Search

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.

Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of some selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology.

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells. DOI: http://dx.doi.org/10.7554/eLife.19276.001, eLife digest Human cells release molecules into their surroundings via membrane-bound packets called exosomes. These molecules can then circulate throughout the body and are protected from degradation. Among the cargos carried by exosomes are small molecules of RNA known as microRNAs, which are involved in regulating gene activity. Only a select subset of the hundreds of microRNAs in a human cell end up packaged into exosomes. This suggests that there might be a specific mechanism that sorts those microRNAs that are destined for export. However, few proteins or other factors that might be involved in this sorting process had been identified to date. Shurtleff et al. set out to identify these factors and started by purifying exosomes from human cells grown in the laboratory and looking for microRNAs that were more abundant in the exosomes than the cells. One exosome-specific microRNA, called miR-223, was further studied via a test-tube based system that uses extracts from cells rather than cells themselves. These experiments confirmed that miR-223 is selectively packed into exosomes that formed in the test tube. Using this system, Shurtleff et al. then isolated a protein called Y-box Protein I (or YBX1 for short) that binds to RNA molecules and found that it was required for this selective packaging. YBX1 is known to be a constituent of exosomes released from intact cells and may therefore be required to sort other RNA molecules into exosomes. Future studies will explore how YBX1 recognizes those RNA molecules to be exported from cells via exosomes. Also, because exosomes have been implicated in some diseases such as cancer, it will be important to explore what role exosome-specific microRNAs play in both health and disease. DOI: http://dx.doi.org/10.7554/eLife.19276.002