Figure 2. OB-Runx2 deficiency and BTZ treatment induce changes in the BM microenvironment in vivo.

BM cells and BMS were harvested from tumor-bearing OB-Runx2^+/+ and OB-Runx2^−/− mice treated for 4 weeks with PBS or BTZ as depicted in Fig. 1. A-H, Immunosuppressive cells and immune effector cells in the BM of tumor-bearing OB-Runx2^+/+ and OB-Runx2^−/− mice were assessed by flow cytometry (n=7 mice/group). A, Representative plots (left) and the percentage of MDSCs (Gr1⁺ CD11b^hi) detected in the BM cell population (right). B, Relative expression of iNOS, arginase 1, and IL-10 in BM MDSCs (Gr1⁺ CD11b^hi). C, Gating strategy for identifying CD8⁺ T cells and CD8⁺ T cells expressing exhaustion markers in the BM cell population. D, Percentage of CD8⁺ T cells detected among all CD3⁺ T cells (CD3⁺CD8⁺) in the BM. E-H, Percentage of BM CD8⁺ T cells expressing PD-1 (E), TIM-3 (F), granzyme B (G), and IFN-γ (H). I, Representative Western blots showing TSP-1 expression in the BMS of tumor-bearing OB-Runx2^+/+ and OB-Runx2^−/− mice after PBS or BTZ treatment (left) and densitometric quantification of TSP-1 normalized to GAPDH (in triplicate) (right). J, Quantification of active TGF-β1 concentration in the BMS of tumor-bearing OB-Runx2^+/+ and OB-Runx2^−/− mice after treatment, measured by ELISA (in duplicate). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. ns, not significant.