The Exosomes-Derived EGFR Ligand Amphiregulin (AREG) Is a New Pro-Osteoclastogenic Factor in Multiple Myeloma

Growing evidence indicates the critical role of the microvesicles to sustain multiple myeloma (MM) cell viability, survival and drug resistance into the bone marrow (BM) microenvironment. Recently it has also been suggested that MM cell-derived exosomes have a role in the induction of osteoclasts (OCs) differentiation and activity. The mechanism by which exosomes might be involved in MM-induced osteoclastogenesis is not known and was investigated in this study focusing on the possible role of Epidermal growth factor receptor (EGFR) ligands because it has been previously published their involvement in osteolytic bone metastasis by breast and lung cancer.

Thus the aim of the study was to investigate the presence of EGFR ligands in exosomes derived from different MM cell lines and primary CD138⁺ cells and to determine whether exosome-enclosed EGFR ligands are able to modulate the tumor microenvironment by affecting the function of OCs.

We firstly analyzed the expression of EGFR ligands at mRNA level on plasmacells (PCs) from patients with different monoclonal gammopathies (11 monoclonal gammopathy of undetermined significance, 133 MM patients at the diagnosis, 9 primary plasma cell leukemia) in a published dataset (accession number GSE16122) and we found that CD138⁺ cells expressed Amphiregulin (AREG) mRNA at high but variable levels among MM patients. Consistently, in a different dataset (GSE2658) of PCs, it has been previously reported that, among the EGFR ligands, AREG is significantly over-expressed by MM cells as compared to normal PCs (GSE5900).

Secondly we isolated exosomes by ultracentrifugation, from MM1S cell line and from BM plasma samples of 4 patients with active MM, and were characterized by western blot analysis of the exosome marker TSG101. We found that AREG was specifically enriched in exosomes derived from both MM1S and plasma samples of 3 patients, as confirmed by its absence in the exosome-deprived conditioned medium of MM1S cell line.

In order to test whether MM-exosomes could potentiate MM-induced osteoclastogenesis through the activation of the EGFR pathway, primary CD14⁺ monocytes purified from peripheral blood (PB) of 3 healthy donors and a murine cell line (RAW264.7) were used as OC models. Cells were treated with (i) exosomes (25 mg/ml) from both MM1S and plasma samples, pre-treated or not with anti-AREG neutralizing antibodies (Abs) (20 mg/ml) or (ii) with recombinant human (rh) AREG (20 mg/ml). Interestingly we found that MM1S exosome treatment induced the activation of EGFR as showed by the increase EGFR phosphorylation in RAW 264.7 cell line; this effect was reverted in the presence of anti-AREG neutralizing Ab. In addition we found that the six days treatment with MM1S exosomes induced a significant increase in the mRNA expression level of SNAIL, a downstream target of EGFR, both in RAW 264.7 and PB CD14⁺. The presence of anti-AREG neutralizing reverted this effect. Finally we showed a significant increased of the expression of OC specifics marker such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP), both in RAW 264.7 and PB CD14⁺ cells after treatment with MM-derived exosomes as compared to the control. The pro-osteoclastogenic effect of MM-derived exosomes was significantly abrogated by exosome pre-treatment with anti-AREG neutralizing Ab suggesting a possible direct effect of MM-exosome derived AREG on OC differentiation. However we also performed treatment of human pre-osteoblasts (PreOBs), obtained from mesenchymal stromal cells, with MM-derived exosomes in the presence or the absence of anti AREG Ab hypothesizing an indirect pro-osteoclastogenic effect mediated by the inhibition of osteoprotegerin (OPG) production by PreOBs.

In conclusion, our data indicate that the EGFR ligands are packed into MM-derived exosomes and involved in OCs differentiation thus highlighting the role of AREG as a potential new player in MM-induced bone resorption.