Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice.
University of Trento researchers designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility.
From plasma of 46 healthy donors, they systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, the researchers detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, they identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, they revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies.
Rapid purification of EVs by NBI preserving their integrity, dispersity, and stability in solution
At pH values >5 the net negative charge of membranous particles makes them susceptible to electrostatic interactions. Using 1–3 mM of chelating agents, TRPS analyses detected particles in the 500–700 nm range of size, selectively eluted from a functionalized stationary phase in contrast to nucleic acids (A260), proteins (A280), or pre-stained ssRNA (RNA Y) pre-incubated with nickel-agarose beads. The procedural steps highlighting the rapid NBI procedure are depicted.
These researchers propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies.