromosomal alterations detected by single nucleotide polymorphism analysis provide a simple but robust parameter to anticipate response to chemotherapy. The proposed chromosomal alteration staging system might therefore help to better predict the clinical course of osteosarcoma patients at the time of initial diagnosis and to adapt neoadjuvant treatment in patients resistant to the current protocols.
Wissenschaftlicher Artikel
Scientific Article
Aubele, M. ; Spears, M. ; Ludyga, N. ; Braselmann, H. ; Feuchtinger, A. ; Taylor, K.J. ; Lindner, K. ; Auer, G. ; Stering, K. ; Höfler, H. ; Schmitt, M. ; Bartlett, J.M.S.
Br. J. Cancer 103, 663-667 (2010)
BACKGROUND: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. METHOD AND RESULTS: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6-HER2 protein-protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). CONCLUSION: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2-PTK6 complexes are of prognostic relevance.
Wissenschaftlicher Artikel
Scientific Article
Rauser, S. ; Marquardt, C. ; Balluff, B. ; Deininger, S.O. ; Albers, C. ; Belau, E. ; Hartmer, R. ; Suckau, D. ; Specht, K. ; Ebert, M.P. ; Schmitt, M. ; Aubele, M. ; Höfler, H. ; Walch, A.K.
J. Proteome Res. 9, 1854-1863 (2010)
Clinical laboratory testing for HER2 status in breast cancer tissues is critically important for therapeutic decision making. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-driven analysis of tissue sections. We hypothesized that MALDI-IMS may determine HER2 status directly from breast cancer tissues. Breast cancer tissues (n = 48) predefined for HER2 status were subjected to MALDI-IMS, and protein profiles were obtained through direct analysis of tissue sections. Protein identification was performed by tissue microextraction and fractionation followed by top-down tandem mass spectrometry. A discovery and an independent validation set were used to predict HER2 status by applying proteomic classification algorithms. We found that specific protein/peptide expression changes strongly correlated with the HER2 overexpression. Among these, we identified m/z 8404 as cysteine-rich intestinal protein 1. The proteomic signature was able to accurately define HER2-positive from HER2-negative tissues, achieving high values for sensitivity of 83%, for specificity of 92%, and an overall accuracy of 89%. Our results underscore the potential of MALDI-IMS proteomic algorithms for morphology-driven tissue diagnostics such as HER2 testing and show that MALDI-IMS can reveal biologically significant molecular details from tissues which are not limited to traditional high-abundance proteins.
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Scientific Article
Echtler, K. ; Stark, K. ; Lorenz, M. ; Kerstan, S. ; Walch, A.K. ; Jennen, L. ; Rudelius, M. ; Seidl, S. ; Kremmer, E. ; Emambokus, N.R. ; von Bruehl, M.L. ; Frampton, J. ; Isermann, B. ; Genzel-Boroviczény, O. ; Schreiber, C. ; Mehilli, J. ; Kastrati, A. ; Schwaiger, M. ; Shivdasani, R.A. ; Massberg, S.
J. Nat. Med. 16, 75-82 (2010)
The ductus arteriosus (DA) is a fetal shunt vessel between the pulmonary artery and the aorta that closes promptly after birth. Failure of postnatal DA closure is a major cause of morbidity and mortality particularly in preterm neonates. The events leading to DA closure are incompletely understood. Here we show that platelets have an essential role in DA closure. Using intravital microscopy of neonatal mice, we observed that platelets are recruited to the luminal aspect of the DA during closure. DA closure is impaired in neonates with malfunctioning platelet adhesion or aggregation or with defective platelet biogenesis. Defective DA closure resulted in a left-to-right shunt with increased pulmonary perfusion, pulmonary vascular remodeling and right ventricular hypertrophy. Our findings indicate that platelets are crucial for DA closure by promoting thrombotic sealing of the constricted DA and by supporting luminal remodeling. A retrospective clinical study revealed that thrombocytopenia is an independent predictor for failure of DA closure in preterm human newborns, indicating that platelets are likely to contribute to DA closure in humans.
Wissenschaftlicher Artikel
Scientific Article
Unger, K. ; Wienberg, J. ; Riches, A. ; Hieber, L. ; Walch, A.K. ; Brown, A. ; O'Brien, P.C. ; Briscoe, C. ; Gray, L. ; Rodriguez, E. ; Jackl, G. ; Knijnenburg, J. ; Tallini, G. ; Ferguson-Smith, M. ; Zitzelsberger, H.
Endocr. Relat. Cancer 17, 87-98 (2010)
Chromosomal copy number alterations and chromosomal rearrangements are frequent mutations in human cancer. Unlike copy number alterations, little is known about the role and occurrence of chromosomal rearrangements in breast cancer. This may be due to the fact that chromosome-based breakpoint analysis is widely restricted to cultured cells. In order to identify gene rearrangements in breast cancer we studied the chromosomal breakpoints in radiation-transformed epithelial breast cell lines using a high-resolution array-based approach using 1Mb BAC arrays. The breakpoints were further narrowed down by FISH with clones from the 32k BAC library. The analysis of the cell lines B42-11 and B42-16 revealed rearrangements of chromosomes 7, 8, 10 and 12. We identified the genes Has2, Grid1, Ret, Cpm, Tbx3, Tbx5, Tuba1a, Wnt1 and Arf3 within the breakpoint regions. Quantitative RT-PCR showed a deregulated expression of all of these candidate genes except for Tbx5 and Tbx3. This is the first study demonstrating gene rearrangements and their deregulated mRNA expression in radiation-transformed breast cells. Since the gene rearrangements occurred in the transformed and tumourigenic cell lines only it is likely that these were generated in conjunction with malignant transformation of the epithelial breast cells and therefore might reflect early molecular events in breast carcinogenesis. Initial studies indicate that these gene alterations are also found in sporadic breast cancers.
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Scientific Article
Shyla, A. ; Hölzlwimmer, G. ; Calzada-Wack, J. ; Bink, K. ; Tischenko, O. ; Guilly, M.N. ; Chevillard, S. ; Samson, E. ; Graw, J. ; Atkinson, M.J. ; Pellegata, N.S.
Int. J. Cancer 126, 2362-2372 (2010)
Pheochromocytomas are neoplasias of neural crest origin that arise from the chromaffin cells of the adrenal medulla. Pheochromocytomas arise with complete penetrance in rats homozygous for a germ-line frameshift mutation of Cdkn1b, encoding the cell cycle inhibitor p27KIP1 (MENX syndrome). We performed a genome-wide scan for allelic imbalance comparing 20 rat pheochromocytoma DNAs with normal rat DNA to better understand the pathobiology of the tumors and to correlate the findings with human pheochromocytoma. We identified allelic imbalance (AI) at candidate regions on rat chromosomes 8 and 19. Interestingly, the regions often lost in rat tumors are syntenic to regions involved in human pheochromocytomas. Fluorescence in situ hybridization analysis further validated the AI data. Sdhd and Rassf1a were analyzed in detail as they map to regions of AI on chromosome 8 and their homologues are implicated in human pheochromocytoma: we found no genetic mutations nor decreased expression. We also analyzed additional candidate genes, that is, rat homologues of genes predisposing to human pheochromocytoma and known tumor-suppressor genes, but we found no AI. In contrast, we observed frequent overexpression of Cdkn2a and Cdkn2c, encoding the cell cycle inhibitors p16INK4a and p18INK4c, respectively. The relative small number of allelic changes we found in rat pheochromocytoma might be related to their nonmalignant status and losses at chromosomes 8 and 19 are events that precede malignancy. Because of the high concordance of affected loci between rat and human tumors, studies of the MENX-associated pheochromocytomas should facilitate the identification of novel candidate genes implicated in their human counterpart.
Wissenschaftlicher Artikel
Scientific Article
2009
Laitinen, I. ; Saraste, A. ; Weidl, E. ; Poethko, T. ; Weber, A.W. ; Nekolla, S.G. ; Leppänen, P. ; Yla-Herttuala, S. ; Hölzlwimmer, G. ; Walch, A.K. ; Esposito, I. ; Wester, H.J. ; Knuuti, J. ; Schwaiger, M.
