Basic StudyImmunization With RANKL Inhibits Osteolytic Bone Metastasis in Breast CancerKim, Bora*,†,‡; Cho, Yong Jin*,†; Park, Mineon*,†; Lim, Wonbong*,†,§ Author Information *Laboratory of Orthopaedic Research †Department of Orthopaedic Surgery, Chosun University Hospital §Department of Premedical Science, College of Medicine, Chosun University, Gwangju ‡Department of Anatomy, Yonsei University College of Medicine, Seoul, Republic of Korea B.K. and Y.J.C. contributed equally. B.K., Y.J.C., and W.L.: designed and performed the research, analyzed and the interpreted data, and wrote the paper. B.K., Y.J.C., and M.P.: analyzed and interpreted the TRAP, RT-PCR, and Western blot data, and helped to draft some of the figures; performed in vitro and in vivo studies under the supervision of W.L., B.K., and Y.J.C.: performed and supervised RT-PCR and Western data analysis; performed micro-CT analysis and contributed to some of the figures. M.P.: assisted with the ELISA assay and performed histologic analysis under the supervision of W.L. Y.J.C.: performed a pathologic review of samples. W.L.: analyzed publicly available datasets and supervised the study. Reprints: Wonbong Lim, Department of Premedical Sciences, College of Medicine, Chosun University, Dong-Gu, Gwangju 501-759, Korea (e-mail: [email protected]). Journal of Immunotherapy: January 2022 - Volume 45 - Issue 1 - p 1-12 doi: 10.1097/CJI.0000000000000393 Buy SDC Metrics Abstract Breast cancer cells often metastasize to bone. Accumulating evidence suggests that inhibiting the receptor activator of nuclear factor-κB ligand (RANKL) not only leads to reduced bone metastasis of breast cancer but also has antitumoral effects. Here, we used mutant receptor activator of nuclear factor-κB ligand (RANKLM) as a vaccine for active immunization to induce antibodies for immunotherapy of bone metastatic cancer. We investigated whether anti-RANKL antibodies inhibit osteolytic bone metastasis in vitro and in a murine model. MC3T3 cells stimulated by MDA-MB-231 culture medium secreted growth differentiation factor-15 (GDF-15), which induced the nuclear factor-κB signaling cascade. In addition, RANKLM treatment-induced reduction of intraosseous growth of MDA-MB-231 cells correlated with decreased GDF-15 expression, a reduced number of osteolytic lesions, and slower tumor progression. In addition, vaccination with RANKLM led to significant improvement in overall survival and skeletal metastasis in tumor-bearing mice. Induction of anti-RANKL antibodies by RANKLM decreased GDF-15 production by deactivating nuclear factor-κB signaling, which in turn inhibited metastasis of MDA-MB-231 cells to bone. Taken together, the results demonstrate a role for RANKLM immunization in preventing bone metastasis of breast cancer. Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.