Characterizing tumor-associated endothelial cells in clear cell renal cell carcinoma via scRNA-seq and Bulk RNA-seq: construction of a prognostic risk signature.

Tumor-associated endothelial cells (TAECs) are integral components of the clear cell renal cell carcinoma (ccRCC) microenvironment, but their gene expression-based prognostic value and functional relevance remain to be defined. Single-cell RNA sequencing (scRNA-seq) data from 7 ccRCC tumors and 5 normal adjacent tissues (GSE156632) were analyzed to identify TAEC subcluster marker genes. A TAEC phenotype score was calculated in the TCGA-ccRCC cohort (n=516) based on the average expression of these markers. Weighted gene co-expression network analysis (WGCNA) was performed to identify gene modules correlated with this score, and module genes were intersected with differentially expressed genes (DEGs) between TCGA tumor and normal tissues. A prognostic risk signature was constructed using LASSO-Cox regression and validated in an external cohort (ICGC RECA-EU + GSE29609). Associations with immune infiltration, pathway activity and drug sensitivity were analyzed. APOLD1-centered pathway analysis was performed using GSVA and PROGENy. Functional validation included qRT-PCR, Western blot, immunofluorescence, Transwell assays, and xenograft models (n=5 mice/group). scRNA-seq revealed 8 distinct TAECs subclusters. Integration with TCGA data identified 61 critical TAECs-related genes after intersecting tumor-related DEGs with WGCNA yellow module genes. A 5-gene prognostic signature (TEK, APOLD1, F2RL3, COL5A3, CD248) was established. The model stratified patients into high/low-risk groups with significant survival differences (P<0.0001), achieving 1-/2-/3-year AUCs of 0.755, 0.713, and 0.736 in the TCGA cohort, with external validation AUCs of 0.712, 0.708, and 0.702 in the merged ICGC/GEO cohort. High-risk tumors exhibited metabolic reprogramming (suppressed PPAR signaling), an immunosuppressive TME (enriched M0 macrophages, Tregs, upregulated non-PD-L1 immune checkpoints), and reduced tumor purity. Low-risk tumors retained fatty acid metabolism and anti-tumor immune activation. Drug sensitivity analysis predicted differential sensitivity to targeted therapies and chemotherapeutics between risk groups. APOLD1 was upregulated in ccRCC endothelia (qRT-PCR/Western blot) and induced by ccRCC cell lines (786-O, Caki-1) culture medium in human umbilical vein endothelial cells (HUVECs). APOLD1-high tumors showed enhanced focal adhesion, TGF-β, MAPK, and PI3K pathway activities, and APOLD1 expression was positively correlated with endothelial and angiogenesis-related genes, including KDR, TEK, FLT1, PECAM1, CDH5, VWF, EMCN, ROBO4, and NOTCH4. In HUVECs, ccRCC cell-conditioned medium induced APOLD1 expression, whereas APOLD1 knockdown attenuated SMAD2/3 and ERK phosphorylation. Functionally, APOLD1 knockdown in HUVECs suppressed ccRCC migration/invasion in vitro and xenograft growth in vivo, whereas APOLD1 overexpression in HUVECs promoted ccRCC migration and invasion, further confirming its pro-tumorigenic role. This study developed a five-gene prognostic signature (TEK, APOLD1, F2RL3, COL5A3, CD248) associated with tumor-associated endothelial cells in clear cell renal cell carcinoma. The signature stratified patients into distinct risk groups with significant differences in overall survival, and its predictive performance was validated in external cohorts. Functional experiments indicated that APOLD1 is upregulated in ccRCC endothelial cells and contributes to tumor progression, suggesting it as a candidate for further investigation. Future studies are needed to translate these findings into clinical applications for high-risk ccRCC patients.