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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. (A) Comparison of the sequences of hRANKL, mRANKL-WT and mRANKL-MT. (B) In vivo experimental flow. Male BALB-c/nu mice (6-weeks-old) were immunised with a subcutaneous injection of test agent (20 μg/kg). (C) Tumour growth and metastases were monitored via in vivo bioluminescence imaging of the reporter activity induced by PC3^Wild, PC3^RANKL+ and PC3^RANKL+ + IM treatment. Data per representative mouse are shown. A large hot-spot of bioluminescence was observed in vivo following inoculation of mice with PC3^RANKL+ cells. Multiple localised and distant metastases were observed in vivo after the injection of PC3^RANKL+ cells into the hearts of nude mice. Coloured bars indicate the bioluminescence signal intensity (photon/s/cm²/steradian). (D) The bioluminescence photon flux that appeared after inoculation of cells into mice is shown graphically. Data from related graphs are displayed as the mean ± SD. (E) Survival and (F) metastasis rate in mice from the SHAM, PC3^Wild, PC3^RANKL+ and PC3^RANKL+ + IM groups.

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