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Background: Triple Negative Breast Cancers (TNBC) are the most aggressive breast cancers and lead to poor prognoses. This is due to a high resistance to therapies, mainly because of the presence of Cancer Stem Cells (CSCs). Plasticity, a feature of CSCs, is acquired through the Epithelial to Mesenchymal Transition (EMT), a process that has been recently shown to be regulated by a key molecule, CD146. Of interest, CD146 is over-expressed in TNBC. Methods: The MDA-MB-231 TNBC cell line was used as a model to study the role of CD146 and its secreted soluble form (sCD146) in the development and dissemination of TNBC using in vitro and in vivo studies. Results: High expression of CD146 in a majority of MDA-MB-231 cells leads to an increased secretion of sCD146 that up-regulates the expression of EMT and CSC markers on the cells. These effects can be blocked with a specific anti-sCD146 antibody, M2J-1 mAb. M2J-1 mAb was able to reduce tumour development and dissemination in a model of cells xenografted in nude mice and an experimental model of metastasis, respectively, in part through its effects on CSC. Conclusion: We propose that M2J-1 mAb could be used as an additional therapeutic approach to fight TNBC.

Additional file 1. Supplementary methods:Peptides and antibodies. Plasmids, siRNA and cell transfection. Western-blot and Immunoprecipitation assays. ELISA experiments. Immunofluorescence experiments. Flow cytometry experiments. Reverse Transcription-quantitative PCR (RT-qPCR). Cell migration assays. Cell Proliferation assays. Transwell invasion assays. Crispr/Cas9 deletion of genes. Experiments on animals and imaging. Immunohistochemistry. Patient cohort. Statistical analysis. Supplementary figures and tables: Supplementary Table 1: Absence of significant impact of age, KPS and steroid dose on progression-free survival or overall survival. Supplementary Table 2: references and application condition of the different antibodies used in the study. Supplementary Figure 1: Expression of CD146 on various types of cancers and effect on overall survival in patients with GBM. Supplementary Figure 2: Glioblastoma cell lines characterization. Supplementary Figure 3: Concentration of VEGF and sCD146 in the culture media of U87, U373, and U118 glioblastoma cell lines. Supplementary Figure 4: Avastin induces proliferation, sCD146 secretion, and EMT/CSC markers in U373 cells. Supplementary Figure 5: Avastin has no effect on CD146-negative U118 glioblastoma cells. Supplementary Figure 6: sCD146 induces U373 cell proliferation, migration and invasion in-vitro and promotes CSC and EMT markers. Supplementary Figure 7: Effect of sCD146 and VEGF on CD146-negative glioblastoma cells. Supplementary Figure 8: sCD146 binds integrin αvβ3 on U373 cells. Supplementary Figure 9: Soluble CD146 induces EMT in U118 cells transfected with integrin αvβ3. Supplementary Figure 10: Knocking-down integrin αvβ3 inhibits sCD146-induced EMT in U87 cells. Supplementary Figure 11: sCD146 mediated its effects on U87 cells through a signalosome containing CD146, αvβ3, and VEGFR2. Supplementary Figure 12: sCD146 mediates its effects on U373 cells through a signalosome containing CD146, αvβ3, and VEGFR2. Supplementary Figure 13: Validating gene knock out in U87 and U373 cells. Supplementary Figure 14: Integrin αvβ3 associates with membrane CD146 and VEGFR2 on HUVECs and binds sCD146. Supplementary Figure 15: CD146/VEGFR2/integrin αvβ3 signalosome mediates sCD146 and VEGF effects in U87 cells and activates common signaling pathways. Supplementary Figure 16: CD146/VEGFR2/integrin αvβ3 signalosome mediates sCD146 and VEGF effects in U373 cells and activates common signaling pathways. Supplementary Figure 17: CD146, integrin αvβ3, and VEGFR2 are essential for mediating sCD146 and VEGF effects on U87 cells. Supplementary Figure 18: CD146, integrin αvβ3, and VEGFR2 are essential for mediating sCD146 and VEGF effects on U373 cells. Supplementary Figure 19: Humanized anti-sCD146 antibody mucizumab significantly decreases U373 cell proliferation, migration and invasion and hampers CSC and EMT in-vitro. Supplementary Figure 20: Humanized anti-sCD146 mucizumab significantly decreases human sCD146 and human VEGF in two different mouse models of glioblastoma. Supplementary Figure 21: Illustrative summary describing the mechanism of resistance to bevacizumab in CD146-positive glioblastoma cells.

International audience; CD146 is a cell adhesion molecule expressed on endothelial cells, as well as on other cells such as mesenchymal stem cells and Th17 lymphocytes. This protein also exists in a soluble form, whereby it can be detected in biological fluids, including the serum or the cerebrospinal fluid (CSF). Some studies have highlighted the significance of CD146 and its soluble form in angiogenesis and inflammation, having been shown to contribute to the pathogenesis of many inflammatory autoimmune diseases, such as systemic sclerosis, mellitus diabetes, rheumatoid arthritis, inflammatory bowel diseases, and multiple sclerosis. In this review, we will focus on how CD146 and sCD146 contribute to the pathogenesis of the aforementioned autoimmune diseases and discuss the relevance of considering it as a biomarker in these pathologies.