{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T02:54:13Z","timestamp":1789008853718,"version":"build-2803163510"},"reference-count":42,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,10,29]],"date-time":"2021-10-29T00:00:00Z","timestamp":1635465600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Extracellular vesicles (EVs), comprising exosomes, microvesicles, and apoptotic bodies, are released by all cells into the extracellular matrix and body fluids, where they play important roles in intercellular communication and matrix remodeling in various pathological conditions. Malignant pleural mesothelioma (MPM) is a primary tumor of mesothelial origin, predominantly related to asbestos exposure. The detection of MPM at an early stage and distinguishing it from benign conditions and metastatic adenocarcinomas (AD) is sometimes challenging. Pleural effusion is often the first available biological material and an ideal source for characterizing diagnostic and prognostic factors. Specific proteins have previously been identified as diagnostic markers in effusion, but it is not currently known whether these are associated with vesicles or released in soluble form. Here, we study and characterize tumor heterogeneity and extracellular vesicle diversity in pleural effusion as diagnostic or prognostic markers for MPM. We analyzed extracellular vesicles and soluble proteins from 27 pleural effusions, which were collected and processed at the department of pathology and cytology at Karolinska University Hospital, representing three different patient groups, MPM (n = 9), benign (n = 6), and AD (n = 12). The vesicles were fractionated into apoptotic bodies, microvesicles, and exosomes by differential centrifugation and characterized by nanoparticle tracking analysis and Western blotting. Multiplex bead-based flow cytometry analysis showed that exosomal markers were expressed differently on EVs present in different fractions. Further characterization of exosomes by a multiplex immunoassay (Luminex) showed that all soluble proteins studied were also present in exosomes, though the ratio of protein concentration present in supernatant versus exosomes varied. The proportion of Angiopoietin-1 present in exosomes was generally higher in benign compared to malignant samples. The corresponding ratios of Mesothelin, Galectin-1, Osteopontin, and VEGF were higher in MPM effusions compared to those in the benign group. These findings demonstrate that relevant diagnostic markers can be recovered from exosomes.<\/jats:p>","DOI":"10.3390\/biom11111606","type":"journal-article","created":{"date-parts":[[2021,11,1]],"date-time":"2021-11-01T11:47:38Z","timestamp":1635767258000},"page":"1606","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Diagnostic and Prognostic Utility of the Extracellular Vesicles Subpopulations Present in Pleural Effusion"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7876-0945","authenticated-orcid":false,"given":"Joman","family":"Javadi","sequence":"first","affiliation":[{"name":"Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9198-0857","authenticated-orcid":false,"given":"Andr\u00e9","family":"G\u00f6rgens","sequence":"additional","affiliation":[{"name":"Division of BCM, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hanna","family":"Vanky","sequence":"additional","affiliation":[{"name":"Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dhanu","family":"Gupta","sequence":"additional","affiliation":[{"name":"Division of BCM, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anders","family":"Hjerpe","sequence":"additional","affiliation":[{"name":"Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samir","family":"EL-Andaloussi","sequence":"additional","affiliation":[{"name":"Division of BCM, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9246-6235","authenticated-orcid":false,"given":"Daniel","family":"Hagey","sequence":"additional","affiliation":[{"name":"Division of BCM, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katalin","family":"Dobra","sequence":"additional","affiliation":[{"name":"Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, 141 52 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Xu, L.L., Yang, Y., Wang, Z., Wang, X.J., Tong, Z.H., and Shi, H.Z. (2018). Malignant pleural mesothelioma: Diagnostic value of medical thoracoscopy and long-term prognostic analysis. BMC Pulm. Med., 18.","DOI":"10.1186\/s12890-018-0619-3"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1177\/1753465816628800","article-title":"Malignant pleural mesothelioma: An update on diagnosis and treatment options","volume":"10","author":"Kondola","year":"2016","journal-title":"Ther. Adv. Respir. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"893","DOI":"10.5858\/arpa.2018-0020-RA","article-title":"Cytopathologic Diagnosis of Epithelioid and Mixed-Type Malignant Mesothelioma: Ten Years of Clinical Experience in Relation to International Guidelines","volume":"142","author":"Hjerpe","year":"2018","journal-title":"Arch. Pathol. Lab. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1097\/MCP.0000000000000593","article-title":"Diagnosis and characterization of malignant effusions through pleural fluid cytological examination","volume":"25","author":"Porcel","year":"2019","journal-title":"Curr. Opin. Pulm. