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

• DOI: 10.1186/s40364-022-00404-1
• Publication Date: 2022-08-06T23:02:43Z
• Authors: Diogo Fortunato; Stavros Giannoukakos; Ana Giménez-Capitán; Michael Hackenberg; Miguel A. Molina-...; Nataša Zarovni
• Keywords: Extracellular Vesicles; Isolation; Liquid biopsy

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, provide accurate timed snapshots of individual pathophysiological status. Since blood plasma circulates through the whole body, it often biofluid choice for biomarker detection in EVs. Blood collection easy minimally invasive, yet reproducible procedures obtain pure EV samples circulating biofluids are still lacking. Here, we addressed central aspects immunoaffinity isolation simple complex matrices, such plasma. Cell-generated spike-in models were isolated purified size-exclusion chromatography, stained with cellular dyes characterized nano flow cytometry. Fluorescently-labelled EVs emerged reliable, high-throughput easily measurable readouts, which employed optimize our immunoprecipitation strategy evaluate its performance. Plasma-derived captured detected using this straightforward protocol, sequentially combining staining specific surface markers, CD9 or CD41. Multiplexed digital transcript data was generated Nanostring nCounter platform evaluated a dedicated bioinformatics pipeline. Beads covalently-conjugated antibodies on their outperformed streptavidin-conjugated beads, coated biotinylated antibodies, immunoprecipitation. Fluorescent spike recovery evidenced target subpopulations efficiently retrieved plasma, enrichment dependent not only matrix composition, but also phenotype. Finally, mRNA profiling experiments proved distinct directly targeting different markers. Furthermore, anti-CD61 beads enclosed expression patterns might associated early-stage lung cancer, contrast CD9, CD63 CD81. The differential clinical value carried within each subset highlights advantages selective isolation. This protocol facilitated extraction clinically useful information Compatible common downstream analytics, readily implementable research tool, tailored truly translational solution routine workflows, fostering inclusion novel liquid biopsy settings.