Mouse Tumor Tissue‐Derived Extracellular Vesicles Induce Angiogenesis Through VEGF Production From Macrophages

Extracellular vesicles (EVs) are nano-sized, spherical, and lipid bilayered particles secreted by most types of cells. Tumor microenvironment is a complex system and highly regulated by dynamic interaction of cancer cells with stromal cells, such as immune cells and endothelial cells. Angiogenesis, a process of new capillary formation from pre-existing vasculatures, plays a critical role in tumorigenesis and cancer progression by providing oxygen and nutrients to proliferating cancer cells. Since EVs have emerged as important mediators of intercellular communication in pathophysiological circumstances, several studies have reported pro-angiogenic activities of EVs derived from in vitro cultured cancer cells and other cells. However, the angiogenic role of EVs directly isolated from in vivo tumor tissues has not been investigated. Here, we isolated EVs directly from mouse primary tumor tissues with high purity and investigated the angiogenic potential of in vivo tumor tissue-derived EVs (tEVs). The purified tumor tEVs showed EV-like features with nano-sized, spherical, and lipid bilayered structures, and enriched with EV marker proteins such as tetraspanins, while being de-enriched with proteins from Golgi apparatus and nucleus. Interestingly, tumor tEVs promoted extensive neovascularization and numerous macrophage infiltrations in vivo Matrigel plug assay. In addition, the angiogenic properties of tumor tEVs were mediated by the infiltrated macrophages through producing vascular endothelial growth factor (VEGF), a pro-angiogenic molecule. These results suggested that tumor tEVs have potent in vivo angiogenic activity by directly promoting macrophage recruitment and activating infiltrated macrophages to produce VEGF. Our study provides the first direct evidence for a key role of infiltrated macrophage VEGF production in tumor tEV-mediated neovascularization in the tumor microenvironment.