Weisser J.,Albert Ludwigs University of Freiburg |
Weisser J.,Austrian Academy of Sciences |
Lai Z.W.,Albert Ludwigs University of Freiburg |
Bronsert P.,Albert Ludwigs University of Freiburg |
And 18 more authors.
BMC Genomics | Year: 2015
Background: Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most abundant resource of archived human specimens in pathology. Such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. In quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness. Results: In the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of FFPE specimens without interference from formalin employed in the FFPE process. Isotopic amine dimethylation of FFPE specimens showed equal labeling efficiency as for cryopreserved specimens. For both FFPE and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. In an initial application, we profiled proteome changes in clear cell renal cell carcinoma (ccRCC) FFPE tissue specimens compared to adjacent non-malignant renal tissue. Our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins. Conclusion: Our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of FFPE specimens and underlines proteome alterations in ccRCC. © 2015 Weißer et al.
Hillebrand L.E.,Albert Ludwigs University of Freiburg |
Hillebrand L.E.,Center for Biological Signalling Studies |
Bengsch F.,Albert Ludwigs University of Freiburg |
Hochrein J.,Albert Ludwigs University of Freiburg |
And 14 more authors.
Oncotarget | Year: 2016
Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The cell population, characterized by the marker profile CD24+CD90+CD45-, showed a high tumorigenicity compared to non-CD24+CD90+CD45- cancer cells in colony formation assays, as well as upon orthotopic transplantation into the mammary fat pad of mice. In addition, these orthotopically grown CD24+CD90+CD45- TICs metastasized to the lungs. The transcriptome of TICs freshly isolated from primary tumors by cell sorting was compared with that of sorted non-CD24+CD90+CD45- cancer cells by RNA-seq. In addition to more established TIC signatures, such as epithelial-to-mesenchymal transition or mitogen signaling, an upregulated gene set comprising several classes of proteolytic enzymes was uncovered in the TICs. Accordingly, TICs showed high intra- and extracellular proteolytic activity. Application of a broad range of protease inhibitors to TICs in a colony formation assay reduced anchorage independent growth and had an impact on colony morphology in 3D cell culture assays. We conclude that CD24+CD90+CD45- cells of the MMTV- PyMT mouse model possess an upregulated proteolytic signature which could very well represent a functional hallmark of metastatic TICs from mammary carcinomas.
Lai Z.W.,Albert Ludwigs University of Freiburg |
Bolm L.,Clinic for Surgery |
Fuellgraf H.,Albert Ludwigs University of Freiburg |
Biniossek M.L.,Albert Ludwigs University of Freiburg |
And 16 more authors.
BMC Cancer | Year: 2015
Background: Ampullary cancer is a relatively rare form of cancer and usually treated by pancreatoduodenectomy, followed by adjuvant therapy. The intestinal subtype is associated with markedly improved prognosis after resection. At present, only few cell lines are available for in vitro studies of ampullary cancer and they have not been collectively characterized. Methods: We characterize five ampullary cancer cell lines by subtype maker expression, epithelial-mesenchymal transition (EMT) features, growth and invasion, drug sensitivity and response to cancer-associated fibroblast conditioned medium (CAF-CM). Results: On the basis of EMT features, subtype marker expression, growth, invasion and drug sensitivity three types of cell lines could be distinguished: mesenchymal-like, pancreatobiliary-like and intestinal-like. Heterogeneous effects from the cell lines in response to CAF-CM, such as different growth rates, induction of EMT markers as well as suppression of intestinal differentiation markers were observed. In addition, proteomic analysis showed a clear difference in intestinal-like cell line from other cell lines. Conclusion: Most of the available AMPAC cell lines seem to reflect a poorly differentiated pancreatobiliary or mesenchymal-like phenotype, which is consistent to their origin. We suggest that the most appropriate cell line model for intestinal-like AMPAC is the SNU869, while others seem to reflect aggressive AMPAC subtypes. © 2016 Lai et al.
Strohmeier O.,Hsg Imit Institute For Mikro Und Informationstechnik |
Strohmeier O.,Albert Ludwigs University of Freiburg |
Lassmann S.,Albert Ludwigs University of Freiburg |
Lassmann S.,Comprehensive Cancer Center Freiburg |
And 12 more authors.
