The role of NOTCH2 in the extracellular vesicles-mediated angiogenesis and osteoclastogenesis in multiple myeloma

Introduction. Multiple myeloma (MM) is an incurable hematological neoplasm mainly due to the interaction with a highly supportive bone marrow (BM) niche. The aberrant expression of the NOTCH2 oncogene in MM cells contributes to MM pathological communication with the BM cells leading to tumor angiogenesis and osteoclastogenesis, two key steps in MM progression. Recently, extracellular vesicles (EV) shed by tumor cells have been identified as key players in the communication between tumor and microenvironment. This work explores the tumorigenic effect of MM-derived EV (MM-EV) and the role of NOTCH2 in EV-mediated communication. Methods. Size and concentration of MM-EV from MM cell lines, RPMI8226 and OPM2, were characterized by nanoparticle tracking analysis along with morphology by transmission electron microscopy and MM-EV uptake by target endothelial cells and osteoclasts (fluorescent microscopy and flow cytometry). MM-EV content and ability to transfer NOTCH2 was assessed by Western blot analysis. To assess the role of NOTCH2 in EV-mediated communication, we used EV from MM cells knocked down for Notch2 (MMN2KD-EV). The difference in MM-EV and MMN2KD-EV size and concentration was assessed together their ability to activate Notch signaling in recipient cells by two reporter assays performed on HeLa cells and on a Notch-reporter Tg(T2KTp1bglob:hmgb1-mCherry)jh transgenic zebrafish embryo. The osteoclastogenic ability of MM-EV and MMN2KD-EV was assessed on the RAW264.7 cell line and the angiogenic activity by a tube formation assay on human pulmonary artery endothelial cells. Results. MM-EV carry and transfer NOTCH2 in a paracrine way, inducing NOTCH signaling activation in recipient cells. Moreover, MM-EV display angiogenic and osteoclastogenic potential depending on the presence of NOTCH2. NOTCH signaling blockade by γ-Secretase inhibitor can interrupt MM-EV mediated pathological communication resulting in angiogenesis and osteoclastogenesis. Conclusion. MM-EV promote the progression of MM by promoting tumor angiogenesis and osteoclastogenesis. NOTCH2 plays a key role in EV-mediated communication in the BM microenvironment. Thereby targeting NOTCH activation may represent a suitable strategy to hamper the pro-tumorigenic activity of MM-EV.