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Here, we comprehensively isolated and characterized sEV from human MM cell lines (HMCL) and patient\u2010derived plasma (psEV) by specific EV\u2010marker enrichment and morphology. Importantly, we demonstrate that HMCL\u2010sEV are readily internalised by stromal cells to functionally modulate proliferation. psEV were isolated using various commercial approaches and pre\u2010analytical conditions (collection tube types, storage conditions) assessed for sEV yield and marker enrichment. Functionally, MM\u2010psEV was shown to regulate stromal cell proliferation and migration. In turn, pre\u2010educated stromal cells favour HMCL adhesion. psEV isolated from patients with both pre\u2010malignant plasma cell disorders (monoclonal gammopathy of undetermined significance [MGUS]; smouldering MM [SMM]) and MM have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre\u2010malignant sEV in disease progression. Proteomic profiling of MM\u2010psEV (305 proteins) revealed enrichment of oncogenic factors implicated in cell migration and adhesion, in comparison to non\u2010disease psEV. This study describes a protocol to generate morphologically\u2010intact and biologically functional sEV capable of mediating the regulation of stromal cells, and a model for the characterization of tumour\u2010stromal cross\u2010talk by sEV in MM.<\/jats:p>","DOI":"10.1002\/pmic.202000119","type":"journal-article","created":{"date-parts":[[2021,2,14]],"date-time":"2021-02-14T10:56:15Z","timestamp":1613300175000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Human myeloma cell\u2010 and plasma\u2010derived extracellular vesicles contribute to functional regulation of stromal cells"],"prefix":"10.1002","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0114-6058","authenticated-orcid":false,"given":"Antonia","family":"Reale","sequence":"first","affiliation":[{"name":"Myeloma Research Group Australian Centre for Blood Diseases Monash University\/Alfred Health  Melbourne Victoria 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