A great deal of interest has developed around evidence of a role for or a marker of extracellular vesicles (EVs)/exosomes and metastatic cancer. However, the strength of a functional connection between EVs and cancer has been hampered by inadequate characterization of EVs and a lack of mechanistic details describing the means by which molecular constituents are incorporated into target cells. Here we consider the mechanisms by which EVs may mediate intercellular communication through ligand-receptor interactions or membrane fusion at the surface of or within recipient cells. We highlight common pitfalls in EV purification procedures and describe how multistep methods combined with quantitative evaluation of EV purification are critical for attributing functional effects to EVs. We explain current limitations in our understanding of the functional internalization of EVs and discuss relevant biological and biochemical controls that may be applied to help strengthen the case for a meaningful effect on target cells.


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