Myeloma Cells Cultivate Megakaryocytes To Promote Lytic Bone Disease.
Authors: Yaccoby Shmuel , Saha Rinku , Kozlowska Ewa , Ge Yun , Ling Wen , Cottler-Fox Michele , Barlogie Bart , Tricot Guido 
Published: April 2020

Abstract
The development of lytic bone lesions in myeloma (MM) is caused by the activation of osteoclasts through direct and indirect interactions with MM cells and by inactivation of osteoblasts by MM cells. Megakaryocytes have been shown to promote osteoblast differentiation and produce critical factors associated with osteoclast activity. Cells of megakaryocytic linage are often expanded in myelomatous bone. The aim of this study was to investigate the impact of myeloma cells on regulation of bone remodeling by megakaryocytes. Culture of megakaryocytes was prepared by incubating mobilized peripheral blood in IMDM media supplemented with BSA, thrombopoeitin (TPO), IL-6 and IL-3. Following removal of all non-adherent cells, the remaining adherent megakaryocytes (&gt;90%) highly expressed c-MPL, CD41a and factor VIII, and had various degree of ploidy. Mesenchymal stem cells (MSCs), osteoclast precursors and CD138-selected MM cells were isolated as previously described (Pittenger et al., Science, 1999; Yaccoby et al., Cancer Res., 2004). Our study revealed that MSCs expressed TPO and that expression of TPO was upregulated 5 fold in MSCs after co-culture with MM cells, as determined by quantitive real time PCR (qRT-PCR). To study the effect on osteoblastogenesis, MSCs were co-cultured with megakaryocytes in the presence and absence of primary MM cells (n=5), in media lacking growth factors. Megakaryocytes promoted osteoblast differentiation as determined by elevated expression of alkaline phosphatase, an effect that was completely abrogated by adding MM cells to the cultures. To study the involvement of megakaryocytes on osteoclastogenesis, we initially examined expression of RANKL and OPG by these cells cultured with and without MM cells, using qRT-PCR. OPG and RANKL were expressed by megakaryocytes and gene expression of both factors was upregulated in co-cultured megakaryocytes. However, whereas OPG was insignificantly upregulated by 1.6±0.3 fold, RANKL expression was increased by 5.1±0.4 folds after co-culture with MM cells (p&lt;0.01). RANKL:OPG expression ratio in cultures of megakaryocytes with and without MM cells were 10.2±2.8 and 2.9±1.6, respectively (p&lt;0.03). This suggests that in the absence of osteoblasts, megakaryocytes are a substantial source of RANKL in myelomatous bone, whereas OPG may serve as a MM cell survival factor by neutralizing TRAIL (Shipman & Croucher, Cancer Res., 2003). The numbers of multinucleated osteoclasts counted after culturing of osteoclast precursors with MM+megakaryocytes, MM cells and megakaryocytes were 453±159, 273±95 and 291±104, respectively (n=3, p&lt;0.04, co-culture vs. MM cells). We conclude that MM cells prevent osteoblast differentiation promoted by megakaryocytes and induce megakaryocytes to increase RANKL:OPG ratio. These effects contribute to the uncoupling processes of osteoclastic resorption and osteoblastic bone formation, induction of lytic bone disease and myeloma progression.