Single-cell and spatial transcriptomics reveal a DLL4-associated tip-like endothelial niche linked to neoadjuvant chemotherapy non-response in muscle-invasive bladder cancer.

Chemotherapy non-response remains a major challenge in bladder cancer, yet the endothelial states and intercellular communication programs associated with treatment failure remain poorly understood. Here, we integrated single-cell RNA sequencing, spatial transcriptomics, bulk transcriptomic data, computational analyses, and in vitro experiments to characterize endothelial programs associated with chemotherapy response. A non-response-related transcriptional program was preferentially enriched in endothelial cells and was most prominent in the TipEC_2 tip-like endothelial subset. Integrative analyses identified DLL4 as a key feature of this endothelial state. Spatial and ligand-receptor analyses further supported a DLL4-associated tip-like endothelial niche and inferred DLL4-NOTCH3-related communication between endothelial and malignant compartments, together with RBPJ-associated transcriptional activity in DLL4-positive TipEC_2 cells. Consistently, cisplatin-resistant bladder cancer cells showed increased expression of NOTCH3 and downstream transcriptional targets, whereas pharmacological Notch inhibition suppressed downstream HES1 and HEY1 expression and partially restored cisplatin sensitivity. Elevated DLL4 expression was associated with chemotherapy non-response and unfavorable survival. In a separate prognostic analysis, a DLL4-positive TipEC_2-associated risk model stratified patients by overall survival in TCGA-BLCA and GSE13507. Collectively, these findings identify a DLL4-associated tip-like endothelial niche linked to chemotherapy non-response and suggest DLL4-NOTCH3 signaling as a potential molecular link between vascular remodeling and treatment resistance in bladder cancer.