“Exosomes-Mediate Crosstalk in Multiple Myeloma progression and drug Resistance”

Purpose: Exosomes (EXOs) mediate local and systemic cell-tocell communication and regulate cell behavior by transferring mRNA, miRNAs and proteins to recipient cells. Recently, we demonstrated that bone marrow (BM) cancer associated fibroblasts (CAFs) promote tumor progression and drug resistance (DR) in multiple myeloma (MM) (1-3). In this study, we analysed the effect of MM-derived EXOs on CAFs in MGUS to MM transition and, in turn, the effect of CAFs-derived EXOs on endothelial (ECs) and MM cells. Methods: EXOs isolation was performed from conditioned medium (CM) of CAFs purified from BM aspirates of 8 active MM patients and from CM of cultured RPMI8226 and U266 MM cells. Electron microscopy (TEM), dual immunofluorescence-confocal laser-scanning microscopy, western blot (WB), flow cytometry (FC) and qRT-PCR studies were performed to evaluate the CAFsand MM-derived EXOs phenotypes, their miRs content and their mutual effect. Results: TEM analysis of CAFs- and MM-derived EXOs showed a vesicles population with heterogeneous aspect, 50-100 nm sized. WB analysis defined the expression of commonly used EXOs surface markers as CD63, Hsp70 for CAFs and CD63, Hsp70, Alix for MM cells. Confocal microscopy showed the ability both CAFs and MM cells to uptake respectively MM- and CAFs-derived EXOs labelled with fluorescent dyes. Functional studies showed that MM-derived EXOs induce a specific miRNA profile as overexpression of miR-27b-3p, -125b-5p, -214-3p and activated phenotype, as expression of FAP+ and SMA+ antigens, in MGUS and MM CAFs. In turn, MM CAFs released EXOs containing miR-27b-3p, -125b-5p, -214-3p are swallowed by MM cells and ECs. Functional studies also showed that CAFsderived EXOs are able to stimulate angiogenic ability in ECs and to induce bortezomib-resistance in MM cells. Discussions and Conclusions: Overall results suggest an important exosomal crosstalk among tumor cells, CAFs and ECs which lead to BM microenvironmental modifications, favouring MM progression and DR. References 1. Frassanito MA et al. Leukemia 2014; 28: 904-16. 2. Frassanito MA et al. Leukemia 2016; 30: 640-8. 3. Di Marzo L et al. Oncotarget 2016; 7: 60698-711.