Figure 5 from OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

Silencing of OPG in 67NR-primed CD19+ B cells abrogates their regulatory activity and restores 4T1 osteolytic and metastatic phenotypes in vivo. A, Experimental scheme: CD19+ B cells were isolated from the iliac BM of BALB/c mice bearing 67NR tumors on day 11 after implantation. Cells were subjected to gene silencing using lentiviral vectors carrying shRNA targeting Tnfrsf11b (OPG; B 67NR sh OPG) or a nontargeting scrambled control (B 67NR sh scr). Naive mice were used as experimental controls (Nv). B, OPG levels in culture supernatants from CD19+ B cells were assessed by ELISA. C, Experimental setup for in vivo analysis: OPG-silenced (sh OPG) or control-transduced (sh scr) CD19+ B cells were adoptively transferred together with CD3+ T cells from 4T1 tumor-bearing mice into immunocompetent BALB/c mice implanted with 1 × 105 4T1 tumor cells. D, Primary tumor growth was monitored over time. E, Representative images of tumor-bearing mice after adoptive B cell transfer. Photographs were taken at the experimental endpoint to illustrate macroscopic differences in tumor burden among groups. F, Quantification of RANKL levels by ELISA in supernatants of in vitro restimulated LNs and BM cells. G, Metastatic burden in draining LNs and BM was assessed by clonogenic assays. Data are presented as mean ± SD from two independent experiments (n = 5 mice/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.