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      Extracellular Vesicles: Current Analytical Techniques for Detection and Quantification

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          Abstract

          Since their first observation, understanding the biology of extracellular vesicles (EV) has been an important and challenging field of study. They play a key role in the intercellular communication and are involved in important physiological and pathological functions. Therefore, EV are considered as potential biomarkers for diagnosis, prognosis, and monitoring the response to treatment in some diseases. In addition, due to their properties, EV may be used for therapeutic purposes. In the study of EV, three major points have to be addressed: 1. How to isolate EV from cell culture supernatant/biological fluids, 2. how to detect them, and 3. how to characterize and quantify. In this review, we focus on the last two questions and provide the main analytical techniques up-to-date for detection and profiling of EV. We critically analyze the advantages and disadvantages of each one, aimed to be of relevance for all researchers working on EV biology and their potential applications.

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          Most cited references53

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          Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

          The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality.
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            Exosomal microRNA: a diagnostic marker for lung cancer.

            To date, there is no screening test for lung cancer shown to affect overall mortality. MicroRNAs (miRNAs) are a class of small noncoding RNA genes found to be abnormally expressed in several types of cancer, suggesting a role in the pathogenesis of human cancer. We evaluated the circulating levels of tumor exosomes, exosomal small RNA, and specific exosomal miRNAs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer (AJCC) disease stage to validate it as an acceptable marker for diagnosis and prognosis in patients with adenocarcinoma of the lung. To date, 27 patients with lung adenocarcinoma AJCC stages I-IV and 9 controls, all aged 21-80 years, were enrolled in the study. Small RNA was detected in the circulating exosomes. The mean exosome concentration was 2.85 mg/mL (95% CI, 1.94-3.76) for the lung adenocarcinoma group versus 0.77 mg/mL (95% CI, 0.68-0.86) for the control group (P < .001). The mean miRNA concentration was 158.6 ng/mL (95% CI, 145.7-171.5) for the lung adenocarcinoma group versus 68.1 ng/mL (95% CI, 57.2-78.9) for the control group (P < .001). Comparisons between peripheral circulation miRNA-derived exosomes and miRNA-derived tumors indicated that the miRNA signatures were not significantly different. The significant difference in total exosome and miRNA levels between lung cancer patients and controls, and the similarity between the circulating exosomal miRNA and the tumor-derived miRNA patterns, suggest that circulating exosomal miRNA might be useful as a screening test for lung adenocarcinoma. No correlation between the exosomal miRNA levels and the stage of disease can be made at this point.
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              High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

              Background Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. Methodology/Principal Findings We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. Conclusions/Significance We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.
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                Author and article information

                Journal
                Biomolecules
                Biomolecules
                biomolecules
                Biomolecules
                MDPI
                2218-273X
                28 May 2020
                June 2020
                : 10
                : 6
                : 824
                Affiliations
                [1 ]Department of Physical and Analytical Chemistry, University of Oviedo, 33006 Oviedo, Spain; serranoesther@ 123456uniovi.es (E.S.-P.); oliveiramyriam@ 123456outlook.es (M.O.-R.); moyanoamanda@ 123456uniovi.es (A.M.)
                [2 ]Instituto Universitario de Biotecnología de Asturias, University of Oviedo, 33006 Oviedo, Spain; matosmaria@ 123456uniovi.es (M.M.); gutierrezgemma@ 123456uniovi.es (G.G.)
                [3 ]Department of Chemical and Enviromental Engineering, University of Oviedo, 33006 Oviedo, Spain
                [4 ]Department of Physics & IUTA, University of Oviedo, Campus de Viesques, 33204 Gijón, Spain; salvadormaria@ 123456uniovi.es (M.S.); rivas@ 123456uniovi.es (M.R.)
                Author notes
                [* ]Correspondence: cblanco@ 123456uniovi.es ; Tel.: +34-985106230
                Author information
                https://orcid.org/0000-0002-6980-0554
                https://orcid.org/0000-0002-6865-7397
                https://orcid.org/0000-0002-2186-0574
                https://orcid.org/0000-0002-9776-9013
                Article
                biomolecules-10-00824
                10.3390/biom10060824
                7357140
                32481493
                990073ee-26b4-47e9-a1b4-9ec277b07400
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 29 April 2020
                : 25 May 2020
                Categories
                Review

                extracellular vesicles,characterization,quantification,elisa,lateral flow immunoassay,nanoparticle tracking analysis,asymmetrical-flow field-flow fractionation

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