Expression Profiles of Co-Inhibitory Receptors in Non-Urothelial Bladder Cancer: Preclinical Evidence for the Next Generation of Immune Checkpoint Inhibitors

Abstract

Immune checkpoint inhibition is a cornerstone of bladder cancer therapy, but its efficacy in non-urothelial subtypes of bladder cancer is limited, and the prognosis remains poor. Therefore, we investigated the potential of the immune checkpoint molecules TIM-3, TIGIT, and LAG-3 in squamous-cell carcinoma (SCC) and adenocarcinoma (ADENO) of the urinary bladder. Tumor-infiltrating lymphocytes (TILs) showed a high expression of TIM-3 and TIGIT in both SCC and ADENO, while LAG-3-positive TILs were absent in ADENO and present in 46% of SCC. Quantitative analysis revealed age-independent expression of TIM-3 in SCC (r = -0.001, p = 0.997) and ADENO (r = 0.135, p = 0.549), with increasing age correlating with higher expression of TIGIT (r = 0.157, p = 0.242) and LAG-3 (0.106, p = 0.436) in the SCC cohort and of TIGIT (r = 0.276, p = 0.214) in the ADENO cohort. Male patients showed increased TIGIT scores in ADENO (p < 0.01). Of note, a high infiltration of TIM-3-TILs (p = 0.048) correlated with worse progression-free survival in SCC. These results highlight the differential expression of co-inhibitory receptors in non-urothelial bladder cancer subtypes and provide preclinical evidence for new therapeutic targets. Biomarker testing prior to clinical trials is essential for identifying the most suitable patients for targeted immunotherapy.

Keywords: LAG-3; TIGIT; TIM-3; adenocarcinoma; bladder cancer; coinhibitory receptor; squamous-cell carcinoma; variant histology.

Figure 1

Impact of age (A,D,G), sex (B,E,H), and local tumor stage (C,F,I) on expression patterns of coinhibitory receptors (TIM-3: (A–C); TIGIT: (D–F); LAG-3: (G–I)) in variant histologies. Red indicates ADENO samples and blue SCC samples in (A,D,G). Red violin plots indicate female and blue male samples in (B,E,H). In (C,F,I), red violin plots indicate <pT3 samples whereas ≥pT3 samples are marked in blue.

Figure 2

Prognostic value for overall and progression-free survival of TIM-3, LAG-3, and TIGIT. Prognostic value of inhibitory coreceptor expression was analyzed for overall survival (OS) and progression-free survival (PFS) for adenocarcinoma (ADENO—(A,B,E,F,I,J)) and squamous-cell carcinoma (SCC—(C,D,G,H,K,L)). The median expression of the respective coreceptor was used as a cut-off. Abbr.: ADENO: adenocarcinoma, SCC: squamous-cell carcinoma, OS: overall survival, PFS: progression-free survival, RC: radical cystectomy.