Disruption of mitochondrial integrity induced by MCL-1 targeting enhances the pro-angiogenic phenotype of breast cancer-associated fibroblasts
Breast cancer-associated-fibroblasts (bCAFs) consist of two pro-tumoral populations:  inflammatory CAFs (iCAF) releasing pro-inflammatory cytokines, and myofibroblastic CAFs (myCAF) known for their extensive production of extracellular matrix proteins and immunosuppressive features. We have previously shown that targeting the anti-apoptotic protein MCL-1 in primary culture of bCAFs directly derived from human samples reduces their myofibroblastic characteristics linked to actomyosin cytoskeleton disorganization and mitochondrial fragmentation. In this study, we explore the involvement of this protein in phenotypic differentiation and plasticity of bCAFs. Single-cell RNA-sequencing analysis reveals a shift from wound-myCAF to IL-iCAFs phenotype expressing genes involved in inflammation like IL-8, IL-1ß, CXCL1, CXCL3, CCL2 and in angiogenesis like VEGF after MCL-1 gene silencing in bCAFs. In vitro, targeting of MCL-1 in bCAFs induces an increase of VEGF secretion associated with enhanced endothelial cell tubulogenesis. In ovo, using chicken chorioallantoic membrane model, we engrafted breast cancer cells and bCAFs to study vascularization. Our data suggest that a low level of MCL-1 expression in bCAF is associated with greater peritumoral vascular density in a VEGF-dependent manner. Overall design: In the study, we explore the role of the anti-apoptotic protein MCL-1 in primary cultures of cancer-associated fibroblasts obtained from treatment-naive patients with invasive breast carcinoma after surgical resection, by genetically targeting of this protein by CRISPR/Cas9.