Fig. 1: scRNA-seq analysis revealed the significant TME remodeling in RCC with TT.

A Schematic diagram of sampling strategy and scRNA-seq analysis workflow for our discovery dataset. B UMAP plot of 163,762 high-quality single cells from 56 freshly collected tissue samples (n = 22 AT, 22 PT, 12 TT samples) collected from 22 RCC patients in our discovery dataset, colored by cell type annotations. Cells were processed using the BD Rhapsody platform following mechanical and enzymatic dissociation, and filtered based on standard quality control criteria (mitochondrial content >20%, gene number <200 or >5000, UMI count <1000 or >20000; see Methods for details). Major cell types were defined based on canonical markers expression following graph-based clustering. C Heatmap showing representative marker genes in each of cell types identified in the discovery scRNA-seq dataset. D Systematic evaluation of alterations in cell-type proportion based on the comparison strategy shown in Supplementary Fig. 2A. Left: Intra-patient comparison of cell-type proportions among AT (n = 14), PT (n = 14), and TT (n = 12) samples from RCC patients with TT (the two-sided paired t-test). Right: Inter-patient comparison of cell-type proportions in the same tissue region (AT or PT) between RCC patients with and without TT (n = 14 and 8, respectively; the two-sided unpaired t-test). The dashed line indicates a false discovery rate (FDR) threshold of 0.2. Full results are provided in Supplementary Data 3. E ssGSEA analysis showing signature scores of fibroblasts (upper) and NK cells (lower) in PTs from patients with (n = 99) and without (n = 431) TT in TCGA-KIRC dataset. Patients were stratified by TT status manually annotated based on the TCGA pathology reports (Supplementary Data 4). 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 fibroblasts (upper) and NK cells (lower) in primary tumors (PTs) from patients with and without TT in the discovery dataset (n = 14 and 8, respectively) and the public scRNA-seq dataset (n = 19, without TT only) from Yu et al.²⁵. As Yu et al.‘s dataset does not include patients with TT, three groups were established: PTs_with_TT (our data), PTs_without_TT (our data) and PTs_without_TT (Yu et al.). P-values were determined using two-sided unpaired t-test to evaluate pairwise difference among the three groups. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. G Representative immunofluorescence images (left) and quantification (right) of fibroblast abundance in tumor sections from RCC patients with (n = 8) and without (n = 8) TT. DCN (green) marks fibroblasts, and DAPI (blue) stains nuclei. Representative images from each clinical subgroup are shown to visualize fibroblast abundance at the tissue level. Fibroblast quantification was performed using QuPath-based cell annotation. This analysis serves as orthogonal validation for Fig. 1D. Scale bar = 200 µm; Scale bar inset = 50 µm. P-values were determined using the two-sided Wilcoxon rank-sum test. Box plots show the distribution of the proportion of DCN⁺ 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. Source data are provided as a Source Data file.