Circ.-Cardiovasc. Imaging 2, 331-338 (2009)
Background-F-18-Galacto-RGD is a positron emission tomography (PET) tracer binding to alpha(v)beta(3) integrin that is expressed by macrophages and endothelial cells in atherosclerotic lesions. Therefore, we evaluated F-18-galacto-RGD for imaging vascular inflammation by studying its uptake into atherosclerotic lesions of hypercholesterolemic mice in comparison to deoxyglucose. Methods and results-Hypercholesterolemic LDLR(-/-)ApoB(100/100) mice on a Western diet and normally fed adult C57BL/6 control mice were injected with F-18-galacto-RGD and H-3-deoxyglucose followed by imaging with a small animal PET/CT scanner. The aorta was dissected 2 hours after tracer injection for biodistribution studies, autoradiography, and histology. Biodistribution of F-18-galacto-RGD was higher in the atherosclerotic than in the normal aorta. Autoradiography demonstrated focal F-18-galacto-RGD uptake in the atherosclerotic plaques when compared with the adjacent normal vessel wall or adventitia. Plaque-to-normal vessel wall ratios were comparable to those of deoxyglucose. Although angiogenesis was not detected, F-18-galacto-RGD uptake was associated with macrophage density and deoxyglucose accumulation in the plaques. Binding to atherosclerotic lesions was efficiently blocked in competition experiments. In vivo imaging visualized F-18-galacto-RGD uptake colocalizing with calcified lesions of the aortic arch as seen in CT angiography. Conclusions- F-18-Galacto-RGD demonstrates specific uptake in atherosclerotic lesions of mouse aorta. In this model, its uptake was associated with macrophage density. F-18-Galacto-RGD is a potential tracer for noninvasive imaging of inflammation in atherosclerotic lesions.
Wissenschaftlicher Artikel
Scientific Article
Rieger, S. ; Senghaas, N. ; Walch, A.K. ; Köster, R.W.
PLoS Biol. 7:e1000240 (2009)
Long distance migration of differentiating granule cells from the cerebellar upper rhombic lip has been reported in many vertebrates. However, the knowledge about the subcellular dynamics and molecular mechanisms regulating directional neuronal migration in vivo is just beginning to emerge. Here we show by time-lapse imaging in live zebrafish (Danio rerio) embryos that cerebellar granule cells migrate in chain-like structures in a homotypic glia-independent manner. Temporal rescue of zebrafish Cadherin-2 mutants reveals a direct role for this adhesion molecule in mediating chain formation and coherent migratory behavior of granule cells. In addition, Cadherin-2 maintains the orientation of cell polarization in direction of migration, whereas in Cadherin-2 mutant granule cells the site of leading edge formation and centrosome positioning is randomized. Thus, the lack of adhesion leads to impaired directional migration with a mispositioning of Cadherin-2 deficient granule cells as a consequence. Furthermore, these cells fail to differentiate properly into mature granule neurons. In vivo imaging of Cadherin-2 localization revealed the dynamics of this adhesion molecule during cell locomotion. Cadherin-2 concentrates transiently at the front of granule cells during the initiation of individual migratory steps by intramembraneous transport. The presence of Cadherin-2 in the leading edge corresponds to the observed centrosome orientation in direction of migration. Our results indicate that Cadherin-2 plays a key role during zebrafish granule cell migration by continuously coordinating cell-cell contacts and cell polarity through the remodeling of adherens junctions. As Cadherin-containing adherens junctions have been shown to be connected via microtubule fibers with the centrosome, our results offer an explanation for the mechanism of leading edge and centrosome positioning during nucleokinetic migration of many vertebrate neuronal populations.
Wissenschaftlicher Artikel
Scientific Article
Hipp, S. ; Walch, A.K. ; Schuster, T. ; Losko, S. ; Laux, H. ; Bolton, T. ; Höfler, H. ; Becker, K.F.
J. Cell. Mol. Med. 13, 3858-3867 (2009)
Reduced E-cadherin expression is associated with tumour progression of many carcinomas, including endometrial cancers. The transcription factor Snail is known as one of the most prominent transcriptional E-cadherin repressors; its regulation in cancer tissues, however, still remains unclear. Here, we report that activation of epidermal growth factor receptor (EGFR) resulted in over-expression of Snail and also identified critical downstream signalling molecules. Stimulation of two endometrial carcinoma cell lines with epidermal growth factor (EGF) lead to an increase of Snail protein expression. In primary human endometrioid endometrial carcinomas Snail protein expression correlated with the activated, phosphorylated form of EGFR (Tyr1086) as revealed by profiling 24 different signalling proteins using protein lysate microarrays. In addition, we observed an inverse correlation between Snail and E-cadherin protein levels in these tumours. Most likely, p38 MAPK, PAK1, AKT, ERK1/2 and GSK-3β are involved in the up-regulation of Snail downstream of EGFR. Snail mRNA expression did not show a correlation with activated EGFR in these tumours. Taken together, profiling of signalling proteins in primary human tissues provided strong evidence that EGFR signalling is involved in Snail protein over-expression.
Wissenschaftlicher Artikel
Scientific Article
Becker, K.F. ; Walch, A.K. ; Ueffing, M.
Virchows Arch. 455, 191-192 (2009)
Proteomics raises high expectations in finding novel and reliable biomarkers for diagnosis, prognosis and therapy prediction. The goal of the 2-day workshop "Protein analysis of tissues-current views and clinical perspectives" was to bring together scientists from multiple areas of protein research interested in tissue analysis.
Wissenschaftlicher Artikel
Scientific Article
Rauser, S. ; Höfler, H. ; Walch, A.K.
Pathologe 30, (Suppl.2), 140-145 (2009)
Die bildgebende Massenspektrometrie ("MALDI Imaging") ist eine neuartige Methode der funktionellen mikroskopischen Bildgebung, welche die Anwendbarkeit der MALDI-TOF- ("matrix-assisted laser desorption/ionization time of flight"-) Massenspektrometrie nun auch bei der Analyse von Gewebeschnitten ermöglicht. Die Methode erlaubt es, Proteine und Peptide, aber auch Wirkstoffe und deren Metabolite in Gewebeschnitten über ihre Massensignale zu lokalisieren. Dazu wird eine Gewebeprobe im Massenspektrometer gescannt und für jeden Messpunkt ein Massenspektrum erzeugt. Die Intensität spezifischer Signale wird von einer Software in Farbsignale umgesetzt. Mithilfe dieser Farbsignale lassen sich Muster erkennen, welche die Verteilung etwa von Proteinen und Peptiden im Gewebe darstellen. Für die praktische Anwendung zeichnen sich derzeit 3 Bereiche ab, die die molekulare Histologie, die Suche nach neuen krankheitsspezifischen Biomarkern sowie die Detektion von Pharmaka und Metaboliten in Geweben umfassen.
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Scientific Article
Lohmann, C. ; Muschaweckh, A. ; Kirschnek, S. ; Jennen, L. ; Wagner, H. ; Häcker, G.
J. Immunol. 182, 4538-4546 (2009)
For the efficient stimulation of T cells by tumor Ag, tumor-derived material has to be presented by dendritic cells (DC). This very likely involves the uptake of dead tumor cells by DC. Cell death in tumors often occurs through apoptosis, but necrotic cell death may also be prevalent. This distinction is relevant because numerous studies have proposed that apoptotic cells have immunosuppressive effects while necrosis may be stimulatory. However, a system has been lacking that would allow the induction of apoptosis or necrosis without side effects by the death stimuli used experimentally. In this study, we present such a system and test its effects on immune cells in vitro. B16 mouse melanoma cells were generated and underwent cell death through the doxycycline-inducible induction of death proteins. In one cell line, the induction of Bim(S), induced rapid apoptosis, in the other line the induction of the FADD death domain induced nonapoptotic/necrotic cell death. Bim(S)-induced apoptosis was associated with the typical morphological and biochemical changes. FADD death domain induced necrosis occurred through a distinct pathway involving RIP1 and the loss of membrane integrity in the absence of apoptotic changes. Apoptotic and necrotic cells were taken up with comparable efficiency by DC. OVA expressed in cells dying by either apoptosis or necrosis was cross-presented to OT-1 T cells and induced their proliferation. These results argue that it is not the form of cell death but its circumstances that decide the question whether cell death leads to a productive T cell response.
Wissenschaftlicher Artikel
Scientific Article
Aubele, M. ; Vidojkovic, S. ; Braselmann, H. ; Ritterswürden, D. ; Auer, G. ; Atkinson, M.J. ; Tapio, S. ; Höfler, H. ; Rauser, S. ; Bartlett, J.M.
Virchows Arch. 455, 117-123 (2009)
In a previous retrospective study, we demonstrated the prognostic value of protein tyrosine kinase 6 (PTK6) protein expression in breast carcinomas. Here, we analyzed PTK6 gene amplification using fluorescence in situ hybridization technique in a cohort of 426 invasive breast carcinomas and compared it with PTK6 expression level as well as with the clinical outcome of patients. Forty-five percent of tumors show increased PTK6 gene copy numbers when compared to normal tissue. Most of these, however, were related to chromosome 20 polysomy (30%), while gene amplification accounted for only 15%. Only "low level" amplification of the PTK6 gene, with up to eight signals per nucleus, was found. The PTK6 cytogenetic status (normal, gene amplification, polysomy 20) was not associated with histopathological parameters or with the protein expression of HER receptors. No statistical association was identified between PTK6 gene status and expression level. Further, the PTK6 gene status does not influence the disease-free survival of patients at a parts per thousand yen240 months. Based on these results, we state that the PTK6 overexpression is not essentially attributed to gene amplification, and the PTK6 protein expression-but not gene status-is of prognostic value in breast carcinomas. PTK6 protein overexpression may result from polysomy 20 in a minority of the tumors. In a marked proportion of tumors, however, the overexpression is likely to be caused by posttranscriptional regulation mechanisms.
