A Novel Ex-Vivo Model of Prolonged Osteoblast Suppression in Multiple Myeloma

Multiple myeloma (MM) is the most frequently involves the skeleton, with over 80% of patients developing osteolytic bone disease (MMBD). Importantly, lytic bone lesions in MM patients rarely heal even during long-term complete remission without detectable myeloma cells. Mesenchymal stem cells from MM patients (MM-MSCs) display distinct gene profiles and impaired osteoblast (OB) differentiation compared with those of normal donors, even after many passages in vitro. We found that MM causes long-term inhibition of OB differentiation by inducing epigenetic changes in the Runx2 promoter in MSCs, which persist in the absence of myeloma cells. Importantly, MSCs from myeloma patients expressed elevated Gfi1, a factor that modifies the chromatin structure of Runx2. However, the molecular mechanisms for MM-induced long-term OB suppression remain poorly understood. Exploring these mechanisms in animal models is costly, slow and complex. Further, it is difficult to replicate the bone marrow microenvironment in vitro.

We have developed an ex-vivo bone organ culture assay (EVOCA) that more closely replicates the marrow microenvironment, to further investigate the molecular mechanisms responsible for the protracted OB suppression in MM and to screen bone anabolic agents for MMBD. 5TGM1 murine MM cells expressing thymidine kinase and GFP (5TGM1-GFI-TK) were co-cultured in EVOCA with mouse long bones (femurs and tibias from 4 weeks old mice). Bones cut longitudinally with marrow in place were cultured in 35 mm dishes +/- 5TGM1-GFI-TK cells (2x10⁵/ml) and 10 % FCS in a 1:1 mix of RPMI/αMEM for 4 days. Conditioned media were collected and the bone resorption marker, c-terminal telopeptide crosslink of type 1 collagen (CTX) was measured by ELISA. CTX levels were increased in 5TGM1-containing EVOCAs compared to cultures without 5TGM1 cells (5.5±0.8 ng/ml vs. 9.8±1.0 ng/ml, p<0.001). 5TGM1-GFI-TK cell can be visualized by florescence microscopy, and HSV-TK expression permits selective cell killing with ganciclovir (Gcv), we then treated EVOCAs +/- 20 µg/ml Gcv on days 4-7 of culture. Myeloma cell survival was monitored by florescence microscopy and assay of IgG2b in the media. Gcv killed 100% of the myeloma cells. EVOCA cultures were continued for 1- 2 weeks more, then bones and outgrowth cells were homogenized, and lysates and RNA assayed for Runx2 and Osterix expression by Western blot and qPCR. Both Runx 2 and Osterix levels were 25% lower in EVOCAs treated with Gcv, and 50% lower compared to EVOCas not cultured with 5TGM1 cells. Runx2 and Osterix levels in EVOCAs cultured with Gcv but no MM cells were similar to levels in vehicle-treated bones. NFATc-1, cathepsin K, RANKL and OPG levels and osteoclast numbers were then measured in EVOCAs at 2 weeks post Gcv treatment. 5TGM1-containing EVOCAs had increased osteoclast numbers (2X) and levels of cathepsin K and NFATc-1. Further, RANKL/OPG ratios were increased in bone extracts from EVOCAs co-cultured with 5TGM1 cells compared with controls, regardless of Gcv treatment.

We next modified EVOCA to evaluate new bone formation. A 0.5mm diameter hole was initially made with a drill bit in intact bones (uncut) and the bones co-cultured +/- 5TGM1-GFP-TK cells (2x10⁵ /ml) 10% FCS in a 1:1 mix of RPMI/αMEM for 4 days. The cultures were then treated +/- Gcv for 3 days. At day 7, no 5TGM1 cells were detectable. The media was then changed to αMEM, and the EVOCAs continued for 1 week. Then 50µg/ml ascorbic acid and 10 mM β-glycerophosphate were added and the cultures continued for 7 more days. Bones were stained with Alizarin Red S to detect new bone formation at the drill site. New bone formation was confirmed by µCT. No new bone formation was detected in bones from EVOCAs initiated with 5TGM1 cells regardless of 5TGM1 removal. In contrast, new bone formation was easily evident in bones not exposed to MM cells.

The results show that increased bone resorption and OB suppression persist in MM-exposed bone even after myeloma cells are removed. The assay should allow us to follow the sequential development of lytic bone lesions, tumor growth, and protracted OB suppression, providing a useful tool for studies of MMBD, and evaluation of anabolic agents and anti-myeloma therapy in an intact marrow microenvironment.