Figure 1 from OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

67NR-derived CD19+ IgD+ IgM+ CD138− B cells are the major source of OPG and inhibit in vitro osteoclastogenesis mediated by soluble factors derived from 4T1 tumor CD3+ T cells. A, Quantification of OPG secretion by total BM cells isolated from distinct skeletal sites: femur, tibia, vertebrae, humerus, and calvaria, of tumor-free, 67NR tumor–bearing, and 4T1 tumor–bearing mice at day 35 after implantation. A total of 107 BM cells/mL were cultured in vitro for 12 hours, and OPG levels in supernatants were measured by ELISA. B, CD19+ B cells isolated from the iliac BM of naive, 67NR-bearing, or 4T1-bearing mice were sorted into CD19+ IgD+ IgM+ CD138− (mature cells) and CD19+ IgD− IgM− CD138+ (plasm-like cells) subsets and were cultured in vitro (106 cells/mL) for 12 hours, and OPG levels in supernatants were measured by ELISA. C, Experimental scheme: (i) 11 days after 4T1 tumor cell injection, CD3+ T cells were isolated from iliac BM and stimulated with naive splenic CD11c+ DCs pulsed with 4T1 sAg. Supernatants were harvested after 72 hours of incubation and referred to as CM T 4T1. (ii) Thirty-five days after 67NR tumor cell injection, CD19+ B cells were isolated from iliac BM and stimulated with 4T1 sAg. Supernatants were harvested after 24 hours of incubation and referred to as CM B 67NR. (iii) OC assays were performed with M-CSF plus either RANKL or CM T 4T1, added to naive total BM cells, in the absence or presence of CM B 67NR. D, Quantification of TRAP+ multinucleated OC cells was determined (magnification 40×). E, Functional assessment of OC-mediated resorption using the BD BioCoat Osteologic Bone Cell Culture System. All data are from three independent experiments and are presented as mean ± SD of 4 to 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 < 0.0001.