Multiple Myeloma Cell-Osteoblast Interaction Results in Impaired Bone Formation.
Authors: Vallet Sonia , Hideshima Teru , Pozzi Samantha , Vaghela Nileshwari , Gharti-Chhetri Gaurav , Mukherjee Siddhartha , Breitkreutz Iris , Kiziltepe Tanyel , Okawa Yutaka , Ikeda Hiroshi , Santo Loredana , Cirstea Diana , Boccadoro Mario , Anderson Kenneth C. , Raje Noopur 
Published: October 2019

Abstract
Osteolytic bone disease is a common complication of multiple myeloma (MM) resulting from uncoupled bone remodeling due to enhanced bone resorption and reduced bone formation. Bone formation is a complex process requiring functionally mature osteoblasts (OB). Mesenchymal stem cells differentiate into mature OB and following an active period of bone matrix synthesis lasting 1–2 weeks, they finally differentiate into inactive bone-lining cells or osteocytes. Although several studies have demonstrated that MM cells inhibit osteoblastogenesis via secretion of DKK1, a Wnt-pathway antagonist, the functional sequelae of interaction of mature OB with MM cells remains to be elucidated. Here, we studied the morphological and functional consequences induced by MM cells interacting with mature OB. Mature OB were generated from MM patients’ bone marrow mononuclear cells by cultivation in differentiation media consisting of αMEM with 20% fetal bovine serum, β-glycerol phosphate (2.16 mg/ml), ascorbic acid (0.05 mg/ml) and dexamethasone (10 nM). These mature OBs were alkaline phosphatase (ALP) positive and secreted and mineralized bone matrix, as demonstrated by Alizarin Red staining. MM cell lines INA6 and MM1.S were co-cultured with mature OB at a 5:1 ratio for 2, 4 and 7 days in OB differentiation media and bone marrow stromal cells (BMSC) were used as negative controls. After 4 days of co-culture, we observed phenotypic changes featured by acquisition of a spindle-like shape with reduced ALP staining in OB. In contrast, OB alone were intensely ALP-positive and cuboidal-shaped cells. Co-culture with INA-6 MM cells induced a reduction in ALP enzymatic activity in a time-dependent manner by 28% (± 10%) at day 2 and 72% (± 5%) at day 4 (p<0.05), respectively, whereas co-culture with MM1.S induced a 38% (± 5%) reduction after 4 days. Other MM cell lines induced similar effects. We then verified OB activity by assessing osteocalcin release and matrix mineralization. Importantly, osteocalcin secretion was completely abrogated in the presence of INA6, while MM1.S reduced it by 50% as early as day 2 (p<0.05). Moreover, Alizarin red staining demonstrated an impairment of matrix mineralization after 7 days of co-culture. Reduced OB function in the presence of MM cells was further confirmed by downregulation of Type-I collagen expression in OB. These effects were associated with only modest (10%) OB apoptosis as demonstrated by APO2.7 staining after 4 days of co-culture compared to OB alone. These phenotypic and functional sequelae on OB were not induced by co-culture supernatants, suggesting the requirement for direct MM cell/OB contact. These results therefore suggest that MM cell/mature OB interactions result in inhibition of bone formation by inactivation of mature OB. Ongoing studies are characterizing the mechanism by which MM cells induce OB inactivation and whether these changes affect the OC compartment. These studies of MM cell-OB interactions will form the basis for evaluation of novel agents with anabolic effects on the bone in the future.