Microchimica Acta | Year: 2014
Point Mutations on the Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified as an important predictive biomarker for response to cancer therapy targeting the epidermal growth factor receptor. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas, and routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs. We report a low-cost, disposable and ready-to-use centrifugal microfluidic cartridge (termed GeneSlice) containing preloaded primers and probes. The GeneSlice cartridge enables the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR. It represents a cost effective alternative to dideoxy-sequencing with a faster time-to-result (~ 2 h versus up to 20 h in case of dd-sequencing). Microfluidic processing of the GeneSlice along with allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler. Intra-chip standard deviation of Cq values on the GeneSlices is negligible (GeneSlice 1: Cq,std.dev. = 0.13; GeneSlice 2: Cq,std.dev = 0.26). In 23 of 24 experiments, the data for genotyping 6 cancer cell lines (n = 4 per cell line) agreed with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n = 3 per patient; one GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrated the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time. © 2013, Springer-Verlag Wien.
Kohler I.,Albert Ludwigs University of Freiburg |
Bronsert P.,Albert Ludwigs University of Freiburg |
Bronsert P.,Comprehensive Cancer Center Freiburg |
Timme S.,Albert Ludwigs University of Freiburg |
And 11 more authors.
Journal of Gastroenterology and Hepatology (Australia) | Year: 2015
Background and Aim: Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive biology and poor prognosis even after resection. Long-term survival is very rare and cannot be reliably predicted. Experimental data suggest an important role of epithelial-mesenchymal transition (EMT) in invasion and metastasis of PDAC. Tumor budding is regarded as the morphological correlate of local invasion and cancer cell dissemination. The aim of this study was to evaluate the biological and prognostic implications of EMT and tumor budding in PDAC of the pancreatic head. Methods: Patients were identified from a prospectively maintained database, and baseline, operative, histopathological, and follow-up data were extracted. Serial tissue slices stained for Pan-Cytokeratin served for analysis of tumor budding, and E-Cadherin, Beta-Catenin, and Vimentin staining for analysis of EMT. Baseline, operative, standard pathology, and immunohistochemical parameters were evaluated for prediction of long-term survival (≥30 months) in uni- and multivariate analysis. Results: Intra- and intertumoral patterns of EMT marker expression and tumor budding provide evidence of partial EMT induction at the tumor-host interface. Lymph node ratio and E-Cadherin expression in tumor buds were independent predictors of long-term survival in multivariate analysis. Conclusions: Detailed immunohistochemical assessment confirms a relationship between EMT and tumor budding at the tumor-host interface. A small group of patients with favorable prognosis can be identified by combined assessment of lymph node ratio and EMT in tumor buds. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.
Klar M.,Boston University |
Michels K.B.,Harvard University |
Michels K.B.,Comprehensive Cancer Center Freiburg |
Michels K.B.,Brigham and Women's Hospital
Journal of Perinatal Medicine | Year: 2014
Background: Despite an increase in the number of cesarean deliveries conducted worldwide, meta-analyses on the long-term effect of cesarean section (CS) on subsequent placental disorders are sparse. Objective: To examine the association between CS and three major types of placental disorders (placental abruption, placenta previa, and placenta accreta with its variants increta/percreta) in subsequent pregnancies. Search strategy: We followed the MOOSE consensus statement for meta-analyses of observational studies and searched the PubMed database for observational studies published between January 1990 and July 2011 for examining the association between CS and placental disorders in subsequent pregnancies, without focusing on the effect of increasing number of CSs. Selection criteria: We included studies which provided adjusted measures of association for multiparous singletonpregnant women with one of the three outcomes and information about prior mode of delivery. Data collection and analysis: Five cohort and 11 case-control studies met the inclusion criteria for this meta-analysis. We combined the results of the included cohort and case-control studies as no significant heterogeneity was found across the studies. Main results: The calculated summary odds ratio was 1.47 (95% confidence interval, CI: 1.44-1.51) for placenta previa, 1.96 (95% CI: 1.41-2.74) for placenta accreta, and 1.38 (95% CI: 1.35-1.41) for placental abruption. Conclusion: In this meta-analysis, cesarean delivery appeared as a consistently reported risk factor for all three major forms of placental disorders in subsequent pregnancies.