Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma

Ilaria Laurenzana,1,* Stefania Trino,1,* Daniela Lamorte,1 Marco Girasole,2 Simone Dinarelli,2 Angelo De Stradis,3 Vitina Grieco,4 Maddalena Maietti,5 Antonio Traficante,5 Teodora Statuto,4 Oreste Villani,6 Pellegrino Musto,6 Alessandro Sgambato,7 Luciana De Luca,4,* Antonella Caivano4,* 1Laboratory...

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Autores principales:	Laurenzana I, Trino S, Lamorte D, Girasole M, Dinarelli S, De Stradis A, Grieco V, Maietti M, Traficante A, Statuto T, Villani O, Musto P, Sgambato A, De Luca L, Caivano A
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2021
Materias:	extracellular vesicles biomarkers hematological malignancies nanoparticle tracking analysis flow cytometry digital pcr Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/017a1bc48faf4c3c8c2ab999d672ca00
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id	oai:doaj.org-article:017a1bc48faf4c3c8c2ab999d672ca00
record_format	dspace
institution	DOAJ
collection	DOAJ
language	EN
topic	extracellular vesicles biomarkers hematological malignancies nanoparticle tracking analysis flow cytometry digital pcr Medicine (General) R5-920
spellingShingle	extracellular vesicles biomarkers hematological malignancies nanoparticle tracking analysis flow cytometry digital pcr Medicine (General) R5-920 Laurenzana I Trino S Lamorte D Girasole M Dinarelli S De Stradis A Grieco V Maietti M Traficante A Statuto T Villani O Musto P Sgambato A De Luca L Caivano A Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma
description	Ilaria Laurenzana,1,* Stefania Trino,1,* Daniela Lamorte,1 Marco Girasole,2 Simone Dinarelli,2 Angelo De Stradis,3 Vitina Grieco,4 Maddalena Maietti,5 Antonio Traficante,5 Teodora Statuto,4 Oreste Villani,6 Pellegrino Musto,6 Alessandro Sgambato,7 Luciana De Luca,4,* Antonella Caivano4,* 1Laboratory of Preclinical and Translational Research, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 2Institute for the Study of the Structure of Matter, National Research Council (CNR), Rome, Italy; 3Institute for Sustainable Plant Protection, National Research Council (CNR), Bari, Italy; 4Laboratory of Clinical Research and Advanced Diagnostics, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 5Unit of Clinical Pathology, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 6Hematology and Stem Cell Transplantation Unit, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 7Scientific Direction, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy*These authors contributed equally to this workCorrespondence: Antonella CaivanoLaboratory of Clinical Research and Advanced Diagnostics, IRCCS CROB, Rionero in Vulture, 85028, Potenza, ItalyTel +39 0972 726395Fax +39 0972 726482Email antonella.caivano@crob.itIntroduction: Extracellular vesicles (EVs) are naturally secreted cellular lipid bilayer particles, which carry a selected molecular content. Owing to their systemic availability and their role in tumor pathogenesis, circulating EVs (cEVs) can be a valuable source of new biomarkers useful for tumor diagnosis, prognostication and monitoring. However, a precise approach for isolation and characterization of cEVs as tumor biomarkers, exportable in a clinical setting, has not been conclusively established.Methods: We developed a novel and laboratory-made procedure based on a bench centrifuge step which allows the isolation of serum cEVs suitable for subsequent characterization of their size, amount and phenotype by nanoparticle tracking analysis, microscopy and flow cytometry, and for nucleic acid assessment by digital PCR.Results: Applied to blood from healthy subjects (HSs) and tumor patients, our approach permitted from a small volume of serum (i) the isolation of a great amount of EVs enriched in small vesicles free from protein contaminants; (ii) a suitable and specific cell origin identification of EVs, and (iii) nucleic acid content assessment. In clonal plasma cell malignancy, like multiple myeloma (MM), our approach allowed us to identify specific MM EVs, and to characterize their size, concentration and microRNA content allowing significant discrimination between MM and HSs. Finally, EV associated biomarkers correlated with MM clinical parameters.Conclusion: Overall, our cEV based procedure can play an important role in malignancy biomarker discovery and then in real-time tumor monitoring using minimal invasive samples. From a practical point of view, it is smart (small sample volume), rapid (two hours), easy (no specific expertise required) and requirements are widely available in clinical laboratories.Keywords: extracellular vesicles, biomarkers, hematological malignancies, nanoparticle tracking analysis, flow cytometry, digital PCR
format	article
author	Laurenzana I Trino S Lamorte D Girasole M Dinarelli S De Stradis A Grieco V Maietti M Traficante A Statuto T Villani O Musto P Sgambato A De Luca L Caivano A
