Single-cell transcriptomic profiling of muscle-invasive bladder cancer reveals differentiation-dependent ADC target dynamics and tumor-immune checkpoint interactions

Antibody-drug conjugates (ADCs) targeting NECTIN4, TACSTD2/TROP-2, and ERBB2/HER2 have demonstrated clinical efficacy in urothelial carcinoma; however, few studies have systematically characterized the single-cell expression landscape of these targets in relation to tumor differentiation states, intrinsic biological programs, or the tumor immune microenvironment. We aimed to construct one of the largest integrated single-cell atlases of muscle-invasive bladder cancer (MIBC) and define the biological determinants of ADC therapeutic vulnerability and identify novel intercellular communication axes with translational relevance. We integrated single-cell RNA sequencing data from 21 MIBC patients (157,449 cells) across three independent cohorts. Consensus molecular subtype classification was performed at single-cell resolution within epithelial populations. Pseudotime trajectory inference using Slingshot was applied to map ADC target expression dynamics along the basal-to-luminal differentiation axis. CellChat analysis was employed to interrogate tumor-immune intercellular communication networks, including a custom-curated NECTIN4-TIGIT ligand-receptor pair. Cross-species validation was performed using normal human (GSE129845; n = 8,697 cells) and mouse (GSE164557; n = 13,058 cells) urothelial datasets. A pseudotime-derived differentiation signature was validated in the TCGA-BLCA cohort ( n = 403) for associations with molecular subtypes, pathway activities, clinicopathologic features, and survival outcomes. Single-cell analysis revealed that NECTIN4, TACSTD2/TROP-2, and ERBB2/HER2 are associated with distinct tumor-intrinsic biological programs. In patient-level analyses, TACSTD2/TROP-2 expression was associated with elevated EMT and ERBB2/HER2 with elevated ABC transporter activity, with modest associations with IFN-γ signaling. Pseudotime trajectory analysis showed that the three targets differ in the breadth along the differentiation axis: TACSTD2/TROP-2 was expressed broadly across the trajectory, ERBB2/HER2 was sharply localized to a narrow window co-localizing with elevated ABC transporter module scores, and NECTIN4 was virtually absent in early basal cells and rose through the partial-EMT transitional zone. CellChat analysis identified NECTIN4-TIGIT as the top-ranked predicted tumor-to-immune inhibitory checkpoint interaction, with tumor epithelial cells serving as the exclusive predicted source of NECTIN4-mediated TIGIT engagement. Interestingly, APP-CD74 emerged as a novel tumor-immune communication axis, with 26 significant intercellular interactions detected; TCGA-BLCA validation demonstrated that APP-high/CD74-low tumors exhibited the worst prognosis (HR = 2.00, 95% CI: 1.31–3.04, p = 0.001), suggesting that disruption of this axis may be associated with worse survival in bladder cancer. Cross-species analysis confirmed that broad TACSTD2/TROP-2 expression, with enrichment in more differentiated cells, is conserved in normal human and mouse urothelium, reflecting a physiological property of urothelial differentiation. In TCGA-BLCA, early-differentiation tumors demonstrated significantly worse overall survival (HR = 1.58, 95% CI: 1.12–2.24, p = 0.009) and were enriched for EMT (ρ = −0.398), IFN-γ response (ρ = −0.430), and proliferation signatures (ρ = −0.502), yet harbored elevated tumor mutational burden, suggesting potential opportunities for immunotherapy. By constructing one of the largest integrated single-cell transcriptomic atlases of MIBC, this study demonstrates that ADC target expression is dynamically regulated along the tumor differentiation trajectory and that the targets differ in the breadth of their expression along that trajectory, with implications for biomarker-guided patient stratification. The identification of NECTIN4-TIGIT as the top-ranked predicted tumor-to-immune inhibitory checkpoint interaction and APP-CD74 as a novel prognostic communication axis provides rationale for combination therapeutic strategies. The pseudotime-derived differentiation signature may serve as a potential biomarker for ADC therapy selection in urothelial carcinoma.