Isolation and characterization of extracellular vesicles for clinical applications in cancer - time for...

Extracellular vesicles (EVs) are nanometer sized lipid enclosed particles released by all cell types into the extracellular space and biological fluids in vivo, and into cell culture media in vitro. An important physiological role of EVs is cell-cell communication. EVs interact with, and deliver, their contents to recipient cells in a functional capacity; this makes EVs desirable vehicles for the delivery of therapeutic cargoes. In addition, as EVs contain proteins, lipids, glycans, and nucleic acids that reflect their cell of origin, their potential utility in disease diagnosis and prognostication is of great interest. The number of published studies analyzing EVs and their contents in the pre-clinical and clinical setting is rapidly expanding. However, there is little standardization as to what techniques should be used to isolate, purify and characterize EVs. Here we provide a comprehensive literature review encompassing the use of EVs as diagnostic and prognostic biomarkers in cancer. We also detail their use as therapeutic delivery vehicles to treat cancer in pre-clinical and clinical settings and assess the EV isolation and characterization strategies currently being employed. Our report details diverse isolation strategies which are often dependent upon multiple factors such as biofluid type, sample volume, and desired purity of EVs. As isolation strategies vary greatly between studies, thorough EV characterization would be of great importance. However, to date, EV characterization in pre-clinical and clinical studies is not consistently or routinely adhered to. Standardization of EV characterization so that all studies image EVs, quantitate protein concentration, identify the presence of EV protein markers and contaminants, and measure EV particle size and concentration is suggested. Additionally, the use of RNase, DNase and protease EV membrane protection control experiments is recommended to ensure that the cargo being investigated is truly EV associated. Overall, diverse methodology for EV isolation is advantageous as it can support different sample types and volumes. Nevertheless, EV characterization is crucial and should be performed in a rigorous manor.