author_facet	Laurenzana I Trino S Lamorte D Girasole M Dinarelli S De Stradis A Grieco V Maietti M Traficante A Statuto T Villani O Musto P Sgambato A De Luca L Caivano A
author_sort	Laurenzana I
title	Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma
title_short	Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma
title_full	Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma
title_fullStr	Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma
title_full_unstemmed	Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma
title_sort	analysis of amount, size, protein phenotype and molecular content of circulating extracellular vesicles identifies new biomarkers in multiple myeloma
publisher	Dove Medical Press
publishDate	2021
url	https://doaj.org/article/017a1bc48faf4c3c8c2ab999d672ca00
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spelling	oai:doaj.org-article:017a1bc48faf4c3c8c2ab999d672ca002021-12-02T15:01:02ZAnalysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma1178-2013https://doaj.org/article/017a1bc48faf4c3c8c2ab999d672ca002021-05-01T00:00:00Zhttps://www.dovepress.com/analysis-of-amount-size-protein-phenotype-and-molecular-content-of-cir-peer-reviewed-fulltext-article-IJNhttps://doaj.org/toc/1178-2013Ilaria Laurenzana,1,* Stefania Trino,1,* Daniela Lamorte,1 Marco Girasole,2 Simone Dinarelli,2 Angelo De Stradis,3 Vitina Grieco,4 Maddalena Maietti,5 Antonio Traficante,5 Teodora Statuto,4 Oreste Villani,6 Pellegrino Musto,6 Alessandro Sgambato,7 Luciana De Luca,4,* Antonella Caivano4,* 1Laboratory of Preclinical and Translational Research, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 2Institute for the Study of the Structure of Matter, National Research Council (CNR), Rome, Italy; 3Institute for Sustainable Plant Protection, National Research Council (CNR), Bari, Italy; 4Laboratory of Clinical Research and Advanced Diagnostics, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 5Unit of Clinical Pathology, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 6Hematology and Stem Cell Transplantation Unit, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy; 7Scientific Direction, Centro di Riferimento Oncologico della Basilicata (IRCCS-CROB), Rionero in Vulture, PZ, Italy*These authors contributed equally to this workCorrespondence: Antonella CaivanoLaboratory of Clinical Research and Advanced Diagnostics, IRCCS CROB, Rionero in Vulture, 85028, Potenza, ItalyTel +39 0972 726395Fax +39 0972 726482Email antonella.caivano@crob.itIntroduction: Extracellular vesicles (EVs) are naturally secreted cellular lipid bilayer particles, which carry a selected molecular content. Owing to their systemic availability and their role in tumor pathogenesis, circulating EVs (cEVs) can be a valuable source of new biomarkers useful for tumor diagnosis, prognostication and monitoring. However, a precise approach for isolation and characterization of cEVs as tumor biomarkers, exportable in a clinical setting, has not been conclusively established.Methods: We developed a novel and laboratory-made procedure based on a bench centrifuge step which allows the isolation of serum cEVs suitable for subsequent characterization of their size, amount and phenotype by nanoparticle tracking analysis, microscopy and flow cytometry, and for nucleic acid assessment by digital PCR.Results: Applied to blood from healthy subjects (HSs) and tumor patients, our approach permitted from a small volume of serum (i) the isolation of a great amount of EVs enriched in small vesicles free from protein contaminants; (ii) a suitable and specific cell origin identification of EVs, and (iii) nucleic acid content assessment. In clonal plasma cell malignancy, like multiple myeloma (MM), our approach allowed us to identify specific MM EVs, and to characterize their size, concentration and microRNA content allowing significant discrimination between MM and HSs. Finally, EV associated biomarkers correlated with MM clinical parameters.Conclusion: Overall, our cEV based procedure can play an important role in malignancy biomarker discovery and then in real-time tumor monitoring using minimal invasive samples. From a practical point of view, it is smart (small sample volume), rapid (two hours), easy (no specific expertise required) and requirements are widely available in clinical laboratories.Keywords: extracellular vesicles, biomarkers, hematological malignancies, nanoparticle tracking analysis, flow cytometry, digital PCRLaurenzana ITrino SLamorte DGirasole MDinarelli SDe Stradis AGrieco VMaietti MTraficante AStatuto TVillani OMusto PSgambato ADe Luca LCaivano ADove Medical Pressarticleextracellular vesiclesbiomarkershematological malignanciesnanoparticle tracking analysisflow cytometrydigital pcrMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol Volume 16, Pp 3141-3160 (2021)

Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma

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