Wissenschaftlicher Artikel
Scientific Article
Ruseva, Z. ; Geiger, P.X. ; Hutzler, P. ; Kotzsch, M. ; Luber, B. ; Schmitt, M. ; Gross, E. ; Reuning, U.
Exp. Cell Res. 315, 1759-1771 (2009)
The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin alphavbeta3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin alphavbeta3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with beta1-integrins, also colocalizes with integrin alphavbeta3. Functionally, elevated KAI1 levels drastically increased integrin alphavbeta3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin alphavbeta3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin alphavbeta3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.
Wissenschaftlicher Artikel
Scientific Article
Ruseva, Z. ; Geiger, P.X. ; Hutzler, P. ; Kotzsch, M. ; Luber, B. ; Schmitt, M. ; Gross, E. ; Reuning, U.
Exp. Cell Res. 315, 1759-1771 (2009)
The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin alphavbeta3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin alphavbeta3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with beta1-integrins, also colocalizes with integrin alphavbeta3. Functionally, elevated KAI1 levels drastically increased integrin alphavbeta3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin alphavbeta3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin alphavbeta3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.
Wissenschaftlicher Artikel
Scientific Article
Peters, D.D. ; Lepikhov, K. ; Rodenacker, K. ; Marschall, S. ; Boersma, A. ; Hutzler, P. ; Scherb, H. ; Walter, J. ; Hrabě de Angelis, M.
Mamm. Genome 20, 664-673 (2009)
In vitro fertilization (IVF) and zona pellucida laser microdissection-facilitated IVF (Laser-IVF) are presently routine procedures in human assisted reproduction. The safety of these methods at the epigenetic level is not fully understood. Studies on mouse Laser-IVF embryos provide evidence that the use of Laser-IVF leads to reduced birth rate, indicating a potential harm of this technique for the embryo. Hence, the aim of this study was to examine the difference in DNA methylation pattern between IVF- and Laser-IVF-derived mouse zygotes. We examined two experimental groups of C3HeB/FeJ oocytes: (1) zona-intact and (2) laser-microdissected oocytes that were fertilized in vitro with freshly collected spermatozoa. Zygotes were fixed 5, 8, and 12 h after fertilization, and indirect immunofluorescence staining was studied using an anti-5-methylcytidine (5-MeC) antibody. The fluorescence intensities of paternal and maternal pronuclei were evaluated using the computer-assisted analysis of digital images. In addition, we performed a semiquantitative RT-PCR analysis of the presence of transcripts of three developmental marker genes, Oct4, Dab2, and Dnmt3b, in IVF- and Laser-IVF-derived blastocysts. We observed no significant differences in methylation status of the paternal genome and in the transcripts of the developmental marker genes after IVF and Laser-IVF. In conclusion, epigenetic patterns and early embryonic development are not altered by laser-assisted IVF techniques and another explanation must be sought for the poor implantation rates observed in mice.
Wissenschaftlicher Artikel
Scientific Article
Xing, X. ; Burgermeister, E. ; Geisler, F. ; Einwächter, H. ; Fan, L. ; Hiber, M. ; Rauser, S. ; Walch, A.K. ; Röcken, C. ; Ebeling, M. ; Wright, M.B. ; Schmid, R.M. ; Ebert, M.P.
Hepatology 49, 979-988 (2009)
Farnesoid X receptor (FXR/Fxr) is a bile acid-regulated nuclear receptor that promotes hepatic bile acid metabolism, detoxification, and liver regeneration. However, the adaptive pathways under conditions of bile acid stress are not fully elucidated. We found that wild-type but not Fxr knockout mice on diets enriched with chenodeoxycholic acid (CDCA) increase their liver/body weight ratios by 50% due to hepatocellular hypertrophy. Microarray analysis identified Hex (Hematopoietically expressed homeobox), a central transcription factor in vertebrate embryogenesis and liver development, as a novel CDCA- and Fxr-regulated gene. HEX/Hex was also regulated by FXR/Fxr and CDCA in primary mouse hepatocytes and human HepG2 cells. Comparative genomic analysis identified a conserved inverted repeat-1-like DNA sequence within a 300 base pair enhancer element of intron-1 in the human and mouse HEX/Hex gene. A combination of chromatin immunoprecipitation, electromobility shift assay, and transcriptional reporter assays demonstrated that FXR/Fxr binds to this element and mediates HEX/Hex transcriptional activation. CONCLUSION: HEX/Hex is a novel bile acid-induced FXR/Fxr target gene during adaptation of hepatocytes to chronic bile acid exposure.
Wissenschaftlicher Artikel
Scientific Article
Sölder, E. ; Kremser, C. ; Rohr, I. ; Hutzler, P.J.S. ; Debbage, P.
Histochem. Cell Biol. 131, 537-551 (2009)
Visualising vascular endothelial cell function in individual blood microvessels allows elucidation of molecular interactions at the vascular wall, the first barrier between blood-borne therapeutic agent and its target. Functional analysis in situ requires sub-micrometer spatial resolution and tagged molecules generating contrast in living blood vessels. Light microscopy fulfills these requirements, particularly if fluorescent tags deliver the contrast. However, vascular arborisations in living organs defy morpho-functional analysis, filling tissues with closely meshed three-dimensional networks which are inaccessible to optical imaging. We protocol here successful morpho-functional analysis of microvascular processing in a living organ, the human placental cotyledon. Fluorescence-tagged tracer was positionally fixed by snap-freezing, frozen sections were cut, freeze-dried and heat-fixed. A brief histochemical procedure then labelled all vascular elements in the sections, providing fluorescence contrast in two colour channels. Mosaic monochromatic images acquired in both channels delivered high-resolution maps of centimeter-wide tissue areas. Quantitative analysis of the images’ greyscale histograms defined objectifiable, reproducible thresholds, used to reduce the images to colour-coded wide-area functional maps tracking placental vascular processing of the tagged molecules. Rapid positional fixing of tracer with reduction of images to maps was combined with ultrastructural tracking to elucidate vascular processing at scales of nanometres and seconds.
Wissenschaftlicher Artikel
Scientific Article
Sölder, E. ; Rohr, I. ; Kremser, C. ; Hutzler, P. ; Debbage, P.L.
Eur. J. Obstet. Gynecol. Reprod. Biol. 144, (Suppl.1), S114-S120 (2009)
Functional analysis of material transfers requires precise statement of residence times in each tissue compartment. For the placenta, neither extractive biochemistry, isotope partitioning, nor mass-based quantitative assays provide adequate spatial resolution to allow the necessary precision. Dual-perfusion assays of material transfer in isolated placental cotyledons provide time-series data for two compartments, the maternal and fetal blood, but fail to distinguish the two cellular compartments (syncytiotrophoblast, fetal endothelium) which actively regulate rates of transfer in each direction for essentially every important molecule type. At present, no definitive technology exists for functional analysis of placental transfer functions. The challenge in developing such a technology lies in the exquisitely small and delicate structures involved, which are scaled at cellular and subcellular sizes (between 50 nm and 50 microm). The only available technologies attaining this high spatial resolution are imaging technologies, primarily light and electron microscopy. To achieve the high-quality images necessary, confocal laser scanning microscopy (CLSM) is required, to provide a uniform optical sectioning plane. In turn, this requires relatively high fluorescence intensities. Design of an adequate technology therefore bases on CLSM imaging fluorochrome-tagged tracers. The temporal resolution necessary to analyse placental material transfers is expected to be of the order of a few seconds, so that conventional wet-fixation protocols are too slow. For adequately rapid fixation, snap-freezing is required. As part of this review we report results obtained from an appropriately designed experimental protocol, analysed by CLSM and transmission electron microscopy (TEM). The images acquired were tested for uniformity of illumination and fluorescence emission strength. Relevant data was encoded in the green channel of the trichrome images obtained, and this was thresholded by application of strict quantitative criteria. The thresholding procedure is suitable for automation and produces reproducible, objectifiable results. Thresholded images were subjected to image calculation procedures designed to highlight image elements (pixels) containing (green) fluorescence associated with the tracer protein; all other sources of fluorescence were visualised in the final images only if no green fluorescence was detectable in that pixel. The resulting images were maps, showing the distribution of tracer molecules at a predefined time interval after perfusion of the tracer into the vital (term) cotyledon. Spatial resolution was routinely better than 1 microm and temporal resolution was approximately 5s. At timepoints up to 10 min after intravital application into the fetal vascular circulation, tracer was associated with capillaries in the villous structures, and no tracer was observed in the syncytiotrophoblast. Clear distinction was achieved between the four tissue compartments relevant to placental transfers, thus providing a novel technology capable of generating high-quality data concerning the regulation of transfers of any molecule that can bear a fluorescent tag. The potential applications of this methodology lie in analyses of factors influencing the rates of fetomaternal and maternofetal exchanges (for example, drugs), and of functional responses of the placental regulation to pathophysiological conditions such as hypoxia.
