An EO771-Derived Metastatic Breast Cancer Model Exhibits RANKL-Associated Osteoimmune Remodeling Before Detectable Bone-Marrow Dissemination

Bone metastases are traditionally regarded as late manifestations of breast cancer progression. However, increasing evidence suggests that systemic skeletal alterations may emerge before overt tumor dissemination, raising the possibility that osteoimmune remodeling may contribute to the establishment of a permissive skeletal microenvironment. Pre-clinical studies from our group demonstrated that metastatic 4T1 breast cancer is associated with the expansion of RANKL-expressing adaptive immune populations and early skeletal alterations in BALB/c mice. Whether similar osteoimmune alterations occur in a distinct syngeneic immunocompetent breast cancer model remained unknown. Here, we generated an EO771-derived metastatic breast cancer population (EO771B) through serial multi-site in vivo selection under orthotopic, hematogenous and bone-associated pressures in immunocompetent C57BL/6 hosts. Compared with its parental counterpart, EO771B exhibited spontaneous dissemination to bone marrow, which was not detected for parental EO771 cells under the experimental conditions evaluated, while dissemination to tumor-draining lymph nodes (dLNs) and primary tumor growth did not differ significantly between the two populations. Microcomputed tomography revealed significant trabecular bone deterioration as early as day 11 following orthotopic inoculation, before bone-marrow dissemination became detectable by the clonogenic assay used in this study, with additional alterations in trabecular microarchitecture observed at later stages of tumor progression. These skeletal changes were accompanied by increased frequencies and/or numbers of RANKL-expressing CD4+ T cells and CD19+ B lymphocytes in the bone marrow, despite the absence of detectable tumor-derived colonies at the early time point. RANKL-expressing CD4+ T-cell populations also increased in tumor-dLNs and the primary tumor microenvironment during tumor progression. Adoptive transfer of tumor-educated leukocytes into lymphocyte-deficient Rag1−/− recipients reproduced trabecular bone deterioration, demonstrating that tumor-educated leukocytes can functionally promote skeletal remodeling, although the specific contribution of lymphocyte-derived RANKL was not directly tested. Collectively, these findings demonstrate that EO771B tumor progression is associated with osteoimmune alterations characterized by increased RANKL-expressing lymphocyte populations and trabecular bone deterioration before bone-marrow dissemination becomes detectable by clonogenic assay. Together with our previous findings in a distinct syngeneic breast cancer model, these results support the occurrence of tumor-associated osteoimmune remodeling across different tumor–host systems and establish the EO771B–C57BL/6 model as a platform for further mechanistic investigation.