Fig. 4

A distinct tumor-associated mesenchymal stroma cell (MSC) in ccRCC bone metastasis displaying CAFs phenotype. A UMAP embedding showing stromal cell subpopulations (left) and cell density difference comparing tumor with benign condition (right). Z score evaluates whether the cells are enriched in tumor (high Z score, red) or benign (low Z score, blue) condition. B Dot plot representing key-marker gene expression of stromal cell types. The color represents scaled average expression of marker genes in each cell type, and the size indicates the proportion of cells expressing marker genes. C Visualization of MSC marker gene expression shown as violin plot. D Bar plot illustrates cell abundance differences between Bone Met (n = 9) and Benign (n = 9) conditions for MSC-1 (left) and MSC-2 (right). Significance was assessed using two-sided Wilcoxon rank sum test. E Heatmap showing scaled average gene expression in MSC-2 across Bone Met and Benign conditions for each patient (column). F UMAP visualization of representative EMT and CAFs signature gene expression in stromal cells. G EMT gene signature score in stromal cells, UMAP visualization of EMT score (left). Violin plots of the EMT gene signature score in Bone Met and Benign MSC-2 cells (right). Significance was assessed using two-sided Wilcoxon rank sum test (****p < 0.0001). H Similar to Fig. 4G, showing CAF gene signature score (****p < 0.0001). I Bar plot showing relative mRNA expression (log fold change) of FAP and FN1 in Benign (n = 5) and Bone Met (n = 7) tissue by RT-qPCR. Data are expressed using the 2 − ∆∆Ct method. Gene expression levels were normalized to the benign control. Statistical significance determined using two-sided t-test. J Kaplan–Meier curves showing ccRCC samples with higher MSC-2 signature gene (COL6A2, FN1, TIMP1, COL3A1, COL1A2) expression have worse progression-free and overall survival (n = 533) in TCGA KIRC data. Patients were stratified into two groups based on the average expression (binary: top 25% versus bottom 25%) of MSC-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). For box plots, center line represents the median and box limits represent upper and lower quartiles, and whiskers depict 1.5 × the interquartile range (IQR)