A total of eight pairs of BLCA tissues and adjacent non-cancerous tissues from Peking University First Hospital-Miyun Hospital were collected between January 2018 and January 2023 (Beijing, China). Each pair of bladder tissue and adjacent non-cancerous tissue came from the same patient. Additionally, 17 pairs of BLCA and adjacent non-cancerous tissues, along with 112 paraffin-embedded BLCA tissue blocks at various stages, were gathered. Data from 112 patients with bladder cancer were incorporated into the Miyun cohort. All patients were pathologically diagnosed with urothelial carcinoma, and the histological characteristics of the samples were confirmed by experienced urological pathologists using hematoxylin-eosin staining. The present study was approved (approval no. 2023-029-001) by the Medical Ethics Committee of Peking University First Hospital-Miyun Hospital (Beijing, China). Clinicopathological characteristics of patients (including sex and age distribution) are listed in Table I.

Transcriptome and clinical data from the The Cancer Genome Atlas (TCGA)-BLCA datasets, encompassing 410 patients with bladder carcinoma, were downloaded from UCSC XENA (https://xena.ucsc.edu/) (17). The Xiantao tool (https://www.xiantao.love/) was used for visualization and analysis of expression differences, prognosis and enrichment (18).

Human ureteral epithelial cells (SV-HUC-1) and human BLCA cell lines (T24, J82, UMUC3 and SW780) were obtained from the American Type Culture Collection and cultured according to the manufacturer's protocols. SV-HUC-1 cells were maintained in F-12K medium (Gibco; Thermo Fisher Scientific, Inc.), while T24, J82, UMUC3 and SW780 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.). All media were supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin G-streptomycin (Sigma-Aldrich; Merck KGaA). Cultures were kept at 37˚C with 5% CO₂.

RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using a reverse transcription system (Tiangen Biotech Co., Ltd.) following the manufacturer's protocol (42˚C for 15 min, 95˚C for 3 min, 4˚C maintenance). RT-qPCR was conducted on the 7500 Reverse transcription PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), with GAPDH as the internal reference. The thermocycling conditions were as follows: pre-denaturation at 95˚C for 3 min, denaturation at 95˚C for 30 sec, annealing extension at 60˚C for 30 sec, deformation and annealing extension for 40 cycles. The dissolution curve was increased by 0.5˚C every 2 cycles to 95˚C. The primer sequences were as follows: IL1RL2 forward, 5'-TCCCGAAGAGTTGTGTTTTGG-3, and reverse, 5'-TGAGTGTGTCAGTATGGCTTGA-3'; and GAPDH forward, 5'-GGAGCGAGATCCCTCCAAAAT-3, and reverse, 5'-GGCTGTTGTCATACTTCTCATGG-3'. The 2^-ΔΔCq method was used for quantification (19).

The primary antibody was incubated at 4˚C overnight, and the secondary antibody was incubated at room temperature for 1 h. Total protein was extracted using NP-40 lysis buffer (Beyotime Institute of Biotechnology) and quantified by the BCA method (Pierce; Thermo Fisher Scientific, Inc.). Proteins were separated by SDS-PAGE (10% gel concentration) and transferred to PVDF membranes. After blocking with 5% skimmed milk for 1 h at room temperature, membranes were incubated with IL1RL2/IL36R antibody (1:1,000; cat. no. 10090; ABclonal Biotech Co., Ltd.) followed by a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000; cat. no. sc-2004/sc-2005; Santa Cruz Biotechnology, Inc.). GAPDH (1:1,000; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.) served as the internal reference. Immunoreactive bands were visualized using the ECL Plus kit (Applygen Technologies Inc.).

Paraffin samples were sliced to a thickness of 5 µm. All paraffin samples were cut by the same pathologist and were of similar thickness. IHC staining was performed using the PV-6000 universal kit (cat. no. IB000088; ZSGB-BIO; OriGene Technologies, Inc.). Sections were heated at 70˚C for 1 h, deparaffinized in xylene and rehydrated (95% anhydrous ethanol), followed by antigen retrieval with citrate repair solution at 110˚C for 10 min. Slides were incubated at room temperature for 20 min away from light) with endogenous peroxidase inhibitor blocking agent and sheep serum (cat. no. ZU-9022). IL1RL2/IL36R antibody (1:500; cat. no. 10090; ABclonal Biotech Co., Ltd.) was applied (incubated overnight at 4˚C in the dark), followed by incubation with a biotinylated secondary antibody (cat. no. 32020; Thermo Fisher Scientific, Inc.; room temperature for 20 min away from light) and staining with the DAB substrate kit (cat. no. ZU-9019; ZSGB-BIO; OriGene Technologies, Inc.). When the background of the slide was brown, reaction was terminated in tap water. Blue was reversed in running tap water for 30 min. Then gradient dehydration with ethanol and xylene was carried out. Finally, it was sealed with neutral resin. IL1RL2 expression was graded based on staining intensity (1, no staining; 2, weak; 3, moderate; 4, strong) and the percentage of reactive cells (1, 0-25%; 2, 26-50%; 3, 51-75%; 4, >75%). The final score (range 1-16) was the product of these variables. The degree of dyeing has several intermediate stages, and the pink brown is also one of them, representing the degree of dyeing. Images were observed using a light microscope.

