Figure 4 from OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

CD3+ T cells are dispensable for the effector function but required for the acquisition of the regulatory phenotype of CD19+ B cells in vivo. A, Experimental scheme: CD19+ B cells and CD3+ T cells were isolated from the iliac BM of naive or 67NR tumor–bearing BALB/c mice at day 11 after implantation and transferred intravenously into immunocompetent BALB/c recipients implanted with 4T1 tumor cells. Animals were grouped as follows: B Nv/T Nv, B 67NR/T Nv, and B 67NR/T 67NR (B and T cells from 67NR-bearing mice). B, The maximum area of primary tumors was determined using digital caliper measures. Tumor growth was monitored longitudinally and is shown as mean tumor volume (mm3) per group at regular intervals. C, On day 28, clonogenic assays were performed in draining LNs and BM. D, Histomorphometric analysis of sagittal iliac bone sections was performed to assess trabecular bone volume, expressed as a percentage of total tissue volume. E, Experimental scheme in which CD19+ B cells were isolated from the iliac BM of B 67NR or B Nude 67NR mice and transferred into 4T1 tumor–bearing immunocompetent BALB/c recipients. F, Quantification of OPG production by in vitro cultured CD19+ B cells isolated from B 67NR and B Nude 67NR donors. OPG levels were measured in culture supernatants by ELISA after 24 hours. G, RANKL levels were measured by ELISA in the supernatants of LN and BM cells restimulated with 4T1 sAg (sAg) in vitro. H, Primary tumor growth kinetics over time in mice receiving B 67NR or B Nude 67NR cells, compared with tumor-only controls. I, Clonogenic assays quantifying metastatic colonies in LNs and BM on day 28 after transfer. Data are expressed 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.