Figure 6 from OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

OPG produced by 67NR-primed CD19+ B cells is required to suppress RANKL production and protect bone structure in vivo. A, Experimental design: CD3+ T cells and CD19+ B cells were isolated from the iliac BM of 4T1 tumor–bearing BALB/c mice on day 11 after implantation. In parallel, CD19+ B cells from 67NR tumor–bearing BALB/c mice were transduced with sh OPG or sh scr. All cell types were adoptively cotransferred into BALB/c SCID recipient mice. On day 21, after transfer, animals were euthanized and analyzed. Naive mice were used as experimental controls (Nv). B, RANKL levels were quantified by ELISA in supernatants of spleen and BM cells restimulated in vitro with 4T1 sAg. C, Histomorphometric analysis was performed on the iliac bones from mice of the different groups. Sagittal sections from demineralized iliac bones were made following conventional methods and stained with H&E. All microscope slides were scanned with a ScanScope GL equipped with a 40× objective. Trabecular bone volume was expressed as a percentage of total tissue volume. D, Micro-CT analysis of iliac bones assessed structural parameters, including BV/TV, total trabecular volume (BV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), alongside representative 3D reconstructions. Data are shown as mean ± SD from two independent experiments with n = 5 mice per group. Statistical comparisons between more than two groups were performed using one-way ANOVA followed by Tukey post hoc test for pairwise comparisons when data were normally distributed. Statistical significance was considered when P < 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.