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

UDI JOSEFINA; URETA DANIELA; LOPEZ MARGARITA; ENGELHARD MONIKA; ALVAREZ ELIDA

Congreso; DGHO Annual Meetin; 2014

DGHO

Introduction: In MM pathogenesis, the interaction of plasma cells and the bone marrow microenvironment plays a crucial role. Exosomes are nanovesicles 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 sought to further elucidate the role of exosomes secreted by both MMCLs and BMSCs as modulators of cell adhesion, bortezomib-induced apoptosis and MM cell migration. Methods: Exosomes were purified from the supernatants of MMCLs and M2-10B4 BMSC by ultracentrifugation after 72 hours of culture with RPMI1640, 0.2% penicillin/streptomycin and 10% exosome-depleted FBS, by differential centrifugation. Purifyand quality of isolated exosomes was confirmed by electron microscopy and western blot for CD63. Exosomal protein concentrations per 5x106 cells were quantified by Bradford assay. CD44 and ICAM1-expression was evaluated by flow cytometry. Apoptosis under bortezomib treatment in the absence/presence of M2-10B4 derived exosomes was assessed after annexin V/7-AAD-staining. Chemotaxis to increasing concentrations of M2-10B4 derived exosomes (100-500μg/ml) was studied using 24-well chemotaxis plates and migrated cells counted by flow cytometry. Results: Representative exosomes secreted by IM-9 cells were identified by electron microscopy, thereby showing a characteristic saucer-like morphology, ranging in diameter between 30 and 120nm. Western blot analyses confirmed the presence of CD63 both on MM cell- and M2-10B4-derived exosomes. Mean exosomal protein concentration was 1.4μg/μl ( confirmar cc es por μl o ml) for M2-10B4 cells 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 derived from MMCLs as compared to basal conditions, but without reaching statistical significance. Conversely, M2-10B4-derived exosomes induced a significant increase in CD44 expression on IM-9 cells (p=0.03), most likely reflecting an increased adhesion to components of the extracellular matrix in the bone marrow and contributing to cell-adhesion mediated drug resistance (CAM-DR). IM-9 apoptotic cells after 24h with bortezomib 10nM decreased from 72% to 61% in the presence of M2-10B4 exosomes and from 74% to 62% in the presence of IM-9 exosomes, suggesting a reduced lower sensitivity of IM-9 cells to bortezomib treatment. Migration of IM-9 cells slightly increased with 200μg/ml M2-10B4 exosomes and decreased with even higher exosome concentrations. Conclusions: This is to the best of our knowledge the first analysis showing the reciprocal effect of exosomes secreted by MMCLs (IM-9, MM.1S, U266, RPMI8226) on the adhesion properties of M2-10B4 BMSCs, and vice versa of M2-10B4-derived exosomes on the adhesion properties of one representative MMCL (IM-9). M2-10B4 derived exosomes also affected bortezomib-induced MM cell death and MM cell migration, thus suggesting their involvement as key players in an autocrine and paracrine bidirectional interaction,influence on treatment resistance and mieloma cells survival.