Wissenschaftlicher Artikel
Scientific Article
Roselli, F. ; Hutzler, P. ; Wegerich, Y. ; Livrea, P. ; Almeida, O.F.
PLoS ONE 4:e6011 (2009)
Disruption of the postsynaptic density (PSD), a network of scaffold proteins located in dendritic spines, is thought to be responsible for synaptic dysfunction and loss in early-stage Alzheimer's disease (AD). Extending our previous demonstration that derangement of the PSD by soluble amyloid-beta (Abeta) involves proteasomal degradation of PSD-95, a protein important for ionotropic glutamate receptor trafficking, we now show that Abeta also disrupts two other scaffold proteins, Homer1b and Shank1, that couple PSD-95 with ionotropic and metabotropic glutamate receptors. Treatment of fronto-cortical neurons with soluble Abeta results in rapid (within 1 h) and significant thinning of the PSD, decreased synaptic levels of Homer1b and Shank1, and reduced synaptic mGluR1 levels. We show that de novo protein synthesis is required for the declustering effects of Abeta on Homer1b (but not Shank1) and that, in contrast to PSD-95, Abeta-induced Homer1b and Shank1 cluster disassembly does not depend on proteasome activity. The regulation of Homer1b and Shank1 by Abeta diverges in two other respects: i) whereas the activity of both NMDAR and VDCC is required for Abeta-induced declustering of Homer1b, Abeta-induced declustering of Shank1 only requires NMDAR activity; and ii) whereas the effects of Abeta on Homer1b involve engagement of the PI-3K pathway and calcineurin phosphatase (PP2B) activity, those on Shank1 involve activation of the ERK pathway. In summary, soluble Abeta recruits discrete signalling pathways to rapidly reduce the synaptic localization of major components of the PSD and to regulate the availability of mGluR1 in the synapse.
Wissenschaftlicher Artikel
Scientific Article
Deplazes, J. ; Fuchs, M. ; Rauser, S. ; Genth, H. ; Lengyel, E. ; Busch, R. ; Luber, B.
Hum. Mol. Genet. 18, 3632-3644 (2009)
Recent evidence suggests a close association between extracellular E-cadherin mutation in diffuse-type gastric carcinoma and the acquisition of a migratory phenotype of tumour cells. To characterise the cellular machinery that mediates the gain of motility of tumour cells with mutant E-cadherin, we turned to the small Rho GTPases Rac1 and Rho because they have been implicated in pathological processes including tumour cell migration and invasion. In the present study, we analyse the activity of Rac1 and Rho in relation to E-cadherin harbouring an in-frame deletion of exon 8, and prove for the first time that the mutation reduces the ability of E-cadherin to activate Rac1 and to inhibit Rho. We provide evidence that the lack of Rac1 activation observed in response to mutant E-cadherin influences the downstream signalling of Rac1, as is shown by the decrease in the binding of the Rac1 effector protein IQGAP1 to Rac1-GTP. Moreover, reduced membranous localisation of p120-catenin in mutant E-cadherin-expressing cells provides an explanation for the lack of negative regulation of Rho by mutant E-cadherin. Further, we show by time-lapse laser-scanning microscopy and invasion assay that the enhanced motility and invasion associated with mutant E-cadherin is sensitive to the inhibition of Rac1 and Rho. Together, these findings present evidence that the mutation of E-cadherin influences Rac1 and Rho activation in opposite directions, and that Rac1 and Rho are involved in the establishment of the migratory and invasive phenotype of tumour cells that have an E-cadherin mutation.
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Scientific Article
Schneider, M. ; Förster, H. ; Boersma, A. ; Seiler, A. ; Wehnes, H. ; Sinowatz, F. ; Neumüller, C. ; Deutsch, M.J. ; Walch, A.K. ; Hrabě de Angelis, M. ; Wurst, W. ; Ursini, F. ; Roveri, A. ; Maleszewski, M. ; Maiorino, M. ; Conrad, M.
FASEB J. 23, 3233-3242 (2009)
Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.
Wissenschaftlicher Artikel
Scientific Article
Herrmann, K. ; Walch, A.K. ; Balluff, B. ; Tänzer, M. ; Höfler, H. ; Krause, B.J. ; Schwaiger, M. ; Friess, H. ; Schmid, R.M. ; Ebert, M.P.
Nat. Rev. Gastroenterol. Hepatol. 6, 170-183 (2009)
Despite substantial improvements in the diagnosis and treatment of many gastrointestinal cancers, particularly colorectal cancer, numerous patients are only diagnosed in advanced stages of disease, which can preclude curative treatment. Screening and early diagnosis of high-risk individuals might be the most promising approach to improve prognosis; however, molecular biomarkers for early diagnosis of most gastrointestinal cancers are not yet available. The prognosis of patients with advanced gastrointestinal cancers has improved through the development of multimodal treatments and the introduction of targeted therapies. Nonetheless, not all patients benefit equally from these treatment approaches, and toxicity can be substantial. The ability to predict whether a patient will respond to therapy early in their treatment for gastrointestinal cancer may be of particular value to stratify and individualize patient treatment strategies. Despite improvement in the understanding of cancer pathogenesis and progression at the molecular level, the molecular changes that underlie treatment response and/or drug resistance are still largely unknown. PET is the first technique to show promise in prediction of response to therapy, and has resulted in promising advancements, particularly in esophageal and gastric cancers. Tissue-based and blood-based molecular biomarkers are still subject to validation. Prediction of response to treatment could ultimately lead to an overall improvement in prognosis.
Review
Review
Khasawneh, J. ; Schulz, M.D. ; Walch, A.K. ; Rozman, J. ; Hrabě de Angelis, M. ; Klingenspor, M. ; Buck, A. ; Schwaiger, M. ; Saur, D. ; Schmid, R.M. ; Klöppel, G. ; Sipos, B. ; Greten, F.R. ; Arkan, M.C.
Proc. Natl. Acad. Sci. U.S.A. 106, 3354-3359 (2009)
Obesity is associated with increased risk for developing pancreatic cancer, and it is suggested that insulin resistance provides the missing link. Here we demonstrate that under the context of genetic susceptibility, a high fat diet (HFD) predisposes mice with oncogenic K-ras activation to accelerated pancreatic intraepithelial neoplasm (PanIN) development. Tumor promotion is closely associated with increased inflammation and abrogation of TNFR1 signaling significantly blocks this process underlining a central role for TNF alpha in obesity-mediated enhancement of PanIN lesions. Interestingly, however, despite increased TNF alpha levels, mice remain insulin sensitive. We show that, while aggravating tumor promotion, a HFD exerts dramatic changes in energy metabolism through enhancement of pancreatic exocrine insufficiency, metabolic rates, and expression of genes involved in mitochondrial fatty acid (FA) beta-oxidation that collectively contribute to improved glucose tolerance in these mice. While on one hand these findings provide significant evidence that obesity is linked to tumor promotion in the pancreas, on the other it suggests alterations in inflammatory responses and bioenergetic pathways as the potential underlying cause.
Wissenschaftlicher Artikel
Scientific Article
Wiech, T. ; Nikolopoulos, E. ; Weis, R. ; Langer, R. ; Bartholomé, K. ; Timmer, J. ; Walch, A.K. ; Höfler, H. ; Werner, M.
Lab. Invest. 89, 385-397 (2009)
We performed genome-wide analysis of copy-number changes and loss of heterozygosity (LOH) in Barrett's esophageal adenocarcinoma by single nucleotide polymorphism (SNP) microarrays to identify associated genomic alterations. DNA from 27 esophageal adenocarcinomas and 14 matching normal tissues was subjected to SNP microarrays. The data were analyzed using dChipSNP software. Copy-number changes occurring in at least 25% of the cases and LOH occurring in at least 19% were regarded as relevant changes. As a validation, fluorescence in situ hybridization (FISH) of 8q24.21 (CMYC) and 8p23.1 (SOX7) was performed. Previously described genomic alterations in esophageal adenocarcinomas could be confirmed by SNP microarrays, such as amplification on 8q (CMYC, confirmed by FISH) and 20q13 or deletion/LOH on 3p (FHIT) and 9p (CDKN2A). Moreover, frequent gains were detected on 2p23.3, 7q11.22, 13q31.1, 14q32.31, 17q23.2 and 20q13.2 harboring several novel candidate genes. The highest copy numbers were seen on 8p23.1, the location of SOX7, which could be demonstrated to be involved in amplification by FISH. A nuclear overexpression of the transcription factor SOX7 could be detected by immunohistochemistry in two amplified tumors. Copy-number losses were seen on 18q21.32 and 20p11.21, harboring interesting candidate genes, such as CDH20 and CST4. Finally, a novel LOH region could be identified on 6p in at least 19% of the cases. In conclusion, SNP microarrays are a valuable tool to detect DNA copy-number changes and LOH at a high resolution. Using this technique, we identified several novel genes and DNA regions associated with esophageal adenocarcinoma.
