P-028: Circulating cytokines present in multiple myeloma patients inhibit the osteoblastic differentiation of adipose and bone marrow stem cells

Abstract

<h3>Background</h3> Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We aimed to identify the cause of this defect. <h3>Materials and Methods</h3> We examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas), in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of MM-ASCs, healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Alizarin red coloration, alkaline phosphatase activity, RUNX2 and osteocalcin expression determined osteoblastic differentiation while oil-red O staining, PPARγ, C/EBPα and CD36 expression reflected the adipogenic capacity. Cytokine arrays were used along with Elisa assays for identification and quantification of relevant cytokines. RNA sequencing was performed on MM and HD-ASCs. <h3>Results</h3> Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas but adipocyte differentiation was unaltered. This defect was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas. They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, in CR-plasmas, the cytokine plasma levels were almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomitantly, MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. In CR-plasmas, the cytokine levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. <h3>Conclusion</h3> We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition, by these new circulating cytokines, was also observed on HD-BM-MSCs, suggesting that this could also be the case on myeloma-BM-MSCs.