Tumoral Hypoxic Extracellular Vesicles Foster a Protective Microenvironment in Triple-Negative Breast Cancer

The highly metastatic triple-negative breast cancer (TNBC) relies on the tumor microenvironment (TME) to maintain phenotypic heterogeneity and progression. Extracellular vesicles from hypoxic TNBC (EVh) have been previously shown to facilitate tumoral invasion; however, their function in the tumor microenvironment remains unclear. We used a novel method to investigate the TME in vitro called multicellular circulating co-culture, to characterize how EVh interferes with tumoral and endothelial cells, fibroblasts, monocytes and macrophages. EVh promoted monocyte differentiation to M2-like macrophages and inhibited macrophage-derived phagocytosis in endothelial and tumoral cells. The protection of endothelial, tumoral and stromal cellular integrity by EVh increased pro-tumoral and pro-angiogenic signaling, collagen matrix synthesis and showed a potential differentiation to cancer-associated fibroblasts. Our findings highlight the critical role of EVh in protecting tumor cells, indicating its cooperation towards a protective TME, which was demonstrated by the multicellular circulating co-culture and conventional co-culture protocols. These findings lead to an adequate system with potential for investigating other tumor-related processes, including circulating tumor cells and metastasis.