Fig 1: OPG promotes lung metastasis by blocking RANKL-RANK signalling on Mo-macs. (a) Gating strategy for RANK-positive cells. Among the live and single cells, most RANK+ cells were CD45 positive. These cells were then subjected to CD11b+F4/80+ staining (Mo-Macs) and Ly6G+Ly6C+ staining (G-MDSCs). n = 3 mice per group. (b, c) Analysis of lung metastasis in B16F10 cells with OPG knockdown. Mice were treated with IgG or an α-CSF1R antibody to deplete Mo-macs in the lung. (b) Representative whole-lung images and statistics of surface metastatic nodules. (c) H&E staining and statistics of the metastatic area. n = 5–6 mice per group. All H&E staining scale bars: 1 mm. (d) Cartoon showing the cancer cell injection strategy used for the WT and Lyz2-iCre;Rankf/f (CKO) mice. (e, f) Analysis of lung metastasis of B16F10 cells with OPG knockdown in WT and CKO mice, as shown in (d). (e), Statistics of pulmonary surface nodules. (f), Statistics of the metastatic tumor area. n = 8 mice per group. All boxplots with all the data points show the minimum, first quartile, median, third quartile, and maximum values, as determined by Mann–Whitney U test.
Fig 2: The RANKL-RANK-CXCL10 signalling axis recruits NK cells to inhibit lung metastasis. (a) Flow cytometry graphs and statistics showing the percentages of CD3-; NK1.1+ NK cells among CD45+ immune cells in the lungs with metastatic nodules generated by control or OPG-KD B16F10 cancer cells. n = 10 mice per group. (b) Number of NK cell in the lungs with metastatic nodules generated by control or OPG-KD B16F10 cancer cells. n = 10 mice per group. (c) Correlation analysis between TNFRSF11B mRNA expression and the activated NK cell signature in lung metastasis samples from the AURORA and RAP cohorts. Statistics was analysed by Pearson correlation analysis. (d–g) Analysis of lung metastases generated by control or OPG-KD B16F10 cells tail vein injected into female C57BL/6 mice. Mice were treated with IgG or an α-NK1.1 antibody. (d, e) Representative lung images and statistics of surface metastatic nodules. (f, g) H&E staining images and statistical analysis of the metastatic areas. n = 5 mice per group. Scale bar: 1 mm. (h) Top DEGs in lung Mo-Macs after treated with either PBS or recombinant RANKL protein (100 ng/mL) for 24 h. A horizontal line was drawn at a adj.p-value of 0.05, and vertical lines represented a fold change of 2. (i) Heat map of comparing chemokine gene expression levels of lung Mo-macs treated with either RANKL or PBS control. Data presented as relative log2 fold change (L2FC). (j, k) Representative whole-lung figures (j) and H&E staining images (k) showing the lung metastases of B16F10 cells overexpressing CXCL10. n = 6 mice per group. Scale bar: 1 mm. (l, m) Analysis of lung metastasis generated by B16F10 cells with OPG knockdown. Mice were treated with vehicle control or AMG-487 (5 mg/kg per mouse) every other day. (l) Statistical analysis of lung surface metastatic nodules and (m) statistical analysis of the metastatic tumor areas. n = 6 mice per group. All boxplots showed the minimum, first quartile, median, third quartile, and maximum of the data points. (a,b) Student's two-sided unpaired t test was used for statistical analysis. (e, g, j, k, l, m) Mann–Whitney U test was used for statistical analysis.
Fig 3: Schematic illustration. Metastatic tumor cells in the lung are stimulated by TGF-β to increase the expression and secretion of OPG. OPG, in turn, suppresses the RANKL-RANK signals on Mo-macs, resulting in decreased CXCL10 production and diminished presence of NK cells in the lung microenvironment. Consequently, this cascade promotes the progression of lung metastasis. Schematic presentation was created with BioRender.com.
Fig 4: OPG promotes lung metastasis in a RANKL-dependent manner. (a, b) Representative whole-lung images (a) and H&E staining images (b) showing the lung metastases of B16F10 cells overexpressing RANKL. n = 6 mice per group. (c, d) Analysis of the lung metastases of B16F10 cells after treatment with recombinant RANKL protein (100 μg/mouse every 3 days). n = 5 mice per group. (e) Cartoon showing the strategy of cancer cell injection of the WT and Rankl−/− mice. (f, g) Analysis of lung metastasis of B16F10 cells with OPG knockdown in WT and Rankl−/− mice, as shown in (e). (f) Statistics of pulmonary surface nodules. (g), Statistics of the metastatic tumor area. n = 5 mice per group. (h) H&E staining of (g). Red arrows indicate the metastatic clones within the lungs of the Rankl−/− mice. H&E staining scale bar: 1 mm. All boxplots showed the minimum, first quartile, median, third quartile, and maximum of the data points. P values were determined by Mann–Whitney U test.
Fig 5: PGDHC promotes lysosomal membrane permeabilization in RANKL‐primed bone marrow‐derived macrophages (BMDMs) in vitro. Representative images of acridine orange staining (AO, green) and nuclei (Hoeschst; blue) (A) and fluorescence intensity quantification (B) in the cytoplasm of RANKL‐primed BMDMs after 48 h stimulation with PGDHC; fluorescence intensity was quantified as the ratio of AO/Hoechst. (C – D) Identification of galectin 3 (Lgals3) and lysosome associated membrane protein 1 (Lamp‐1) proteins associated with the lysosomal membrane permeabilization in the cell lysate of BMDMs after 48 h stimulation with RANKL and PGDHC. (E) Immunofluorescent staining of Lgals‐3 (red), Lamp‐1 (green), and nuclear counterstain (blue). Arrow heads point to areas of colocalization of Lgals‐3 and Lamp‐1. *p < 0.05; **p < 0.01; Scale bars, 50 μm
Supplier Page from BioLegend for Recombinant Mouse TRANCE (RANKL, TNFSF11) (carrier-free)