TY - JOUR
T1 - Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood
AU - Notarangelo, Michela
AU - Zucal, Chiara
AU - Modelska, Angelika
AU - Pesce, Isabella
AU - Scarduelli, Giorgina
AU - Potrich, Cristina
AU - Lunelli, Lorenzo
AU - Pederzolli, Cecilia
AU - Pavan, Paola
AU - la Marca, Giancarlo
AU - Pasini, Luigi
AU - Ulivi, Paola
AU - Beltran, Himisha
AU - Demichelis, Francesca
AU - Provenzani, Alessandro
AU - Quattrone, Alessandro
AU - D'Agostino, Vito G.
PY - 2019/5
Y1 - 2019/5
N2 - 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).
AB - 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).
KW - Alpha
KW - Biomarkers
KW - Cancer
KW - Droplet PCR
KW - Extracellular vesicles
KW - Liquid biopsy
KW - Nickel
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U2 - 10.1016/j.ebiom.2019.04.039
DO - 10.1016/j.ebiom.2019.04.039
M3 - Article
C2 - 31047861
AN - SCOPUS:85064768769
VL - 43
SP - 114
EP - 126
JO - EBioMedicine
JF - EBioMedicine
SN - 2352-3964
ER -