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. Since blood plasma circulates through the whole body, it is often the biofluid of choice for biomarker detection in EVs. Blood collection is easy and minimally invasive, yet reproducible procedures to obtain pure EV samples from circulating biofluids are still lacking. Here, we addressed central aspects of EV immunoaffinity isolation from simple and complex matrices, such as plasma.

METHODS

Cell-generated EV spike-in models were isolated and purified by size-exclusion chromatography, stained with cellular dyes and characterized by nano flow cytometry. Fluorescently-labelled spike-in EVs emerged as reliable, high-throughput and easily measurable readouts, which were employed to optimize our EV immunoprecipitation strategy and evaluate its performance. Plasma-derived EVs were captured and detected using this straightforward protocol, sequentially combining isolation and staining of specific surface markers, such as CD9 or CD41. Multiplexed digital transcript detection data was generated using the Nanostring nCounter platform and evaluated through a dedicated bioinformatics pipeline.

RESULTS

Beads with covalently-conjugated antibodies on their surface outperformed streptavidin-conjugated beads, coated with biotinylated antibodies, in EV immunoprecipitation. Fluorescent EV spike recovery evidenced 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. Finally, mRNA profiling experiments proved that distinct EV subpopulations can be captured by directly targeting different surface markers. Furthermore, EVs isolated with anti-CD61 beads enclosed mRNA expression patterns that might be associated to early-stage lung cancer, in contrast with EVs captured through CD9, CD63 or CD81. The differential clinical value carried within each distinct EV subset highlights the advantages of selective isolation.

CONCLUSIONS

This EV isolation protocol facilitated the extraction of clinically useful information from plasma. Compatible with common downstream analytics, it is a readily implementable research tool, tailored to provide a truly translational solution in routine clinical workflows, fostering the inclusion of EVs in novel liquid biopsy settings.