Proteomic and Biological Analysis of Myeloma Cell Derived Extracellular Vesicles

Intercellular communication between multiple myeloma (MM) cells and the normal bone marrow stroma leads to a modification of the bone marrow microenvironment, which favors tumor progression. New developments in extracellular vesicle (EV) research suggest that this diverse population of vesicles, released by cancer cells including MM cells, express transmembrane proteins and carry cargo that can modify recipient cells in myriad ways. In particular, tumor-derived EVs have been shown to create permissive microenvironments that lead to metastatic colonization by circulating tumor cells. As we have previously shown that MM EVs can enhance proliferation of recipient MM cells, we hypothesize that MM EVs can potentially play a much larger role in MM development and progression. In order to understand the scope of potential EV roles in MM, we executed a comprehensive proteomic analysis of the cargo of MM EVs. We isolated EVs from patient derived MM cell lines that represent the most common genetic variants of this tumor, and used in vitro generated plasma cells (IVPCs) as a reference population. In a first pass analysis, we selected proteins that were expressed ≥3 fold higher in MM EVs than in IVPC EVs, and this analysis identified 306 proteins. Of interest, included in the 306 differentially proteins were several involved in the regulation of cell adhesion such as members of the a disintegrin and metalloprotease domain (ADAM8, 9, 10, 13, 15 and 22) family, which are also associated with inhibition of cell proliferation. Moreover, CEACAM1, PTPRK, and CDH2, which are also linked to cellular adhesion, were also expressed at a higher level in MM EVs. Various proteins linked to myeloma cell biology were likewise found to be over-represented in MM EVs, and these include BCMA, ITGAV, ITGB5, IL6ST (gp130), CD276 (B7-H3), and CD28. As we are most interested in biologically relevant and actionable proteins present uniquely in MM derived EVs, we filtered from our data proteins that were also present in EVs from IVPCs. We further filtered out proteins known to be present in EVs from normal cells. This filtering strategy reduced the number of interesting candidates to 8, which included CD28, MET, TRKC, and ADAM15. Thus far, we have confirmed the presence of these 4 proteins in a panel of MM EVs, and we are currently in the process of validating additional candidate cargo proteins for their biological role in enhancing tumor cell proliferation and/or protection from apoptosis. In summary, EV proteomic analysis of cell lines representing MM genetic subtypes can lead to the identification of biologically relevant proteins transported systemically by EVs as well as suggest novel biomarkers that are easily detectable in plasma and may permit earlier recognition of disease progression in patients with MM.