Multiple myeloma-derived exosomes are enriched of amphiregulin (AREG) and activate the epidermal growth factor pathway in the bone microenvironment leading to osteoclastogenesis

Background: Multiple myeloma (MM) is a clonal plasma cell malignancy associated with osteolytic bone disease. Recently, the role of MM-derived exosomes in the osteoclastogenesis has been demonstrated although the underlying mechanism is still unknown. Since exosomes-derived epidermal growth factor receptor ligands (EGFR) are involved in tumor-associated osteolysis, we hypothesize that the EGFR ligand amphiregulin (AREG) can be delivered by MM-derived exosomes and participate in MM-induced osteoclastogenesis. Methods: Exosomes were isolated from the conditioned medium of MM1.S cell line and from bone marrow (BM) plasma samples of MM patients. The murine cell line RAW264.7 and primary human CD14 + cells were used as osteoclast (OC) sources. Results: We found that AREG was specifically enriched in exosomes from MM samples and that exosomes-derived AREG led to the activation of EGFR in pre-OC, as showed by the increase of mRNA expression of its downstream SNAIL in both RAW264.7 and CD14 + cells. The presence of neutralizing anti-AREG monoclonal antibody (mAb) reverted this effect. Consequently, we showed that the effect of MM-derived exosomes on osteoclast differentiation was inhibited by the pre-treatment of exosomes with anti-AREG mAb. In addition, we demonstrated the ability of MM-derived AREG-enriched exosomes to be internalized into human mesenchymal stromal cells (MSCs) blocking osteoblast (OB) differentiation, increasing MM cell adhesion and the release of the pro-osteoclastogenic cytokine interleukin-8 (IL8). Accordingly, anti-AREG mAb inhibited the release of IL8 by MSCs suggesting that both direct and indirect effects are responsible for AREG-enriched exosomes involvement on MM-induced osteoclastogenesis. Conclusions: In conclusion, our data indicate that AREG is packed into MM-derived exosomes and implicated in OC differentiation through an indirect mechanism mediated by OBs.