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Background In the past decade, immunotherapy using immune checkpoint inhibitors (especially targeting the PD-1/PD-L1 axis) has been demonstrated as a promising strategy for the treatment of cancers that do not respond to classical chemoradiotherapy. Given that cancer cells have the potential to express many immunosuppressive molecules other than PD-L1, the combination of immune checkpoint inhibitors with other drugs thwarting tumor immunosuppressive microenvironment could represent a promising strategy. Among these immunosuppressive molecules, RANKL, a member of the TNF superfamily, which mainly affects the immune system and bone remodeling, has been shown to be a key factor promoting the progression of breast cancer. In addition, RANKL induces the formation of tolerogenic dendritic cells and Treg cells, which promotes immunotolerance to the tumor. The aim of this research project is to study the impact of several RANKL inhibitors on triple negative breast cancer and to analyze the efficiency of their association with anti-PD-1/PD-L1 agents. Materials and Methods We studied RANKL and PD-L1 expression in several murine and human breast cancer cell lines by immunohistochemistry. The secretion of RANKL was analyzed by ELISA. Inhibitors of RANKL were then tested in vitro. We selected several RANKL inhibitors: anti-RANKL antibody, RANK-Fc, Isoliquiritigenin and Gallocatechin gallate. The efficacy of these inhibitors was indirectly evaluated with the murine macrophage RAW264.7 cell line which undergoes, in the presence of RANKL, an osteoclast differentiation (TRAP and Cathepsin K expression). The efficacy of RANKL inhibitors was then evaluated, in this model, by RT-qPCR. Apoptosis and proliferation of the cancer cell lines in the presence of the inhibitors were also analyzed. Results RANKL/PD-L1 expression profile on specimens from each breast cancer subtypes showed that both immunosuppressive molecules are expressed by all breast cancers with a significantly more intense immunoreactivity for triple negative breast cancers. Most of the cell lines expressed both proteins. We found that RANKL is secreted in their extracellular environment. RANKL inhibitors are efficient and will be tested in vivo. Conclusions Several murine triple negative breast cancer cell lines will be sub-cutaneously injected in mice and the efficacy of both RANKL and PD-L1 inhibitors will be evaluated (separately or in combination). The infiltration of tumor microenvironment by different immune cell populations, the presence of metastasis and the tumor growth will be analyzed. Disclosure Information C. Pilard: None. P. Roncarati: None. E. Hendrick: None. A. Lebeau: None. D. Bruyere: None. T. Lerho: None. M. Ancion: None. C. Reynders: None. P. Delvenne: None. M. Herfs: None. P. Hubert: None. |
