Defining a Murine Model To Study Bone Disease in Multiple Myeloma (MM).
Authors: Neri Paola , Tassone Pierfrancesco , Shen Zhenxin , Patel Nipun , Fajardo Robert , Fulciniti Mariateresa , Prabhala Rao , Shammas Masood , Blotta Simona , Hideshima Teru , Chauhan Dharminder , Muller John , Venuta Salvatore , Snyder Brian D. , Goldring Steven R. , Anderson Kenneth C. , Munshi Nikhil C. 
Published: October 2019

Abstract
Multiple Myeloma (MM) is associated with significant skeletal changes including ospeoporosis and bone lesions in more than 80% of patients. Bone resorption is caused by an enhanced osteoclast number and activation. However, molecular and cellular basis for these effects are not completely understood. Here we describe a model that allows us to evaluate in vivo, the relationship and interactions between MM cells and cellular elements of bone formation and resorption. In this model, the BM stroma and IL-6-dependent human MM cell line (INA-6) was injected in a human bone chip implanted into SCID mice. Bone chip in the control mice were injected with media alone. At different time points (days 1, 7 and 30) following injection, myelomatous and control bone chips were retrieved, fixed in paraformaldehyde and evaluated by micro-computed tomography (μCT) and histological examinations for effects of MM cells on bone compartment. The μCT is a non-invasive quantitative imaging method that can asses the degree and extent of bone disease as well as quantify changes in bone structure, that may reflect the effect of interaction between MM cells and bone elements. Additionally, we have measured the levels of human osteocalcin in murine serum to reflect the remodelling of human bone in mice. We have observed that only at day 30 after the MM cell injections, lesions in myelomatous bone is observed in mice; while no significant changes in the matched control bones are seen. By 3-D histomorphometric parameters, calculated from μCT (bone volume fraction= bone volume/total volume), the quantitative measurement confirmed that at day 30, a decreased bone volume fraction (314) was observed in bones with MM compared with normal bones (394), due to reduction of bone density in the trabecular bone. Moreover histological examination of the same bones revealed a significant increase in number of multinucleated TRAP-expressing osteoclasts in mice engrafted with MM versus control (52 versus 8 per field, p= 0.011). Also compared to serum from control mice, animals with bone containing myeloma cells had significantly elevated markers of bone remodelling. To confirm the validity of this model to study effect of MM cells on bone as well as to show its utility for evaluation of novel agents active in MM bone disease, we treated mice with Zoledronic acid at 0,6 mg/kg, weekly for 2 months. As evaluated by μCT, X-rays and TRAP staining Zoledronic acid was able to significantly inhibit the development of bone lesions in treated mice compared to untreated mice. Additionally, , we also observed that Zoledronic acid was also able to retard the MM tumor growth as measured by human IL-6sR released by INA-6 cells in mice sera corroborating its reported anti-MM activity. This model therefore provides a reproducible and predictable in vivo system to study biology of bone disease in MM and to identify and evaluate therapeutic targets and agents targeting MM bone disease.