Development of lytic lesions in mice injected with 5TGM1-GFP-TK MM cells results in persistent OB suppression after culturing BMSCs in vitro. Mice were injected intratibially with 20 μL saline with or without 10⁵ 5TGM1-GFP-TK (5TGM1) cells and compared with uninjected controls. Lytic lesions were allowed to develop for the indicated time periods. At the end of each time point, the tibias were dissected, and micro-QCT and fluorescent images were obtained. (A) Micro-QCT images of right tibiae obtained from mice sacrificed at 0, 2, 3, and 4 weeks after their injection with 5TGM1-GFP-TK cells or at 4 weeks after saline injection. (B) Fluorescent images of the injected tibias taken using the LT-9MACIMSYSPLUS Fluorescence Imaging System. (C-D) BMSCs were recovered from these tibiae, treated with ganciclovir until no GFP⁺ MM cells were visible (10 days) and expanded (3 weeks), before starting OB differentiation by culturing with or without BMP2 (50 ng/mL) in either α-MEM or OB differentiation medium (OB med). (C) At day 5, protein lysates and RNAs were isolated for measurement of ALP activity and quantitative PCR analysis of Bsp, Ocn, Runx2, and Osx expression relative to the uninjected mice BMSCs (using 2^−ΔΔCt analysis). GAPDH, reference gene. (D) At day 21, mineralization was assayed by alizarin red. (E) 5TGM1-GFP-TK cells and BMSCs (with the MM cells removed as in panel C and was photographed using a light box with no magnification) isolated from 4-week injected mice and controls were analyzed by quantitative PCR for expression of TNF-α, IL-7, and DKK1 and the data graphed relative to the BMSCs from uninjected mice using 2^−ΔΔCt. GAPDH, reference gene. In 5TGM1-GFP-TK cells, relative to GAPDH using ΔC_t analysis, relative fold mRNA expression was: TNF-α (48 ± 7), IL-7 (35 ± 12), and DKK1 (1 ± 0.3).