Wissenschaftlicher Artikel
Scientific Article
Reiter, R. ; Gais, P. ; Steuer-Vogt, M.K. ; Boulesteix, A.L. ; Deutschle, T. ; Hampel, R. ; Wagenpfeil, S. ; Rauser, S. ; Walch, A.K. ; Bink, K. ; Jütting, U. ; Neff, F. ; Arnold, W. ; Höfler, H. ; Pickhard, A.
Acta Otolaryngol. 129, 205-213 (2009)
Numerical and structural centrosome abnormalities play a critical role in the tumor progression of in head and neck squamous cell carcinoma (HNSCC) and may provide useful information as a prognostic factor for these patients. Objectives. Centrosome alterations are often linked with aneuploidy, cell transformation, and tumor progress. We investigated centrosome abnormalities in HNSCC and correlated these variables to clinicopathological parameters and clinical follow up data of the patients. Methods. Retrospective analysis of numerical and structural alterations of centrosomes in tumor tissues and corresponding normal epithelium (n=50 and 31, respectively). Immunohistochemistry was performed using an anti-gamma-tubulin antibody. Image acquisition was done by an Orthoplan microscope, centrosomes were segmented interactively, and area as well as mean optical density was measured. Aneuploidy was evaluated by fluorescence in situ hybridization in a subset of cases (n=29). Results. Numerical and structural centrosome abnormalities differed significantly between normal squamous epithelium and tumor cells (both P<0.0001). Especially numerical centrosome abnormalities were significantly associated with T category and tumor stage (both P<0.0001) and the occurrence of distant metastasis (P=0.002 and P=0.019, respectively). Numerical centrosome abnormalities correlated also with disease free survival of the patients (P=0.032) as well as shorter overall survival (P=0.003).
Wissenschaftlicher Artikel
Scientific Article
2008
Ying, S. ; Pettengill, M. ; Latham, E.R. ; Walch, A.K. ; Ojcius, D.M. ; Häcker, G.
J. Infect. Dis. 198, 1536-1544 (2008)
The obligate intracellular development of Chlamydia suggests that the bacteria should be vulnerable to premature host cell apoptosis, but because Chlamydia-infected cells are apoptosis resistant, this has never been able to be tested. We have devised a system to circumvent the apoptotic block imposed by chlamydial infection. When the proapoptotic protein BimS was experimentally induced, epithelial cells underwent apoptosis that was not blocked by chlamydial infection. Apoptosis during the developmental cycle prevented the generation of infectious bacteria and caused transcriptional changes of bacterial genes and loss of intracellular ATP. Intriguingly, although apoptosis resulted in destruction of host cell structures and of the Chlamydia inclusion, and prevented generation of elementary bodies, BimS induction in the presence of a caspase inhibitor allowed differentiation into morphologically normal but noninfectious elementary bodies. These data show that chlamydial infection renders host cells apoptosis resistant at a premitochondrial step and demonstrate the consequences of premature apoptosis for development of the bacteria.
Wissenschaftlicher Artikel
Scientific Article
Schillinger, U. ; Wexel, G. ; Hacker, C. ; Kullmer, M. ; Koch, C. ; Gerg, M. ; Vogt, S. ; Ueblacker, P. ; Tischer, T. ; Hensler, D. ; Wilisch, J. ; Aigner, J. ; Walch, A.K. ; Stemberger, A. ; Plank, C.
Pharm. Res. 25, 2946-2962 (2008)
Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration. METHODS: Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL(R) and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally,chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model. RESULTS: The optimized glue is based on the fibrinogen component of TISSUCOL(R), a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)- DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (>70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group). CONCLUSIONS: COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.
Wissenschaftlicher Artikel
Scientific Article
May, F. ; Matiasek, K. ; Vroemen, M. ; Caspers, C. ; Mrva, T. ; Arndt, C. ; Schlenker, B. ; Gais, P. ; Brill, T. ; Buchner, A. ; Blesch, A. ; Hartung, R. ; Stief, C. ; Gansbacher, B. ; Weidner, N.
Eur. Urol. 54, 1179-1187 (2008)
Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regenerative capacity. The present study evaluates whether the transplantation of GDNF-overexpressing Schwann cells may enhance regeneration of bilaterally transected erectile nerves in rats. METHODS: Silicon tubes seeded with either GDNF-overexpressing or GFP-expressing Schwann cells were implanted into the gaps between transected cavernous nerve endings. Six (10 study nerves) or 12 wk (20 study nerves) postoperatively, erectile function was evaluated by relaparotomy, electrical nerve stimulation, and intracavernous pressure recording, followed by ultrastructural evaluation of reconstructed nerves employing bright-field and electron microscopy. Additional animals were either sham-operated (positive control; 20 study nerves) or received bilateral nerve transection without nerve reconstruction (negative control; 20 study nerves). RESULTS: The combination of GDNF delivery and Schwann cell application promoted an intact erectile response in 90% (9 of 10) of grafted nerves after 6 wk and in 95% (19 of 20) after 12 wk, versus 50% (5 of 10) and 80% (16 of 20) of GFP-expressing Schwann cell grafts (p=0.02). The functional recovery was paralleled by enhanced axonal regeneration in GDNF-overexpressing Schwann cell grafts, as indicated by larger cross-sectional areas and a significantly higher percentage of neural tissue compared with GFP-transduced controls. CONCLUSIONS: These findings demonstrate that the time required to elicit functional recovery of erectile nerves can be reduced by local delivery of GDNF. In terms of clinical application, this enhanced nerve repair might be critical for timely reinnervation of the corpus cavernosum as a prerequisite for functional recovery in men.
Wissenschaftlicher Artikel
Scientific Article
Riener, M.O. ; Nikolopoulos, E. ; Herr, A. ; Wild, P.J. ; Hausmann, M. ; Wiech, T. ; Orlowska-Volk, M. ; Lassmann, S. ; Walch, A.K. ; Werner, M.
Hum. Pathol. 39, 1621-1629 (2008)
Tubular breast carcinoma is a highly differentiated carcinoma with an excellent prognosis. Distinct genetic alterations in tubular breast carcinoma cells have been described, especially broad genetic losses on the q-arm of chromosome 16. These are more common in lobular breast carcinoma and low-grade ductal carcinoma in situ than in ductal breast carcinoma and high-grade ductal carcinoma in situ. To further delineate the molecular changes involved in tubular breast carcinoma more precisely, we examined 23 formalin-fixed and paraffin wax-embedded tissue samples (21 of tubular breast carcinoma and 2 of nonneoplastic breast epithelium) by microarray-based comparative genomic hybridization focusing on 287 genomic target clones of oncogenes and tumor suppressor genes. The results obtained from all nonneoplastic tissue samples of breast epithelium indicate no DNA copy number changes. In the tubular breast carcinoma samples, the highest frequencies for DNA sequence copy number losses were detected for CDH13 (in 86% of the samples) and MSH2, KCNK12 (in 52% of the samples). The highest frequencies of DNA sequence copy number gains were detected for HRAS and D13S319XYZ (each in 62% of the samples). Using principal component analysis, 3 subgroups of tubular breast carcinomas showing relative genetic changes were identified. For validation, the most frequent DNA copy number loss for CDH13 (18/21) was confirmed using fluorescence in situ hybridization in 4 of 5 tubular breast carcinomas analyzed. The newly identified genes with considerable copy number changes may include so far unknown candidate genes for the development and progression of tubular breast carcinoma, such as CDH13. The study provides the starting point for further delineating their detailed influence on the pathogenesis of tubular breast carcinoma.
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Scientific Article
Winkelheide, U. ; Engelhard, K. ; Kaeppel, B. ; Winkler, J. ; Hutzler, P. ; Werner, C. ; Kochs, E.
