Routes of cell entryEndocytosis is a key mechanism driving EV entry into cells. Mammalian cells depend on a diversity of endocytic mechanisms that occur concurrently, including clathrin-dependent and clathrin–independent mechanisms as well as macropinocytosis. Specialized phagocytic cells are furthermore capable of ingesting other cells and/or particles via phagocytosis. Interestingly, all of these different endocytic mechanisms have been implicated in EV internalization, which is likely a result of the large diversity of EV donor and recipient cell pairs that have been reported, as reviewed in5. The mechanism of internalization may be relevant to the fate of EVs and their cargo. For example, when studying EV-mediated small RNA delivery, a poor correlation between EV/small RNA uptake efficiency and functional small RNA transfer (assessed via target gene knockdown) was observed among different cell types, including phagocytic Kupffer cells75. It is thus tempting to speculate that different EV internalization mechanisms may lead to different functional outcomes: degradation versus functional transfer of EV cargo.Intracellular trafficking and signallingIn general, endocytosis in mammalian cells leads to internalization of cargo from the plasma membrane, and transport from early endosomes to either recycling endosomes through which the cargo is recycled back to the plasma membrane, or to late endosomes and finally lysosomes where it is degraded. EVs seem to follow a similar path, with evidence suggesting that EVs, or at least the dyes or labels that are used to visualize them, eventually end up in lysosomes69,76. Accumulation of EVs in lysosomes may also reflect the final destination of EVs that have signalled at the surface of the recipient cells. While lysosomes are often seen as sites for breaking down biomolecules (and therefore considered non-functional EV destinations), increasing evidence suggests they also function in various cellular processes such as plasma membrane homeostasis, signalling and energy metabolism. Indeed, lysosomal catabolism of EVs by endothelial cells has been suggested to provide these cells with trophic support59. Thus, lysosomes may serve as a site of functionality for EVs, and may not be simply a compartment for their destruction.At the same time, there is overwhelming evidence that EVs are capable of transferring their luminal cargo, including RNA, into the cytosol of recipient cells, which indicates that EVs are capable of escaping the endolysosomal pathway. How EVs induce such endosomal escape is largely unknown, however there is some evidence that this occurs through EVs fusing with late endosomal membranes77,78, potentially triggered by acidification along the endolysosomal axis79,80. Whether there are intrinsic differences in EV subtypes which are destined for lysosomal degradation or cargo release through fusion, or whether EV fate is dictated by the characteristics of the recipient cell remains unknown