Fig 2: Combination of doxorubicin or paclitaxel-based chemotherapy with RANKL inhibitors displays substantial anticancer efficacy. (A) Mouse TNBC 67NR cells were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Tumor-bearing mice were treated with one single dose of chemotherapy (doxorubicin (2.5 mg/kg) or paclitaxel (10 mg/kg)), with RANKL inhibitors (RANK-Fc (1 mg/kg) and GCG (10 mg/kg), treatment at 3-day intervals) or with the combined treatment regimen. PBS-treated mice were used as controls. The mean tumor volumes±SEM are represented. (B) The tumor growth inhibition induced by doxorubicin, paclitaxel, RANK-Fc or GCG alone as well as by the combination regimens was determined. To do so, the volume of each harvested tumor was compared with the average tumor volume obtained in the control group. The means±SEM (plus each individual data point) are represented. (C) Single growth curves of 67NR tumors treated with mono or combination therapy. (D) Following mouse euthanasia, tumors were retrieved and enzymatically dissociated before flow cytometry analysis. Scatter dot plots showing the total number of (CD45+) immune cells per milligram of tumor as well as the percentage of each individual immune cell population (neutrophils, granulocytic and monocytic MDSC, DC, pDC, M1-like and M2-like macrophages, CD4+ and CD8+ T cells) among CD45+ cells in the different treated groups. The increased proportion of CD4+/CD8+ T lymphocytes (associated with the reduction of both granulocytic MDSC and M2-like macrophages) following chemotherapy should be noticed. The tumor-infiltrating immune cells were analyzed in 5 mice per condition. (E) Scatter dot plots illustrating the percentage of intratumor CD4+ and CD8+ Treg cells among total CD4+ and CD8+ populations in the different treatment groups. The activation status of DC (F) and pDC (G) was determined by analyzing the expression of several surface markers (CD80, CD86, I-A/I-E, ILT3 (DC) and ICOSL (pDC)) by flow cytometry. Data represent the mean fluorescent intensity (MFI)±SEM of 5 independent analyzes in each group (each individual data point is shown). Regardless of neoadjuvant chemotherapy, reduced proportions of CD4+/CD8+ Treg cells associated with increased activation of antigen-presenting cells were observed following RANKL blockade. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). P values were determined using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test (D, E, F, G) or Bonferroni post-test (B). ANOVA, analysis of variance; DC, dendritic cell; GCG, gallocatechin gallate; MDSC, myeloid-derived suppressor cell; PBS, phosphate-buffered saline; pDC, plasmacytoid dendritic cell; RANKL, receptor activator of nuclear factor κB ligand; TNBC, triple-negative breast cancer; Treg, T regulatory.

Fig 3: RANKL promotes the acquisition of a tolerogenic functionality by dendritic cells. (A) Influence of RANKL on DC migration in a Boyden chamber assay. Two different concentrations (100 and 500 µg/L) were tested. Data represent the means±SEM of four independent experiments performed in sextuplicate. (B) Representative FACS histograms showing the expression (normalized mean fluorescent intensity (MFI)) of CD80, CD83, CD86, MHC class I (HLA-ABC), MHC class II (HLA-DR), CCR7 and ILT3 on cell surface of DC cultured in the presence (red) or absence (gray) of 0.5 µg/mL RANKL during 3 days before LPS stimulation. Overlays were created using FlowJo software (V.10). (C) Expression of the aforementioned cell-surface molecules on DC pretreated with RANKL before stimulation with LPS. All data were normalized to LPS-stimulated DC (red line). Data represent the relative MFI±SEM of five independent experiments (each individual data point is shown). The significant reduction of all DC maturation markers studied (associated with the increased expression of both CCR7 and ILT3) in the case of RANKL exposure should be noticed. (D) CD4+ T cells were mixed with DC that were either pretreated with RANKL for 72 hours or not. After 6 days of co-culture, the expression of various immune tolerance markers (Foxp3, CD25, CD69, Helios, neuropilin-1) by T lymphocytes was determined by RT-qPCR. Results represent the means±SEM of three independent experiments performed in duplicate. (E) Suppressor activity of T cells originally primed by DC pretreated or not with RANKL. Allogeneic CD4+ T cells were co-cultured during 6 days with DC, either pretreated with RANKL or not. Cell mixture from the first mixed lymphocyte reaction (considered as potential “suppressor” cells) was then mixed with both freshly isolated T cells and other DC. Cell viability/proliferation was finally evaluated by MTT assay. Data represent the means±SEM of four independent experiments. (F) Cytokines/chemokines secreted by RANKL-untreated DC and DC pretreated with RANKL were detected using cytokine array. The respective MIF, CCL5, CXCL10, IL-16, Serpin E1 and IL-6 spots are highlighted (red boxes). The reduced secretions of these proinflammatory or pivotal molecules for DC functionality should be noticed. (G) Phosphorylation status of mTOR, S6 kinase and S6 ribosomal protein was determined by Western blot in DC exposed or not to RANKL. A representative immunoblot from three independent experiments is shown. The protein bands were quantified by densitometric analysis (ImageJ software). (H) Expression of maturation markers (CD80, CD83, CD86 and MHC class I/II) as well as CCR7 and ILT3 on DC pretreated with a mTOR inhibitor (Rapamycin or Torkinib/PP242) before being stimulated with LPS. All data were normalized to LPS-stimulated DC (red line). Data represent the relative MFI±SEM of four independent experiments (each individual data point is shown). The similarity between these latter results and those obtained with DC exposed to RANKL for 72 hours should be noticed. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). P values were determined using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test (A), one sample t-test (C, D, H) or unpaired t-test (E). ANOVA, analysis of variance; DC, dendritic cell; FACS, fluorescence-activated cell sorting;HFM, human fibroblast conditioned medium; LPS, lipopolysaccharide; MHC, major histocompatibility complex; mRNA, messenger RNA; ns, not significant; RANKL, receptor activator of nuclear factor κB ligand; RT-qPCR, quantitative reverse transcription PCR.

