Figure 3 from OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

Early-stage acquisition of a regulatory phenotype by CD19+ B cells from 67NR tumor–bearing mice is required for optimal suppression of 4T1 tumor progression, RANKL production, bone loss, and metastasis. A, Experimental scheme: CD19+ B cells were isolated from the iliac BM of 67NR tumor–bearing BALB/c mice on day 11 after tumor implantation. These cells were transferred intravenously into BALB/c mice injected with 4T1 tumor cells at different time points: day 0, day 7, or day 14 after tumor injection. Mice were euthanized on day 31 for analysis. B, Primary tumor growth was monitored over time, and tumor volume was calculated on the indicated days. C, On day 31, RANKL production was measured by ELISA in supernatants from splenic and BM cell cultures restimulated in vitro with sAg. D, Metastatic burden was evaluated by clonogenic assays in draining LNs and BM. E, Micro-CT analysis of iliac bones was performed to assess structural parameters of trabecular bone, including BV/TV, trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th). Representative 3D reconstructions of trabecular bone are shown below the graphs. Data are shown as mean ± SD (n = 4–5 mice per group), representative of at least two independent experiments. 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; Nv, naive control.