Fig. 3: Stromal remodeling during RCC tumorigenesis and TT formation.

A UMAP plot of mesenchymal cells (n = 13,965 cells) from our discovery dataset (n = 22 patients; n = 22 AT, 22 PT, and 12 TT samples), colored by mesenchymal cell subsets. For the distribution of these cells by tissue region (AT, PT and TT), clinical subgroup (with and without TT), and patient identity, see also Supplementary Fig. 6A, B. B Heatmap showing representative marker genes for each mesenchymal cell subset identified in the discovery dataset. C Systematic evaluation of alterations in the proportions of mesenchymal cell subsets in our discovery dataset, based on our comparison strategy (refer to Supplementary Fig. 2A). For more details, see the legend for Fig. 1D. Full results are provided in Supplementary Data 3. D Schematic diagram of the scRNA-seq workflow used for the validation dataset, which applied a negative selection strategy to enrich for mesenchymal and epithelial cells. E ssGSEA analysis showing the enrichment scores of FAP⁺ fibroblast (upper) and CYSLTR2⁺ fibroblast (lower) signatures—derived from our discovery dataset —in ATs (right; n = 18 and 54, respectively) and PTs (left; n = 99 and 431, respectively) from RCC patients with and without TT in the TCGA-KIRC bulk RNA-seq dataset. P-values were determined using the two-sided Wilcoxon rank-sum test. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. F Box plots showing the proportions of FAP⁺ fibroblasts (upper) and CYSLTR2⁺ fibroblasts (lower) in PTs from patients with and without TT, based on scRNA-seq data from both our discovery dataset (n = 8 and n = 5, respectively; PT samples with <50 mesenchymal cells were excluded) and the Yu et al²⁵. dataset (n = 19, without TT only). Groups were defined as in Fig. 1F, and P-values were calculated using the two-sided unpaired t-test. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. G Stacked bar plots showing the proportions of mesenchymal cell subsets in PTs from each patient in our validation dataset (n = 6). We highlighted FAP⁺ fibroblasts and CYSLTR2⁺ fibroblasts in this plot. Patient IDs were colored by TT status (blue: with TT; red: without TT). H Representative multiplex immunofluorescence images (left) and quantification (right) of FAP⁺ fibroblasts—defined as DCN⁺FAP⁺ double-positive cells—in tumor sections from RCC patients without (n = 8) and with (n = 8) TT. DCN (green) marks fibroblasts, and FAP (red) labels the specific fibroblast subset. FAP⁺ fibroblasts were annotated and quantified using QuPath. Box plots show the distribution of the proportion of DCN⁺FAP⁺ cells across groups. The box represents the interquartile range (IQR, 25th–75th percentile), with the horizontal line indicating the median. Whiskers denote minimum and maximum values. This analysis provides orthogonal validation for Fig. 3C. Scale bar = 200 µm. Scale bar inset = 50 µm. P-values were calculated using the two-sided Wilcoxon rank-sum test. I Violin plot showing the cell2location-inferred proportions of each mesenchymal cell subset within CN15, based on spatial mapping of 48 spatial transcriptomics samples using reference signatures estimated from our discovery dataset. J Representative multiplex immunofluorescence images (left) and quantification (right) of CN15-like regions—refined based on the spatial adjacency of FAP⁺ fibroblasts (DCN⁺FAP⁺, green & red) and EMT-like cancer cells (PLOD2⁺, white)—in tumor sections from RCC patients without (n = 5) and with (n = 5) TT. DCN⁺FAP⁺ double-positive cells and PLOD2⁺ cells were annotated using QuPath. Their interface regions were manually delineated and quantified using Fiji software, followed by normalization to the total area of the tumor section. Box plots show the distribution of the interface area (% tissue area) of DCN⁺FAP⁺ cells and PLOD2⁺ cells across groups. The box represents the interquartile range (IQR, 25th-75th percentile), with the horizontal line indicating the median. Whiskers denote minimum and maximum values. This analysis provides spatial validation for Figs. 2H and 3I. Scale bar = 200 μm; Scale bar inset = 50 μm. P-values were determined using the two-sided Wilcoxon rank-sum test. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. Source data are provided as a Source Data file.