Fig 4: Anti-RANKL antibody, RANK-Fc and Gallocatechin gallate (GCG) exert efficient neutralizing effect on extracellular RANKL without altering cancer cell proliferation, apoptosis and metabolism. (A) Schematic representation of RANKL inhibitors tested in the present study as well as their respective mode of inhibition. (B) Murine RAW 264.7 cells were stimulated with recombinant RANKL alone or in the presence of graded concentrations of each RANKL inhibitor for 6 days. TRAP and CTSK mRNA levels were then determined by RT-qPCR in order to assess the efficiency of each individual inhibitor in blocking RANKL-dependent osteoclastogenesis. Note the significant downregulation of these two osteoclast markers when anti-RANKL antibody, RANK-Fc, GCG and isoliquiritigenin were added in the cell culture media. Results represent the means±SEM of three independent experiments performed in duplicate. (C) In parallel, the neutralizing activity of each RANKL inhibitor was also determined by TRAP staining. Representative pictures of untreated or treated RAW 264.7 cells are shown. (D) Number of multinucleated (≥ 3 nuclei), violet stained (TRAP-positive) cells per mm2 in each culture condition (anti-RANKL antibody (1 µg/mL), RANK-Fc (1 µg/mL), GCG (10 µg/mL), isoliquiritigenin (1.5 µg/mL), L3-3B (10 µg/mL), Ampelopsin H (10 µg/mL)). Results represent the means±SEM of three independent experiments. (E) Proliferation, (F) apoptosis and (G) oxygen consumption rate (OCR) of 4T1 cells in the absence or presence of anti-RANKL antibody (1 µg/mL), RANK-Fc (1 µg/mL), isoliquiritigenin (1.5 µg/mL) and GCG (10 µg/mL) were determined using Incucyte live cell analyzing system, annexin V-propidium iodide staining assay and Seahorse extracellular flux analyzer, respectively. The decrease of OCR when cancer cells were treated with isoliquiritigenin should be noticed. Results represent the means±SEM (each individual data point is shown). The scale bar represents 100 µm. Asterisks indicate statistically significant differences (**p<0.01; ***p<0.001; ****p<0.0001). P values were determined using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test (B, D, E, F). ANOVA, analysis of variance; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone;mRNA, messenger RNA; ns, not significant; RANKL, receptor activator of nuclear factor κB ligand; RT-qPCR, quantitative reverse transcription PCR.

Fig 5: No synergistic antitumor effect is detected in the case of combined RANKL and PD-1 blockade. (A) Mouse TNBC 67NR cells were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Anti-PD-1 antibody was tested alone (10 mg/kg per i.p. injection at days 0, 6 and 13) and in combination with RANKL inhibitors (RANK-Fc (1 mg/kg) and GCG (10 mg/kg), treatment at 3-day intervals). In order to precisely determine the potential additive or synergistic effect of combination therapies, each individual RANKL inhibitor was also used in monotherapy. The mean tumor volumes±SEM are represented. (B) Following mouse euthanasia, the final volume of each resected tumor was determined, allowing the assessment of tumor growth inhibition induced by anti-PD-1 antibody, RANK-Fc or GCG alone as well as by the combination regimens. Each individual tumor volume was compared with the average tumor volume obtained in the control group (PBS-treated mice). The means±SEM (plus each individual data point) are represented. (C) Single growth curves of 67NR tumors treated with mono (anti-PD-1 antibody (n=10), RANK-Fc (n=10), GCG (n=10)) or combination therapy (anti-PD-1 plus RANK-Fc (n=10) or GCG (n=10)). Unlike RANKL inhibitors which have always been well tolerated, it is important to note that the third injection of anti-PD-1 antibody induced 50% mortality in tumor-bearing mice. P values were determined using one-way ANOVA followed by Bonferroni multiple comparison post hoc test (B). ANOVA, analysis of variance; GCG, gallocatechin gallate; i.p., intraperitoneal; PBS, phosphate-buffered saline; PD-1, programmed cell death protein-1; RANKL, receptor activator of nuclear factor κB ligand; TNBC, triple-negative breast cancer.