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. 2021 Jun 9;11:12186. doi: 10.1038/s41598-021-91721-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of immunisation with mutant RANKL on PC3-the innoculated metastatic model. (A) Three-dimensional micro-CT images revealed the trabecular bone architecture of the volume of interest in SHAM and mRANKL-immunized (IMMUNIZATION) mouse femurs (n = 10 images taken in total, one image for each mouse). (B) Bone mineral density (BMD) and (C) Serum calcium Level are shown. No significant differences were observed between two groups. (N.S.) (D) representative H&E stained and immunostained images of RANKL and IL-6 in the PC3^Wild, PC3^RANKL+ and PC3^RANKL+ + IM groups. The metastatic tumor cells or immuno-positive cells were indicated by black arrows. Magnification: × 200. Scale bar = 100 μm. (E) Concentration of RANKL in mouse serum. The mean ± SD values were obtained by densitometry, as shown in the analysis. Significant differences were observed at *p < 0.05 and **p < 0.01 vs. control. (F) Serum samples from mice were obtained after immunisation. Anti-RANKL values in PC3^Wild, PC3^RANKL+ and PC3^RANKL+ + IM groups. The mean ± SD values were obtained by densitometry, as shown in the analysis. Significant differences were observed at *p < 0.05 and **p < 0.01 vs. the control. (G) Anti-RANKL values and (H) RANKL concentration in the serum of RANKL^-immunized mice with metastasis (+) or metastasis (−). Bar graphs show the mean ± standard deviation (SD). Significant differences were observed at *p < 0.05, metastasis (+) vs. no metastasis (−).