Fig. 2

Distinct tumor-associated macrophage subpopulations in ccRCC bone metastasis. A UMAP joint embedding showing myeloid cell subsets. B Comparison of relative cell abundance of myeloid cell subsets between Bone Met (n = 9) and different control fractions (Healthy n = 12, Benign n = 7, Involved n = 4, Distal n = 4). Statistics are accessed with two-sided Wilcoxon rank sum test and BH multiple testing correction. (*p < 0.05, ***p < 0.001, Additional file 1: Table S3). C Box plot showing the percent of Macrophages (CD68 +) of the CD45 + / CD11b + population in Bone Met (n = 4) and Distal (n = 4) by flow cytometry. Statistical significance determined using two-sided t-test (*p < 0.05). D Scaled average expression of M2 signature genes visualized on UMAP embedding. E Representative M2 marker gene expression shown on violin plot. F UMAP joint embedding showing integrated analysis of myeloid cells from ccRCC primary tumor and bone metastasis tumor. G Violin plot showing representative marker gene expression across three macrophage subpopulations. H Box plot comparing proportion of macrophage populations across bone metastatic ccRCC (n = 9), primary ccRCC (n = 14), and adjacent normal tissue (n = 9). Statistics are accessed with two-sided Wilcoxon rank sum test and BH multiple testing correction. (*p < 0.05, ***p < 0.001). I Dot plots showing cytokine gene expression across different macrophage subsets. The color represents scaled average expression of marker genes in each cell type, and the size indicates the proportion of cells expressing marker genes. J, K Gating strategy for enrichment of TREM2 + SPP1 + macrophages. Labels above the flow plots refer to the parent population in the percentages are of the parent gate (J). Box plot showing the percent of TREM2 + /SPP1 + cells for the CD45 + / CD11b + population in Bone Met (n = 4) and Distal (n = 4). Statistical significance was determined using two-sided t-test (K). L Kaplan–Meier curves showing ccRCC samples with higher Macro-2 signature gene (SPP1, FABP5, CCL18, CXCL5, CCL7) expression have worse overall survival (top; n = 533) and progression-free survival (bottom; n = 435) in TCGA KIRC data. Patients were stratified into two groups based on the average expression (binary: top 25% versus bottom 25%) of Macro-2 signatures. p value was evaluated using Log-rank test. Bootstrap resampling was performed on signature genes and p-value was calculated using the 95% reproducibility power p-value (see the “Methods” section)