Exosomes Secreted By Multiple Myeloma Cell Lines (MMCLs) and Bone Marrow Stromal Cells (BMSCs) Reciprocally Modulate Cell Adhesion and Impact on MM Cell Migration and Bortezomib-Induced Apoptosis

Introduction: In MM pathogenesis, the interaction of plasma cells and the bone marrow microenvironment plays a crucial role. Exosomes are secreted by nearly all cell types, including cancer cells, and have been implicated in intercellular communication, drug resistance and tumor progression. We therefore purified, characterized and studied the influence of exosomes secreted by MMCLs and BMSCs on cell adhesion, bortezomib-induced apoptosis and cell migration.

Methods: Exosomes were purified from the supernatants of MMCLs (IM-9, MM.1S, U266, RPMI8226) and M2-10B4 BMSCs after 72h of culture with RPMI1640+10% exosome-depleted FBS by differential centrifugation. Purity and quality of exosomes was confirmed by electron microscopy and western blot for CD63. Exosomal protein concentration per 5x10⁶ cells was quantified by Bradford. CD44 and ICAM1 were evaluated by flow cytometry. Apoptosis was assessed via annexin V/7-AAD-staining. Chemotaxis to M2-10B4 exosomes (100-500μg/ml) was studied using 24-well chemotaxis chambers. Migrated cells were counted by flow cytometry.

Results: Exosomes from IM-9 cells were identified by electron microscopy, thereby showing a characteristic saucer-like morphology, ranging in diameter between 30 and 120nm. Both MM cell and M2-10B4 exosomes expressed CD63. Mean exosomal protein concentration was 1.4μg/μl for M2-10B4 and varied for MMCLs between 1.2 and 1.7μg/μl. M2-10B4 cells expressed higher levels of both adhesion molecules CD44 and ICAM-1 when incubated with exosomes from MMCLs compared to basal conditions, however, not reaching statistical significance. Conversely, M2-10B4 exosomes induced a significant increase in CD44 expression on IM-9 cells (p=0.03), reflecting an increased adhesion to components of the extracellular matrix that might contribute to cell-adhesion mediated drug resistance. IM-9 apoptotic cells after 24h with 10nM bortezomib decreased from 72% to 61% in the presence of M2-10B4 exosomes and from 74% to 62% with IM-9 exosomes, suggesting a lower sensitivity to bortezomib. Migration of IM-9 cells slightly increased with 200μg/ml M2-10B4 exosomes and decreased with higher exosome concentrations.

Conclusions: Our findings underline the role of M2-10B4 exosomes on adhesion and migration of one representative MMCL (IM-9), and the impact of M2-10B4 and IM-9 exosomes on bortezomib cytotoxicity in this MMCL, suggesting their involvement in a paracrine/autocrine loop modulating treatment response and MM cell survival.