Public data sources

TCGA-KIRC (renal clear cell carcinoma), TCGA-KIRP (papillary renal cell carcinoma) and TCGA-KICH (chromophobe renal cell carcinoma) Level 3 high-throughput RNA sequencing data, mutation data, and clinical information were acquired from the UCSC XENA platform (https://xenabrowser.net/datapages/). The advanced RCC cohorts were derived from previously published studies [16].

Pan-cancer evaluation

We utilized the R package “TCGAplot” (version 4.0.0) [17], which integrates expression profiles and follow-up information from the TCGA Pan-Cancer cohort. This package enabled us to perform differential expression analysis of TROAP in both non-matched and matched samples within the Pan-Cancer dataset, using the Wilcoxon test for statistical validation. Additionally, we conducted Cox regression analysis to evaluate the prognostic significance of TROAP.

Prognostic analysis

To evaluate the prognostic significance of TROAP, we utilized the R packages “timeROC” (0.4), “survival” (3.5-5) (https://CRAN.R-project.org/package=survival) and “survminer” (0.4.9). These tools were used to calculate the area under the receiver operating characteristic (ROC) curve (AUC), and to perform Cox regression and Kaplan-Meier (KM) analyses. For the KM survival analysis, TROAP expression levels were categorized as high or low based on the optimal cut-off value.

Functional enrichment

We assessed the correlation between TROAP expression and other mRNA expressions using correlation analysis. The “clusterProfiler” R package (version 4.8.1) [18] was employed for Gene Set Enrichment Analysis (GSEA) to perform functional enrichment.

Immunohistochemistry

In this study, RCC tissue samples were obtained from Shanghai Zhuoli Biotech Company. The tissue microarray (HKid-CRCC150CS-01) consisted of a total of 148 valid samples derived from patients diagnosed with KIRC. This tissue microarray includes 74 cases of KIRC and 74 matched normal tissue adjacent to the tumor (NAT). The collection of tissue samples was ethically approved by the Ethics Committee of Shanghai Zhuoli Biotech Company (SHLLS-BA-22101102). Prior to embedding in paraffin, samples were fixed in 4% formaldehyde. Each tissue block was sectioned into 4 μm thick slices, treated with a 1% H2O2 solution, and blocked with non-immunogenic goat serum. The sections were incubated overnight at 4 °C with primary antibodies, followed by a 30-minute room temperature incubation with biotinylated secondary antibodies to bind to the primary antibodies. The specific immunohistochemistry (IHC) staining procedure was consistent with our previous studies [19]. Scoring for immunoreactive cells was based on the percentage of positive staining cells and the intensity of staining, calculated by adding the percentage score and the intensity score. The scoring for the percentage of immunoreactive cells was defined as follows: 0 (0%), 1 (1–10%), 2 (11–50%), and 3 (> 50%). Staining intensity was visually scored and categorized as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The antibody used was an anti-TROAP antibody (Novus Biologicals, NBP1-92532).

Immune cell infiltration

The immune cell infiltration in TCGA-KIRC, TCGA-KIRP, and TCGA-KICH datasets was analyzed using the “IOBR” R package (version 0.99.9) [20]. Specifically, we employed the xCELL algorithm within the “IOBR” package to compute Type 2 T helper (Th2) cell scores for each sample. Spearman correlation analysis was then performed to evaluate the relationship between TROAP expression and Th2 cell infiltration.

Western blotting

Cell lysates were generated by harvesting cells and lysing them in RIPA buffer supplemented with protease inhibitors. Proteins from these lysates were separated using SDS-PAGE and then transferred onto PVDF membranes. These membranes were initially blocked using 5% non-fat milk before being incubated with a primary anti-TROAP antibody (Novus Biologicals, NBP1-92532) a, followed by incubation with an anti-β-ACTIN-Rb secondary antibody (Novus Biologicals). Subsequent to antibody binding, the membranes were washed three times in PBST, each wash lasting 10 min, in preparation for detection. β-ACTIN, utilized as a normalization control, was also added at a 1:5000 dilution (Novus Biologicals). The intensities of the protein bands were quantified using Image J software.

Cell culture and transfection

The Caki-1 and 786-O cell lines were acquired from Beina Chuanglian Biotechnology Institute (Beijing, China). Prior to use, all cell lines underwent mycoplasma testing and were authenticated via short tandem repeat (STR) profiling. The cells were maintained at 37 °C in an atmosphere containing 5% CO2. Transfections were executed with GP-transfect-Mate (GenePharma, Suzhou, China), adhering to the supplied protocol [21]. Both a negative control (NC) and TROAP-specific siRNA (Table S1, Ribobio, Guangzhou, China) were introduced to the RCC cells as per the experimental design.

Cell counting Kit-8 assay

A 96-well plate was set up with roughly 4,000 cells that had been transiently transfected. This setup was replicated across five wells for each condition. The cells were incubated for time intervals of 2, 24, 48, and 72 h, after which 100 µL of a 1:9 dilution of CCK-8 solution in culture medium was administered to each well. Following an additional incubation period of 2 h, the optical density (OD) was measured at 450 nm using a spectrophotometer to assess cell viability.

Cell migration assay

To evaluate cell migration, we seeded about 40,000 transiently transfected cells in the upper chamber of a transwell apparatus using serum-free medium, while the lower chamber was filled with complete medium. The cells were incubated at 37 °C in an environment containing 5% CO₂ for a duration of 48 h. Post-incubation, cells were washed with saline, fixed with paraformaldehyde, and stained with 0.1% crystal violet. The stained cells were then visualized and quantified using microscopy.

Colony formation assay

Cells were plated at a density of 1,000 cells per well in 6-well plates and incubated at 37 °C with 5% CO₂ for a period of two weeks. Upon completion of the incubation period, the colonies were rinsed twice with cold phosphate-buffered saline (PBS), fixed using 4% paraformaldehyde for 15 min, and subsequently stained with 1% crystal violet for 20 min at ambient temperature. After staining, the colonies that were clearly visible were enumerated.

Mutation analysis

We utilized the R package ‘maftools’ to explore potential differences in gene mutation profiles between high and low TROAP expression groups [22]. This analysis allowed us to quantitatively assess the genomic variations and understand the mutational landscape associated with varying levels of TROAP expression.

Statistical analysis

Spearman’s method was employed for correlation assessments, while continuous variables were compared using the Wilcoxon rank-sum test. Categorical variables were analyzed using either the Chi-squared test or Fisher’s exact test as appropriate.