Data were analyzed using GraphPad Prism 9.0 (Dotmatics) or SPSS 20.0 (IBM). Results are expressed as the mean ± SD. Continuous variables were compared using paired and unpaired Student's t-test or one-way ANOVA followed by Bonferroni's post hoc test (for multiple-group comparison) single factor analysis. Pearson's chi-square test or Fisher's exact test were used for correlation analysis. Survival rates were analyzed using the Kaplan-Meier method and log-rank P-tests. Prognostic correlations between clinicopathological and IHC data were assessed by univariate and multivariate Cox regression analyses. ^*P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression of IL1RL2 in BLCA, IL1RL2 mRNA expression was examined using data from the TCGA database. As illustrated in Fig. 1A, IL1RL2 expression in BLCA tissues from TCGA was significantly elevated compared with normal tissues. Data from TCGA indicated upregulated expression of IL1RL2 mRNA in high-stage and high-grade BLCA tissues (Fig. 1B and C). Additionally, IL1RL2 mRNA was also highly expressed in patients with BLCA and lymph node metastasis (Fig. 1D).

RT-qPCR was employed to assess IL1RL2 mRNA levels specifically in BLCA cell lines. The results revealed higher IL1RL2 mRNA expression in the tumor cell lines compared with SV-HUC-1 cells, as depicted in Fig. 2A. Meanwhile, the mRNA expression level of IL1RL2 in BLCA tissues was higher than that in adjacent normal tissues (Fig. 2C). Furthermore, western blot analysis demonstrated an upregulation of IL1RL2 protein expression in both BLCA cell lines (Fig. 2B) and tissues (Fig. 2D). To further understand the IL1RL2 presence in BLCA tissues, IHC was performed on 17 pairs of BLCA tissues and adjacent normal tissues. The results revealed that IL1RL2 was highly expressed in BLCA tissues compared with adjacent normal tissues (Fig. 2E and F).

IHC scoring based on different staining intensities (Fig. 3A) and areas was performed. The average IHC score of IL1RL2 expression in patients with high-stage and high-grade BLCA was significantly higher than in low-stage (P<0.001; Fig. 3B) and low-grade BLCA (P<0.01; Fig. 3C; Table I). Patients with BLCA with lymph node metastasis demonstrated a significantly higher average IHC score of IL1RL2 expression compared with non-metastatic patients (P<0.001; Fig. 3D).

To evaluate the diagnostic potential of IL1RL2, the differential expression in BLCA vs. adjacent normal tissues was analyzed, generating receiver operator characteristic (ROC) curves using data from the TCGA database and a cohort of 112 patients with BLCA. The ROC curves revealed that IL1RL2 expression distinguished tumors from adjacent normal tissues with an AUC of 0.700 (95% CI: 0.579-0.821) in TCGA and 0.647 (95% CI: 0.497-0.797) in Miyun chart database (Fig. 4A and B). IHC analysis of 112 patients with BLCA at different stages and grades revealed a correlation between OS time and IL1RL2 expression (Fig. 4C). For OS analysis, in univariate analysis, IL1RL2 expression was negatively correlated with OS (95% CI: 0.072-0.552; P=0.002); and smoking (95% CI: 1.091-22.834; P=0.038), age (95% CI: 1.035-1.175; P=0.003) and lymph node metastasis (95% CI: 0.024-0.502; P=0.004) were also factors influencing OS (Table II). In multivariate analysis, smoking (95% CI: 1.024-28.661; P=0.047), age (95% CI: 1.028-1.190; P=0.007) and IL1RL2 expression (95% CI: 0.089-0.988; P=0.048) were associated with poorer OS. A line chart predicting survival was constructed based on smoking, age and IL1RL2 (Fig. 4D). For RFS analysis, in univariate analysis, sex (95% CI: 0.116-0.939; P=0.038) and pathological stage (95% CI: 0.136-0.989; P=0.047) were factors influencing RFS, while IL1RL2 was not a factor for RFS (Table SI). In multivariate analysis, sex (95% CI: 0.332 (0.116-0.949; P=0.0039) was associated with poorer RFS, suggesting that sex may be an independent prognostic factor for patients with BLCA. In TCGA database, High levels of IL1RL2 expression were associated with lower disease-specific survival and progression-free survival (Fig. 4E and F).

To explore the functional implications of IL1RL2 in BLCA, enrichment analysis of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed using genes co-expressed with IL1RL2 obtained from the TCGA BLCA database. This analysis aimed to uncover the biological roles associated with IL1RL2 in BLCA. In GO enrichment analysis, cell-cell junction, epidermis development and MAP kinase activity were enriched (Fig. 5A). KEGG enrichment highlighted MAPK signaling pathway and Salmonella infection (Fig. 5A). Additionally, Gene Set Enrichment Analysis (GSEA) in BLCA indicated that elevated IL1RL2 expression was linked to several critical processes that promote tumorigenesis, including tumor immune infiltration-related processes such as CD8 T cells and memory CD4 (Fig. 5B), suggesting that IL1RL2 may facilitate bladder tumor development. Protein-protein interaction analysis revealed that potential targets of IL1RL2 were primarily genes associated with cell proliferation and migration (Fig. 5C). Immuno-infiltration analysis of IL1RL2 in BLCA revealed potential associations with plasmacytoid dendritic cells (pDC) and T follicular helper cells (TFH) (Fig. 5D). Overall, these results suggested that IL1RL2 may regulate tumor development through modulation of the tumor immune microenvironment and MAPK signaling pathway.