Novel In Vivo Model of Multiple Myeloma Bone Disease Using ß-Tricalcium Phosphate Artificial Bone Grafts
Authors: Koulnis Miroslav , Eda Homare , Santo Loredana , Siu Ka Tat , Ramachandran Janani , Panaroni Cristina , Tanaka Takaaki , Raje Noopur S. 
Published: October 2019

Abstract
Model systems to study Multiple Myeloma (MM) related bone disease exist but have a number of limitations. Disseminated MM models have variable cell homing and do not precisely recapitulate the human microenvironment interactions with myeloma cells. Severe combined immunodeficiency (SCID) mice engrafted with human fetal bone (SCID-hu) have been used by us, and are able to recapitulate the human bone marrow microenvironment. The fetal bone chips are however difficult to obtain, and vary in size and shape, complicating inter-sample comparison. Similarly, the poly-ε-caprolactone polymeric scaffold, previously used to seed murine or human stromal compartment, may not correctly reproduce bone destruction and inhibition of osteogenesis by MM as seen in patients, making this model difficult to test therapies targeting the MM niche.
β-tricalcium phosphate (β-TCP) is a biocompatible and biodegradable bone graft substitute that is uniform in structure and easily available, and may be a viable alternative to overcome SCID-hu difficulties in modeling MM bone disease. Here, we utilized β-TCP bone graft substitute to develop a novel in vivo MM model where β-TCP permits the development of the bone microenvironment, supports MM development, and is technically feasible and highly reproducible. Using this model, we aim to better understand the biology of the niche in MM by genetically modifying its components and by testing new niche-targeting therapies.
Our initial results show that osteogenesis takes place in the β-TCP bone graft, and the implant is supportive of MM tumor growth. Inter-scapular subcutaneous implantation of β-TCP alone, or co-implantation with human-derived stromal cell line HS27A in immunocompromised recipients resulted in the expression of osteogenic markers Runx2, alkaline phosphatase (ALP), Col1A1, and Osteocalcin (OCN), as well as a marker of bone resorption. Further, implants supported the growth of human-derived MM1.S and murine 5TGM1 cells, as visualized directly in vivo by serial luciferase bioluminescence imaging (BLI) and by immunohistochemistry.
Modifying the niche compartment in Cre/iDTR animals with MM disease is an exciting novel strategy to understand which niche component in vivo may be targeted to suppress MM development. Mouse strains with promoter-specific Cre recombinase that induces the expression of the diphtheria toxin (DT) receptor (iDTR) can be utilized to selectively ablate a cell population of interest in vivo, via intraperitoneal DT injection. Here, we first utilized OCN-Cre/iDTR mice to test the deletion of mature osteoblasts in β-TCP artificial bone graft post-implantation. Our data show a dose-dependent reduction in osteoblastic markers OCN, ALP, Runx2, Sclerostin, Osteoprotegerin and RANKL. Importantly, DT ablation of osteoblasts in the β-TCP implant resulted in a significantly increased 5TGM1 tumor growth, as judged by BLI and tumor weight. Our data show that the mature osteocalcin-positive niche population is protective against MM disease. Ongoing studies of the β-TCP mouse model will address the relative contribution of various osteogenic populations to the course of MM development in vivo, and test the efficacy of novel MM drugs.

Disclosures
Raje: BMS: Consultancy; Amgen: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Onyx: Consultancy; BMS: Consultancy; AstraZeneca: Research Funding; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Millenium: Consultancy; Eli Lilly: Research Funding; Novartis: Consultancy; Acetylon: Research Funding; Millenium: Consultancy; Novartis: Consultancy; Acetylon: Research Funding.