Disregulated Osteogenesis By Mesenchymal Stromal Cells (MSC) After In Vivo Exposure To Multiple Myeloma: Studies In a Novel Humanized Mouse Model For Bone Remodelling In Myeloma

Multiple myeloma (MM), a B-cell neoplasm characterized by a clonal expansion of malignant plasma cells in the bone marrow (BM), is accompanied by osteolytic lesions and/or diffuse osteopenia in up to 90% of the patients. Even after successful treatment, these MM-induced bone lesions do not normalize. We hypothesized that this might be caused by MM-induced irreversible impairment of the osteoblast function in the BM microenvironment. To study this bone remodeling processes in MM we used a recently developed, humanized mouse model of MM that allows engraftment and outgrowth of patient MM (pMM) cells in a humanized BM niche. To this end, ceramic scaffolds are seeded with culture-expanded human mesenchymal stromal cells (MSCs) from human BM, differentiated in vitro to osteoblasts for 1 week, then implanted subcutaneously in immune-deficient RAG2^-/-g_c^-/--mice and after 6-8 weeks a layer of human bone is deposited on the surface of the scaffolds. Following the injection of luciferase-GFP gene marked primary MM cells (pMM), this results in homing and outgrowth of pMM in the scaffolds (Groen et al., Blood 2012). Here we describe a modification of this in vivo model, by co-implanting MSC loaded scaffolds, with pMM cells adhered to the hybrid scaffolds, at one side of the mouse, and with hybrid scaffolds only (without pMM) at the other side of the mouse. At this contra-lateral location bone formation can take place undisturbed (i.e., not affected by the presence of MM) and serves as an internal control for the osteogenic potential of the osteoblasts. Thus this model allows us to study bidirectional interactions between pMM cells and the osteoblast and the resulting inhibition of osteogenesis. Here we report that outgrowth pMM cells indeed resulted in on average 50-75% decrease in bone formation, and, using bioluminescence imaging, we found an inverse correlation between the size of the tumor and the amount of bone formation: with increasing tumor size, the amount of bone formed was less. Human AML growing in the scaffolds (serving as control) does not influence the bone forming process. At the end of the experiment when we analyzed gene expression in the human stromal cells (CD73+ CD90+ CD105+) that we cultured from scaffolds containing pMM tumors, we found a significant reduction in expression of transcripts for alkaline phosphatase (ALP), collagen1A1 (colA1), osteoglycin (OGN), osteomodulin (OMD), and abnormal spindle-like microcephaly associated (ASPM), genes that have been implicated in osteogenesis. These data suggest that pMM cells interfere with the osteogenic differentiation of MSCs in the context of an in vivo biocompatible scaffold engineered to simulate the human BM microenvironment. Taken together, our data show that co-implanting MSCs together with the pMM cells can serve as a model to study the effect of pMM cells on osteogenesis, which provides a tool to unravel the mutual interaction between MM cells and the bone marrow microenvironment.