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"419853","DOI":"10.1155\/2014\/419853","article-title":"Diagnostic and prognostic value of soluble syndecan-1 in pleural malignancies","volume":"2014","author":"Mundt","year":"2014","journal-title":"Biomed Res. Int."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1002\/dc.23271","article-title":"Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma: Complementary Statement from the International Mesothelioma Interest Group, Also Endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology","volume":"43","author":"Hjerpe","year":"2015","journal-title":"Diagn. Cytopathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"122","DOI":"10.30699\/ijp.14.2.122","article-title":"The Role of Immunohistochemistry Studies in Distinguishing Malignant Mesothelioma from Metastatic Lung Carcinoma in Malignant Pleural Effusion","volume":"14","author":"Halimi","year":"2019","journal-title":"Iran. J. Pathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2879","DOI":"10.1002\/cam4.2928","article-title":"Clinicopathological significance of microRNA-21 in extracellular vesicles of pleural lavage fluid of lung adenocarcinoma and its functions inducing the mesothelial to mesenchymal transition","volume":"9","author":"Watabe","year":"2020","journal-title":"Cancer Med."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Stok, U., Blokar, E., Lenassi, M., Holcar, M., Frank-Bertoncelj, M., Erman, A., Resnik, N., Sodin-Semrl, S., Cucnik, S., and Pirkmajer, K.P. (2020). Characterization of Plasma-Derived Small Extracellular Vesicles Indicates Ongoing Endothelial and Platelet Activation in Patients with Thrombotic Antiphospholipid Syndrome. Cells, 9.","DOI":"10.3390\/cells9051211"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1016\/j.matbio.2017.10.003","article-title":"Extracellular vesicles are integral and functional components of the extracellular matrix","volume":"75","author":"Rilla","year":"2019","journal-title":"Matrix Biol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Lee, J.S., Hur, J.Y., Kim, I.A., Kim, H.J., Choi, C.M., Lee, J.C., Kim, W.S., and Lee, K.Y. (2018). Liquid biopsy using the supernatant of a pleural effusion for EGFR genotyping in pulmonary adenocarcinoma patients: A comparison between cell-free DNA and extracellular vesicle-derived DNA. BMC Cancer, 18.","DOI":"10.1186\/s12885-018-5138-3"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/nrd3978","article-title":"Extracellular vesicles: Biology and emerging therapeutic opportunities","volume":"12","author":"Samir","year":"2013","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Guo, M., Wu, F., Hu, G., Chen, L., Xu, J., Xu, P., Wang, X., Li, Y., Liu, S., and Zhang, S. (2019). Autologous tumor cell-derived microparticle-based targeted chemotherapy in lung cancer patients with malignant pleural effusion. Sci. Transl. Med., 11.","DOI":"10.1126\/scitranslmed.aat5690"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/s41556-018-0250-9","article-title":"Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication","volume":"21","author":"Mathieu","year":"2019","journal-title":"Nat. Cell Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1083\/jcb.201211138","article-title":"Extracellular vesicles: Exosomes, microvesicles, and friends","volume":"200","author":"Raposo","year":"2013","journal-title":"J. Cell Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1161\/ATVBAHA.117.309795","article-title":"Extracellular Vesicles Secreted by Atherogenic Macrophages Transfer MicroRNA to Inhibit Cell Migration","volume":"38","author":"Nguyen","year":"2018","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1247\/csf.25.317","article-title":"Domain analysis of the tetraspanins: Studies of CD9\/CD63 chimeric molecules on subcellular localization and upregulation activity for diphtheria toxin binding","volume":"25","author":"Ryu","year":"2000","journal-title":"Cell Struct. Funct."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"E968","DOI":"10.1073\/pnas.1521230113","article-title":"Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes","volume":"113","author":"Kowal","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1080\/14789450.2017.1260450","article-title":"Proteomic insights into extracellular vesicle biology\u2014Defining exosomes and shed microvesicles","volume":"14","author":"Greening","year":"2017","journal-title":"Expert Rev. Proteom."},{"key":"ref_20","first-page":"e55057","article-title":"Isolation and Characterization of Microvesicles from Peripheral Blood","volume":"119","author":"Menck","year":"2017","journal-title":"J. Vis. Exp."