The role of Notch pathway in the pro-tumorigenic activity of extracellular vesicles in multiple myeloma

Introduction: Multiple myeloma (MM) is characterized by the tight interaction between MM cells and bone barrow (BM) niche, resulting in tumor progression. MM cells overexpressed Notch 2 and Jagged 1 and 2, triggering Notch pathway activation on BM population and their pro-tumorigenic activity. Extracellular vesicles (EV) represent novel pro-tumorigenic players in the MM microenvironment. In this work we assess the tumorigenic effect of MM-derived EVs and the role played by the Notch pathway in EV-mediated communication between MM cells and the BM cells. Methods: EVs from MM cell lines (MM-EVs) or MM cell lines constitutively inhibited for Jagged1/2 (MMJ1/2KD-EVs) or Notch2 (MMN2KD-EVs) were characterized for Notch2 and Jagged1 and 2 content by Western blot and for size and number by nanoparticle tracking analysis and electronic transmission microscopy. The transfer of HA-tagged Notch2 via EVs was evaluated by an engineered system of HEK293 sending and receiving cells. Notch pathway activation was evaluated in vivo by injecting MM-EVs in the duct of Cuvier of 2 days post fertilization Notch-reporter Tg(T2KTp1bglob:hmgb1-mCherry)jh transgenic zebrafish embryos. The pro-tumorigenic effect of MM-EVs, MMJ1/2KD-EVs and MMN2KD-EVs were assessed in vitro by measuring the osteoclastogenic potential, the ability to induce human endothelial cells to organize tubular structures and assessing changes in stromal cell-mediated drug resistance. Results: MM-EVs carry Notch2 Jagged1 and 2 and transfer them to recipient cells; Notch members levels depend on their expression in MM cells. The analysis of the functional effects indicates that MM-EVs interact and activate Notch pathway in receiving cells in vitro and in vivo and display a pro-tumorigenic effect. MM-EVs show osteoclastogenic effect and angiogenic ability and boost drug resistance induced by the BM stromal cells HS5. All these effects are lost when EVs are produced by MMJ1/2KD and MMN2KD cells. Conclusions: These results provide the first evidence that targeting the Notch pathway may be a valid therapeutic strategy to hamper the pro-tumorigenic role of EV in MM progression.