Selective isolation of extracellular vesicles from minimally processed human plasma as a translational strategy for liquid biopsies

Abstract

Background: Intercellular communication is mediated by extracellular vesicles (EVs), as they enclose selectively packaged biomolecules that can be horizontally transferred from donor to recipient cells. Because all cells constantly generate and recycle EVs, they provide accurate timed snapshots of individual pathophysiological status. Although EVs can be isolated from any biofluid, blood plasma circulates through the whole body and it potentially transports EVs derived from most organs, making it the biofluid of choice in most studies. Blood collection is easy and minimally invasive, yet reproducible procedures to obtain pure bulk EV samples and specific EV subtypes from blood are still lacking. Here, we addressed central aspects of EV immunoaffinity isolation from simple and complex matrices, such as plasma. Methods/Results: Fluorescent EV spiking allowed us to demonstrate that target EV subpopulations can be efficiently retrieved from plasma, and that their enrichment is dependent not only on complex matrix composition, but also on the EV surface phenotype. Additionally, we found that plasma-derived EVs can be captured and detected using a simple protocol, which sequentially combines isolation and staining of specific surface markers. Finally, we conducted mRNA profiling experiments to prove that distinct EV subpopulations can be captured by directly targeting different surface markers. Furthermore, platelet-derived EVs encapsulated mRNA expression patterns that might be associated to early-stage lung cancer, which demonstrated that each EV subset conferred a differential clinical value, highlighting the advantages of selective isolation.Conclusions: In summary, our EV isolation protocol facilitated the extraction of clinically useful information from plasma. Compatible with common downstream analytics, it is a readily implementable tool that was tailored to provide a truly translational solution in routine clinical workflows, fostering the inclusion of EVs in novel liquid biopsy settings.