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1038\/cr.2014.44","article-title":"Double-stranded DNA in exosomes: A novel biomarker in cancer detection","volume":"24","author":"Thakur","year":"2014","journal-title":"Cell Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2377","DOI":"10.2337\/db17-1308","article-title":"Altered Extracellular Vesicle Concentration, Cargo, and Function in Diabetes","volume":"67","author":"Freeman","year":"2018","journal-title":"Diabetes"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"19413","DOI":"10.1038\/srep19413","article-title":"Plasma extracellular RNA profiles in healthy and cancer patients","volume":"6","author":"Yuan","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1164\/ajrccm.162.3.9910050","article-title":"Volume and cellular content of normal pleural fluid in humans examined by pleural lavage","volume":"162","author":"Noppen","year":"2000","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1042","DOI":"10.1378\/chest.106.4.1042","article-title":"Mononuclear cells in exudative malignant pleural effusions: Characterization of pleural phagocytic cells","volume":"106","author":"Gjomarkaj","year":"1994","journal-title":"Chest"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1038\/ncb1596","article-title":"Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells","volume":"9","author":"Valadi","year":"2007","journal-title":"Nat. Cell Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"156","DOI":"10.4161\/cam.1.3.5114","article-title":"Cell to cell signalling via exosomes through esRNA","volume":"1","author":"Lotvall","year":"2007","journal-title":"Cell Adh. Migr."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1038\/nature14581","article-title":"Glypican-1 identifies cancer exosomes and detects early pancreatic cancer","volume":"523","author":"Melo","year":"2015","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Javadi, J., Dobra, K., and Hjerpe, A. (2020). Multiplex Soluble Biomarker Analysis from Pleural Effusion. Biomolecules, 10.","DOI":"10.3390\/biom10081113"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1326","DOI":"10.3389\/fimmu.2018.01326","article-title":"Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures","volume":"9","author":"Wiklander","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e12142","DOI":"10.1002\/jev2.12142","article-title":"Extracellular vesicles are the primary source of blood-borne tumour-derived mutant KRAS DNA early in pancreatic cancer","volume":"10","author":"Hagey","year":"2021","journal-title":"J. Extracell. Vesicles"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1007\/978-1-4939-7253-1_13","article-title":"Analysis of Extracellular Vesicles Using Fluorescence Nanoparticle Tracking Analysis","volume":"1660","author":"Tannetta","year":"2017","journal-title":"Methods Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1016\/j.nano.2011.04.003","article-title":"Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis","volume":"7","author":"Dragovic","year":"2011","journal-title":"Nanomedicine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e13367","DOI":"10.1111\/aji.13367","article-title":"Preparation and characterization of extracellular vesicles","volume":"85","author":"Tiwari","year":"2021","journal-title":"Am. J. Reprod. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1038\/ncb1725","article-title":"Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells","volume":"10","author":"Meehan","year":"2008","journal-title":"Nat. Cell Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"356","DOI":"10.1038\/s41419-018-0391-6","article-title":"The role of osteopontin in the progression of solid organ tumour","volume":"9","author":"Zhao","year":"2018","journal-title":"Cell Death Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1038\/nrc1527","article-title":"Galectins as modulators of tumour progression","volume":"5","author":"Liu","year":"2005","journal-title":"Nat. Rev. Cancer"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1016\/j.cytogfr.2013.07.003","article-title":"Vascular galectins: Regulators of tumor progression and targets for cancer therapy","volume":"24","author":"Thijssen","year":"2013","journal-title":"Cytokine Growth Factor Rev."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"276","DOI":"10.2174\/1871520611313020014","article-title":"The role of mesothelin in tumor progression and targeted therapy","volume":"13","author":"Tang","year":"2013","journal-title":"Anticancer Agents Med. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1276","DOI":"10.1097\/MPA.0b013e318221bed8","article-title":"Co-expression of mesothelin and CA125 correlates with unfavorable patient outcome in pancreatic ductal adenocarcinoma","volume":"40","author":"Einama","year":"2011","journal-title":"Pancreas"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"10093","DOI":"10.1074\/jbc.C112.444562","article-title":"Heparanase regulates secretion, composition, and function of tumor cell-derived exosomes","volume":"288","author":"Thompson","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"89","DOI":"10.5858\/arpa.2017-0124-RA","article-title":"Guidelines for Pathologic Diagnosis of Malignant Mesothelioma 2017 Update of the Consensus Statement From the International Mesothelioma Interest Group","volume":"142","author":"Husain","year":"2018","journal-title":"Arch. Pathol. Lab. Med."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/11\/11\/1606\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:23:16Z","timestamp":1760167396000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/11\/11\/1606"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,29]]},"references-count":42,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2021,11]]}},"alternative-id":["biom11111606"],"URL":"https:\/\/doi.org\/10.3390\/biom11111606","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,29]]}}}