Large extracellular vesicles regulate endothelial angiogenic potential via paracrine and autocrine signaling.

Angiogenesis, a process associated with tumor growth and development, is often linked to advanced disease and poor clinical outcomes. Tumor cells establish a proangiogenic microenvironment through the release of paracrine signaling mediators, including extracellular vesicles (EVs). EVs have been shown to facilitate intercellular communication and encompass a diverse range of secreted vesicles, including small EVs, which range in size from ∼60 to 100 nm, and large EVs (L-EVs), which are even more diverse and range from 200 nm to >1 μm in size. Despite advancements in anti-angiogenic cancer therapies, such as bevacizumab, late-stage tumors, including advanced melanomas, exhibit mixed clinical responses. In this study, we elucidate a unique role for melanoma-derived L-EVs in promoting bevacizumab-insensitive endothelial angiogenic phenotypes. L-EV-mediated increase in endothelial tube formation is sensitive to the effects of sorafenib, a multikinase inhibitor, but not SU5416, a selective vascular endothelial growth factor (VEGF)-receptor inhibitor. We also demonstrate that melanoma L-EVs contain VEGF as luminal cargo and induce paracrine effects by modulating the endothelial EV secretome. The release from endothelial cells of soluble VEGFs, EVs, and proangiogenic cytokines, such as interleukin-8, macrophage migration inhibitor factor, and plasminogen activator inhibitor-1, drives sustained endothelial tube formation through autocrine signaling. Finally, we show that EV subtypes have distinct effects on the acquisition of angiogenic phenotypes, and their roles vary with tumor cell type. These findings provide new insight into the mechanisms of angiogenic therapy resistance in melanoma and demonstrate the differential functions of EV subtypes in angiogenesis across tumor types.