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The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry.

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BackgroundBreast cancer is the most common malignancy with the highest incidence rates among women worldwide. Triple-negative breast cancer (TNBC) represents the major phenotype of basal-like molecular subtype of breast cancer, characterized by higher incidence in young women and a very poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs playing significant role in the pathogenesis of many cancers including breast cancer. Therefore, miRNAs are also potential prognostic and/or predictive biomarkers in triple-negative breast cancer patients.MethodsThirty-nine TNBC patients with available formalin-fixed paraffin-embedded (FFPE) tissues were enrolled in the study. MiR-34a, miR-34b, and miR-34c were analyzed using qRT-PCR and correlated to clinico-pathological features of TNBC patients.ResultsExpression levels of miR-34b significantly correlate with disease free survival (DFS) (p = 0.0020, log-rank test) and overall survival (OS) (p = 0.0008, log-rank test) of TNBC patients. No other significant associations between miR-34a, miR-34b, and miR-34c with available clinical pathological data were observed.ConclusionsMiR-34b expression negatively correlates with disease free survival and overall survival in TNBC patients. Thus, miR-34b may present a new promising prognostic biomarker in TNBC patients, but independent validations are necessary.

Asporin has been reported as a tumor suppressor in breast cancer, while asporin-activated invasion has been described in gastric cancer. According to our in silico search, high asporin expresion associates with significantly better relapse free survival (RFS) in patients with low-grade tumors but RFS is significantly worse in patients with grade 3 tumors. In line with other studies, we have confirmed asporin expression by RNA scope in situ hybridization in cancer associated fibroblasts. We have also found asporin expression in the Hs578T breast cancer cell line which we confirmed by quantitative RT-PCR and western blotting. From multiple testing, we found that asporin can be downregulated by bone morphogenetic protein 4 while upregulation may be facilited by serum-free cultivation or by three dimensional growth in stiff Alvetex scaffold. Downregulation by shRNA inhibited invasion of Hs578T as well as of CAFs and T47D cells. Invasion of asporin-negative MDA-MB-231 and BT549 breast cancer cells through collagen type I was enhanced by recombinant asporin. Besides other investigations, large scale analysis of aspartic acid repeat polymorphism will be needed for clarification of the asporin dual role in progression of breast cancer.