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

The highly metastatic triple-negative breast cancer (TNBC) relies on the tumour microenvironment (TME) to maintain phenotypic heterogeneity and progression. Extracellular vesicles from hypoxic TNBC (EVh) have been previously shown to facilitate tumoural invasion; however, their function in the TME remains unclear. We used a novel method to investigate the TME in vitro called multicellular circulating co-culture, to characterise how EVh interferes with tumoural and endothelial cells, fibroblasts, monocytes and macrophages. EVh promoted monocyte differentiation to M2-like macrophages and inhibited macrophage-derived phagocytosis in endothelial and tumoural cells. The protection of endothelial, tumoural and stromal cellular integrity by EVh increased pro-tumoural and pro-angiogenic signalling, collagen matrix synthesis and showed a potential differentiation to cancer-associated fibroblasts. Our findings highlight the critical role of EVh in protecting tumour 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 tumour-related processes, including circulating tumour cells and metastasis.