Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (similar to 1% spontaneous rate at 1h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (similar to 30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion. Extracellular vesicles mediate cell-cell communication, however, their capacity to deliver their content within acceptor cells is unclear. Here, the authors develop a quantitative assay and show that release of extracellular vesicle contents requires endosomal acidification and may involve membrane fusion.