Neurocrit. Care 9, 387-393 (2008)
This study compares the effect of mild and severe cerebral ischemia on neuronal damage and neurogenesis. METHODS: Sixteen Sprague-Dawley rats, anesthetized with 0.8 vol% halothane in O(2)/air, were subjected to forebrain ischemia by bilateral common carotid artery occlusion plus hemorrhagic hypotension (mean arterial blood pressure = 40 mmHg) for 8 (mild) or 13 (severe) min. Four non-ischemic animals were investigated as naïve controls. Bromodeoxyuridine (50 mg/kg), a marker of new cells, was administrated for seven consecutive postischemic days. After 28 days, animals were perfused with 4% paraformaldehyde and the brains were sliced. Histopathological damage of the hippocampus and the volume of the dentate gyrus were assessed by HE-staining. With immunohistochemistry BrdU-positve cells were detected in the dentate gyrus. The amount of new generated neurons was identified by double-immunofluorescence-staining of BrdU and neuronal marker (NeuN). RESULTS: In the CA-1 region of the hippocampus, mild ischemia induced damage up to 10% (HE-index 0.8 +/- 1.2) and severe ischemia up to 50% (HE-index 2.1 +/- 1.4). There was no histopathological damage in naïve control animals. The amount of new neurons was increased by 250% after mild insult and by 160% after severe insult compared to the naïve control animals. CONCLUSIONS: These data indicate that histopathological damage depends on the severity of the ischemic insult and that forebrain ischemia activates generation of new neurons. A mild ischemic challenge appears to be a more potent neurogenic stimulus than severe ischemia. The new neurons survive at least 28 days. This may relate to delayed histopathological and functional recovery after cerebral ischemia.
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Schmidt, R. ; Bültmann, A. ; Fischel, S. ; Gillitzer, A. ; Cullen, P. ; Walch, A.K. ; Jost, P. ; Ungerer, M. ; Tolley, N.D. ; Lindemann, S. ; Gawaz, M. ; Schömig, A. ; May, A.E.
Circ. Res. 102, 302-309 (2008)
In atherosclerosis, circulating platelets interact with endothelial cells and monocytes, leading to cell activation and enhanced recruitment of leukocytes into the vascular wall. The invasion of monocytes is accompanied by overexpression of matrix metalloproteinases (MMPs), which are thought to promote atherosclerosis and trigger plaque rupture. Following interaction with itself, the extracellular matrix metalloproteinase inducer (EMMPRIN) induces MMP synthesis via a little-known intracellular pathway. Recently, we showed upregulation of EMMPRIN on monocytes during acute myocardial infarction. EMMPRIN also stimulates secretion of MMP-9 by monocytes and of MMP-2 by smooth muscle cells, indicating that it may be an important regulator of MMP activity. Expression of EMMPRIN on platelets has not been described until now. Here, we demonstrate that resting platelets show low surface expression of EMMPRIN, which is upregulated by various platelet stimulators (flow cytometry). EMMPRIN is located in the open canalicular system and in alpha granules of platelets (according to electron microscopy and sucrose gradient ultracentrifugation). Platelet stimulation with recombinant EMMPRIN-Fc induced surface expression of CD40L and P-selectin (according to flow cytometry), suggesting that EMMPRIN-EMMPRIN interaction activates platelets. Coincubation of platelets with monocytes induced EMMPRIN-mediated nuclear factor kappaB activation (according to Western blot) in monocytes with increased MMP-9 (zymography), interleukin-6, and tumor necrosis factor-alpha secretion (according to ELISA) by monocytes. In conclusion, EMMPRIN displays a new platelet receptor that is upregulated on activated platelets. Binding of EMMPRIN to platelets fosters platelet degranulation. Platelet-monocyte interactions via EMMPRIN stimulate nuclear factor kappaB-driven inflammatory pathways in monocytes, such as MMP and cytokine induction. Thus, EMMPRIN may represent a novel target to diminish the burden of protease activity and inflammation in atherosclerosis.
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Aubele, M. ; Walch, A.K. ; Ludyga, N. ; Braselmann, H. ; Atkinson, M.J. ; Luber, B. ; Auer, G. ; Tapio, S. ; Cooke, T. ; Bartlett, J.M.
Br. J. Cancer 99, 1089-1095 (2008)
The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (P
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Wiech, T. ; Nikolopoulos, E. ; Hausmann, M. ; Walch, A.K. ; Werner, M. ; Fisch, P.
Breast J. 14, 487-491 (2008)
We report a case of an invasive ductal breast carcinoma with significant heterogeneity: a HER-2+ tumor component was densely infiltrated by T-cells, whereas the HER2- tumor component, including two axillary lymph node metastases, showed much fewer tumor infiltrating lymphocytes. Array comparative genomic hybridization of dissected tumor cells from both components revealed many shared chromosomal aberrations but also unique alterations of the HER2+ tumor cell population besides HER2 amplification. We found a clonally dominated T-cell receptor rearrangement of the tumor infiltrating lymphocytes in the HER2+, but not in the HER2- tumor component. Thus, in this case HER2 overexpression is associated with a marked infiltration by T-cells suggesting a specific T-cell response against the HER2+ tumor cell population.
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Walch, A.K. ; Rauser, S. ; Deininger, S.O. ; Höfler, H.
Histochem. Cell Biol. 130, 421-434 (2008)
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.
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Bauer, V.L. ; Braselmann, H. ; Henke, M. ; Mattern, D. ; Walch, A.K. ; Unger, K. ; Baudis, M. ; Lassmann, S. ; Huber, R. ; Wienberg, J. ; Werner, M. ; Zitzelsberger, H.
J. Mol. Med. 86, 1353-1365 (2008)
It is well established that genetic alterations may be associated to prognosis in tumor patients. This study investigates chromosomal changes that predict the clinical outcome of head and neck squamous cell carcinoma (HNSCC) and correlate to characteristic clinicopathological parameters. We applied comparative genomic hybridization (CGH) to tissue samples from 117 HNSCC patients scheduled for radiotherapy. Genomic aberrations occurring in more than five patients were studied for impact on locoregional progression (LRP)-free survival. p values were adjusted by the Hochberg-Benjamini procedure and significant aberrations and clinical variables subjected to a stepwise backwards Cox proportional model. Significant alterations were further analyzed by array-CGH and fluorescence in situ hybridization (FISH). In multivariate survival analysis gains on 1q and 16q predict reduced LRP-free survival independently from known prognostic factors. Cluster analysis separated the HNSCC cases into two groups (cluster 1 and 2) that are characterized by significant differences for imbalances in 13 chromosomal regions. Moreover, it became apparent that cluster 1 correlates to nonanemic patients, while cluster 2 represents predominantly anemic cases. Array-CGH pinpoints 16q24.3 to be the region of interest on chromosome 16 which was further verified by FISH analysis where an increased copy number of FANCA, a member of the Fanconi anemia/breast cancer pathway, could be identified. This study demonstrates that chromosomal gains on 1q and 16q as well as chromosomal loss on 18q represent prognostic markers in HNSCC and that these alterations may explain to some extent the dismal course of a subgroup of patients.
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Zischka, H. ; Lichtmannegger, J. ; Jägemann, N. ; Jennen, L. ; Hamöller, D. ; Huber, E. ; Walch, A.K. ; Summer, K.H. ; Göttlicher, M.
In: Posch, A.* [Eds.]: 2D Page: Sample Preparation and Fractionation. Totowa, NJ: Humana Press, 2008. 333-348 (Methods Mol. Biol. ; 424)
This protocol describes the purification of mitochondria from rat liver with the aid of zone electrophoresis in a free flow device (ZE-FFE). Starting from liver homogenate, cell debris and nuclei are removed by low speed centrifugation. A crude mitochondrial fraction is obtained by medium speed centrifugation and is further purified by washing followed by a Nycodenz® gradient centrifugation. Lysosomes and microsomes are located at the upper parts of the gradient, whereas mitochondria are found in the medium part of the gradient. A subsequent purification step with ZE-FFE efficiently removes remaining lysosomes and microsomes and, importantly, damaged mitochondrial structures. The resulting purified mitochondria can be concentrated by centrifugation and used for further experiments. Finally, possible modifications of this protocol with respect to the isolation of pure lysosomes are discussed.
Zischka, H. ; Larochette, N. ; Hoffmann, F. ; Hamöller, D. ; Jägemann, N. ; Lichtmannegger, J. ; Jennen, L. ; Müller-Höcker, J. ; Roggel, F. ; Göttlicher, M. ; Vollmar, A.M. ; Kroemer, G.
Anal. Chem. 80, 5051-5058 (2008)
A pathological increase of the permeability of the mitochondrial membranes may culminate in the irreversible rupture of the mitochondrial outer membrane. Such a permeability transition is lethal because it results in the release of death-inducing molecules from mitochondria and/or metabolic failure. Current methods to assess this outer membrane damage are mostly indirect or scarcely representative of the overall mitochondrial population. Here we present an analytical and preparative approach using free flow electrophoresis to directly distinguish rat liver mitochondria that have undergone the permeability transition from unaffected organelles or from organelles that are damaged to a minor degree. Mitochondrial populations, which considerably differ in outer membrane integrity or cytochrome c content, were separated by this means. We further show that the relative abundance of each population depends on the dose of the permeability transition inducer and the duration of the treatment time. Finally, we have employed this approach to investigate the impairment of mitochondria that were isolated from livers subjected to ischemia/reperfusion damage.
