Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from bloodResearch in context
Michela Notarangelo,
Chiara Zucal,
Angelika Modelska,
Isabella Pesce,
Giorgina Scarduelli,
Cristina Potrich,
Lorenzo Lunelli,
Cecilia Pederzolli,
Paola Pavan,
Giancarlo la Marca,
Luigi Pasini,
Paola Ulivi,
Himisha Beltran,
Francesca Demichelis,
Alessandro Provenzani,
Alessandro Quattrone,
Vito G. D'Agostino
Affiliations
Michela Notarangelo
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Chiara Zucal
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Angelika Modelska
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Isabella Pesce
Cell Analysis and Separation Core Facility (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Giorgina Scarduelli
Advanced Imaging Core Facility (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Cristina Potrich
Fondazione Bruno Kessler (FBK), Laboratory of Biomolecular Sequence and Structure Analysis for Health, Trento, Via Sommarive 14, Trento 38123, Italy
Lorenzo Lunelli
Fondazione Bruno Kessler (FBK), Laboratory of Biomolecular Sequence and Structure Analysis for Health, Trento, Via Sommarive 14, Trento 38123, Italy
Cecilia Pederzolli
Fondazione Bruno Kessler (FBK), Laboratory of Biomolecular Sequence and Structure Analysis for Health, Trento, Via Sommarive 14, Trento 38123, Italy
Paola Pavan
Immunohematology and Cell Factory Unit, Meyer Children's University Hospital, Viale Pieraccini 24, Florence 50139, Italy
Giancarlo la Marca
Department of Experimental and Clinical Biomedical Sciences, Centro di Eccellenza Denothe, Aou Meyer University of Florence, Viale Pieraccini 6, 50139, Italy
Luigi Pasini
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via Piero Maroncelli 40, Meldola 47014, Italy
Paola Ulivi
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via Piero Maroncelli 40, Meldola 47014, Italy
Himisha Beltran
Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA
Francesca Demichelis
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Alessandro Provenzani
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Alessandro Quattrone
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy
Vito G. D'Agostino
Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy; Corresponding author at: Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy.
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). Keywords: Cancer, Biomarkers, Extracellular vesicles, Liquid biopsy, Nickel, Alpha, Droplet PCR
