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Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.


ABSTRACT: BACKGROUND: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. METHODS:We 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. FINDINGS:From plasma of 46 healthy donors, we 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, we 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, we 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, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. INTERPRETATION:We 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. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).

SUBMITTER: Notarangelo M 

PROVIDER: S-EPMC6558028 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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<h4>Background</h4>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.<h4>Methods</h4>We 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 bioma  ...[more]

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