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Unger, K. ; Malisch, E. ; Thomas, G. ; Braselmann, H. ; Walch, A.K. ; Jackl, G. ; Lewis, P. ; Lengfelder, E. ; Bogdanova, T. ; Wienberg, J. ; Zitzelsberger, H.
Oncogene 27, 4592-4602 (2008)
The aim of this study is to investigate additional genetic alterations in papillary thyroid carcinomas (PTCs) with known RET/PTC rearrangements. We applied array-based comparative genomic hybridization (array CGH) to 33 PTC (20 PTC from adults, 13 post-Chernobyl PTC from children) with known RET/PTC status. Principal component analysis and hierarchical cluster analysis identified cases with similar aberration patterns. Significant deviations between tumour-groups were obtained by statistical testing (Fisher's exact test in combination with Benjamini-Hochberg FDR-controlling procedure). FISH analysis on FFPE sections was applied to validate the array CGH data. Deletions were found more frequently in RET/PTC-positive and RET/PTC-negative tumours than amplifications. Specific aberration signatures were identified that discriminated between RET/PTC-positive and RET/PTC-negative cases (aberrations on chromosomes 1p, 3q, 4p, 7p, 9p/q, 10q, 12q, 13q and 21q). In addition, childhood and adult RET/PTC-positive cases differ significantly for a deletion on the distal part of chromosome 1p. There are additional alterations in RET/PTC-positive tumours, which may act as modifiers of RET activation. In contrast, alterations in RET/PTC-negative tumours indicate alternative routes of tumour development. The data presented serve as a starting point for further studies on gene expression and function of genes identified in this study.
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Paschen, S.A. ; Christian, J.G. ; Vier, J. ; Schmidt, F. ; Walch, A.K. ; Ojcius, D.M. ; Häcker, G.
J. Cell Biol. 182, 117-127 (2008)
Chlamydiae replicate in a vacuole within epithelial cells and commonly induce cell damage and a deleterious inflammatory response of unknown molecular pathogenesis. The chlamydial protease-like activity factor (CPAF) translocates from the vacuole to the cytosol, where it cleaves several cellular proteins. CPAF is synthesized as an inactive precursor that is processed and activated during infection. Here, we show that CPAF can be activated in uninfected cells by experimentally induced oligomerization, reminiscent of the activation mode of initiator caspases. CPAF activity induces proteolysis of cellular substrates including two novel targets, cyclin B1 and PARP, and indirectly results in the processing of pro-apoptotic BH3-only proteins. CPAF activation induces striking morphological changes in the cell and, later, cell death. Biochemical and ultrastructural analysis of the cell death pathway identify the mechanism of cell death as nonapoptotic. Active CPAF in uninfected human cells thus mimics many features of chlamydial infection, implicating CPAF as a major factor of chlamydial pathogenicity, Chlamydia-associated cell damage, and inflammation.
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Hipp, S. ; Walch, A.K. ; Schuster, T. ; Höfler, H. ; Becker, K.-F.
Clin. Exp. Metastasis 25, 679-683 (2008)
Over-expression of the zinc finger transcription factor Snail leads to down-regulation of the epithelial proteins E-cadherin and Cytokeratin 18 and to upregulation of the mesenchymal protein Vimentin. The aim of our study was to characterize for the first time Snail protein expression in formalin-fixed primary tumour tissues using protein lysate microarrays and correlate Snail with E-cadherin, Cytokeratin 18, and Vimentin protein abundances. In a first feasibility study, we examined 17 formalin-fixed endometrioid adenocarcinomas by protein lysate microarrays. Snail expression showed a statistical significant inverse correlation with the expression of E-cadherin (P < 0.001). A trend for correlation between Snail and Cytokeratin 18 (P = 0.043) and the tumour grade (P = 0.074) was seen. For Snail and Vimentin no correlation was found (P = 0.384). In conclusion, our results fit to the proposed function of Snail as a transcriptional repressor of E-cadherin and Cytokeratin 18 in primary human carcinomas and demonstrate the usefulness of protein lysate microarrays for the precise determination of proteins involved in epithelial-mesenchymal-transition.
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Hense, B.A. ; Gais, P. ; Jütting, U. ; Scherb, H. ; Rodenacker, K.
J. Plankton Res. 30, 587-606 (2008)
Automated identification and quantification of algae in microscopic images is a tool that allows high taxonomic resolution with reasonable technical efforts. However, in samples containing various non-algal objects, this is still not a satisfactorily solved problem. We show that autofluorescence information improves discrimination of algae from non-algal objects as well as phycoerythrin (PE) containing algae from others. We analyse the stability of the autofluorescence to estimate its constraints. Cold and dark storage of glutaraldehyde fixed samples maintains autofluorescence sufficiently for 3 weeks. Under repeated excitations, chlorophyll a (Chl a) or PE autofluorescence show an exponential decrease followed by an intermediate maximum. A peak also occurs in emission wavelength ranges without chlorophyll and PE fluorescence. The unspecific autofluorescence causing the peaks is at least partly identical with the blue–green fluorescence (BGF) in plant cells. BGF interferes with identification of algae, thus correction of pigment autofluorescence with such unspecific fluorescence allows a more reliable algal discrimination procedure. A classification scheme for discrimination of Chl a and PE-containing algae shows a high performance in a test with natural samples. Integration of fluorescence and bright-field image information provides a powerful tool for phytoplankton analysis in complex samples.
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Fend, F. ; Tzankov, A. ; Bink, C. ; Seidl, S. ; Quintanilla-Martinez, L. ; Kremer, M. ; Dirnhofer, S.
Prog. Histochem. Cytochem. 42, 203-252 (2008)
Histopathological examination of a bone marrow (BM) trephine biopsy is an integral part of the diagnostic work-up of patients with haematological disorders and other diseases which may afflict hematopoiesis. Until recently, the dramatic increase in modern immunological and molecular techniques which have been added to the diagnostic repertoire of clinical haematology has largely bypassed the BM trephine. In recent years, however, many of the technical obstacles preventing application of these techniques to BM biopsies have been surmounted, and immunohistochemistry, fluorescence in situ hybridization and polymerase chain reaction (PCR)-based molecular techniques for examination of DNA and RNA have successfully been applied to conventionally processed BM trephines. This review tries to give an overview of techniques suitable for trephine biopsies, as well as diagnostic and research applications.
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Matiasek, K. ; Gais, P. ; Rodenacker, K. ; Jütting, U. ; Tanck, J.J. ; Schmahl, W.
Anat. Histol. Embryol. 37, 205-213 (2008)
Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.
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Walch, A.K. ; Seidl, S. ; Hermannstädter, C. ; Rauser, S. ; Deplazes, J. ; Langer, R. ; von, Weyhern, C.H. ; Sarbia, M. ; Busch, R. ; Feith, M. ; Gillen, S. ; Höfler, H. ; Luber, B.
Mod. Pathol. 21, 544-552 (2008)
Rho GTPases are a family of major regulators of E-cadherin-mediated cell adhesion that are implicated in the carcinogenic process by deregulated expression of the family members itself or of upstream modulators or downstream effectors. Combined investigation of the Rho GTPase Rac1, the effector protein IQGAP1 and the activator Tiam1 in relation to expression or mutation of E-cadherin in gastric adenocarcinomas has not been reported. The aim of the study was to determine the expression and prognostic significance of Rac1, IQGAP1, Tiam1 and E-cadherin in gastric adenocarcinomas. Gastric carcinomas of 76 patients were investigated immunohistochemically in a tissue microarray study for expression of Rac1, IQGAP1, Tiam1 and E-cadherin. Correlations with clinical and follow-up data were examined. Moderate or strong reactivity for Rac1 was observed in 46% and for Tiam1 in 56% of tumors. Expression of IQGAP1 was present in 59% and of E-cadherin in 87% of tumors. While Rac1 and E-cadherin expression were not related to prognosis, a trend was observed between a lack of IQGAP1 expression (log-rank 0.088) as well as presence of Tiam1 (log-rank 0.097) and favorable prognosis in Kaplan-Meier survival analysis. Expression of Rac1 was positively linked to IQGAP1 expression (P=0.007, r=0.343) and tended to be inversely associated with expression of E-cadherin (P=0.055, r=-0.245). In conclusion, we observed deregulated expression of Rac1, IQGAP1, Tiam1 and E-cadherin in gastric cancer. We present evidence that either upregulation (for Rac1 and IQGAP1) or downregulation (for Tiam1 and E-cadherin) occurs. Rac1 and E-cadherin expression were not related to prognosis, while trends pointing to favorable prognosis of patients with Tiam1 expression and a lack of IQGAP1 expression were observed. These results indicate that the investigated regulators of E-cadherin-mediated cell adhesion play a role in gastric carcinogenesis.
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Villmann, T. ; Schleif, F.M. ; Kostrzewa, M. ; Walch, A.K. ; Hammer, B.
Brief. Bioinform. 9, 129-143 (2008)
In the present contribution we propose two recently developed classification algorithms for the analysis of mass-spectrometric data-the supervised neural gas and the fuzzy-labeled self-organizing map. The algorithms are inherently regularizing, which is recommended, for these spectral data because of its high dimensionality and the sparseness for specific problems. The algorithms are both prototype-based such that the principle of characteristic representants is realized. This leads to an easy interpretation of the generated classifcation model. Further, the fuzzy-labeled self-organizing map is able to process uncertainty in data, and classification results can be obtained as fuzzy decisions. Moreover, this fuzzy classification together with the property of topographic mapping offers the possibility of class similarity detection, which can be used for class visualization. We demonstrate the power of both methods for two exemplary examples: the classification of bacteria (listeria types) and neoplastic and non-neoplastic cell populations in breast cancer tissue sections.
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Bremm, A. ; Walch, A.K. ; Fuchs, M. ; Mages, J. ; Duyster, J. ; Keller, G. ; Hermannstädter, C. ; Becker, K.F. ; Rauser, S. ; Langer, R. ; von, Weyhern, C.H. ; Höfler, H. ; Luber, B.
Cancer Res. 68, 707-714 (2008)
Mutations of the tumor suppressor E-cadherin and overexpression of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) are among the most frequent genetic alterations associated with diffuse-type gastric carcinoma. Accumulating evidence suggests a functional relationship between E-cadherin and EGFR that regulates both proteins. We report that somatic mutation of E-cadherin is associated with increased activation of EGFR followed by enhanced recruitment of the downstream acting signaling components growth factor receptor binding protein 2 and Shc, and activation of Ras. Reduced complex formation of mutant E-cadherin - with an in frame deletion of exon 8 in the extracellular domain resulting in reduced adhesion and increased motility - with EGFR was observed compared with wild-type E-cadherin. We conclude that reduced binding of mutant E-cadherin to EGFR in a multicomponent complex or reduced stability of the complex may enhance EGFR surface motility, thereby facilitating EGFR dimerization and activation. Furthermore, reduced surface localization due to enhanced internalization of mutant E-cadherin compared with the wild-type protein was observed. The internalization of EGFR was decreased in response to epidermal growth factor stimulation in cells expressing mutant E-cadherin, suggesting that mutation of E-cadherin also influences the endocytosis of EGFR. Moreover, we show increased activation of EGFR in gastric carcinoma samples with mutant E-cadherin lacking exons 8 or 9. In summary, we describe activation of EGFR by mutant E-cadherin as a novel mechanism in tumor cells that explains the enhanced motility of tumor cells in the presence of an extracellular mutation of E-cadherin.
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Lisse, T.S. ; Thiele, F. ; Fuchs, H. ; Hans, W. ; Przemeck, G.K.H. ; Abe, K. ; Rathkolb, B. ; Quintanilla-Martinez, L. ; Hölzlwimmer, G. ; Helfrich, M. ; Wolf, E. ; Ralston, S.H. ; Hrabě de Angelis, M.
PLoS Genet. 4:e7 (2008)
Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2) that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1) gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proalpha1(I) chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and -3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.
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Bink, K. ; Haralambieva, E. ; Kremer, M. ; Ott, G. ; Beham-Schmid, C. ; de, Leval, L. ; Peh, S.C. ; Laeng, H.R. ; Jütting, U. ; Hutzler, P. ; Quintanilla-Martinez, L. ; Fend, F.
Haematologica 93, 623-626 (2008)
Primary extramedullary plasmacytoma is an indolent neoplasm that infrequently converts to multiple myeloma. Since cytogenetic data on extramedullary plasmacytoma are lacking, we studied 38 cases of this type of neoplasm by fluorescence in situ hybridization. Fourteen cases (37%) contained IGH breaks, including six with a t(4;14) translocation. No translocations t(11;14), t(14;16), t(8;14), nor breaks involving MALT1, BCL6 or FOXP1 were found. Loss of 13q (40%), as well as chromosomal gains (82%) were common. There was no correlation between chromosomal alterations and clinical features or local relapse. Cytogenetically, extramedullary plasmacytoma and multiple myeloma are closely related. However, the distribution of IGH translocation partners, with the notable absence of t(11;14), is different. Key words: extramedullary plasmacytoma, multiple myeloma, cytogenetics, IGH translocation, fluorescence in situ hybridization.
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Slotta-Huspenina, J. ; Koch, I. ; Richter, M. ; Bink, K. ; Kremer, M. ; Specht, K. ; Krugmann, J. ; Quintanilla-Martinez, L. ; Fend, F.
Leuk. Res. 32, 79-88 (2008)
Multiple myeloma (MM) frequently shows overexpression of cyclin D1, either due to a t(11;14)(q13;q32) translocation, or in association with polysomy 11. The predominant expression of a cyclin D1 mRNA isoform lacking the 3'-untranslated region (Delta3'UTR) is associated with higher total cyclin D1 mRNA levels, increased proliferation and poor prognosis in mantle cell lymphoma, and can be caused by genetic alterations of the 3'UTR region. The role of this cyclin D1 isoform in MM is unknown. We therefore quantified levels of total and Delta3'UTR cyclin D1 mRNA by real-time RT-PCR in cytogenetically characterized cyclin D1+MM primary cases, and cyclin D1+cell lines. Both long and Delta3'UTR cyclin D1 transcripts were expressed in 35/41 MM cases, but none of the samples showed complete loss of the long transcript or genomic alterations of the 3'UTR. Predominance of the Delta3'UTR mRNA was associated with higher cyclin D1 levels in cases with t(11;14), but did not correlate with the proliferation rate, suggesting a different role of this isoform in MM.
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Fuchs, M. ; Hermannstädter, C. ; Hutzler, P. ; Häcker, G. ; Haller, F. ; Höfler, H. ; Luber, B.
Exp. Cell Res. 314, 153-163 (2008)
E-Cadherin-mediated cell-cell adhesion plays a key role in epithelial cell survival and loss of E-cadherin or beta-catenin expression is associated with invasive tumor growth. Somatic E-cadherin mutations have been identified in sporadic diffuse-type gastric carcinoma. Here, we analysed the fate of E-cadherin with an in frame deletion of exon 8 compared to wild-type E-cadherin and the involved signalling events during cisplatin-induced apoptosis. We report that mutant E-cadherin was more readily cleaved during apoptosis than the wild-type form. Also beta-catenin, an important binding partner of E-cadherin, was processed. E-cadherin cleavage resulted in disconnection of the actin cytoskeleton and accumulation of E-cadherin and beta-catenin in the cytoplasm. Inhibitor studies demonstrated that E-cadherin cleavage was caused by a caspase-3-mediated mechanism. We identified the Akt/PKB and the ERK1/2 signalling pathways as important regulators since inhibition resulted in increased E-cadherin cleavage and apoptosis. In summary, we clearly demonstrate that somatic E-cadherin mutations affect apoptosis regulation in that way that they can facilitate the disruption of adherens junctions thereby possibly influencing the response to cisplatin-based chemotherapy. Elucidating the mechanisms that regulate the apoptotic program of tumor cells can contribute to a better understanding of tumor development and potentially be relevant for therapeutic drug design.
Wissenschaftlicher Artikel
Scientific Article
2006
Hahn, U.K. ; Aichler, M. ; Boehm, R. ; Beyer, W.
Vaccine 24, 4595-4597 (2006)
Currently available live spore vaccines against anthrax in animals have many drawbacks, one of which is their presumed inability to induce a long lasting immunity. In the present study we compared the immunological memory after a protein vaccination with DNA vaccinations in sheep. The antigen used was the protective antigen (PA83) of Bacillus anthracis. Sheep were vaccinated three times with either PA83 plus alhydrogel, or with one of four different plasmid DNA formulations, which all encoded either the full-length PA83 or its domain 4. Two pDNA formulations included Vaxfectin adjuvant, the other two were injected in PBS without adjuvant. Initially, the antibody titres of protein vaccinated sheep were significantly higher than the titres of pDNA vaccinated sheep. After 5 months, however, the antibody titres of protein vaccinated sheep had dropped remarkably, while the titres of all four pDNA vaccinated groups were either stable or had increased. Humoral responses of sheep immunised with pDNA formulated with Vaxfectin adjuvant were higher than the responses of the corresponding groups that received pDNA in PBS only.
Wissenschaftlicher Artikel
Scientific Article
Takenaka, S. ; Karg, E.W. ; Kreyling, W.G. ; Lentner, B. ; Möller, W. ; Behnke-Semmler, M. ; Jennen, L. ; Walch, A.K. ; Michalke, B. ; Schramel, P. ; Heyder, J.
Inhal. Toxicol. 18, 733-740 (2006)
The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 mu g/m 3 (4 x 10(6)/cm(3) , 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.
Wissenschaftlicher Artikel
Scientific Article