Dissecting the tumor microenvironment of advanced urothelial carcinoma pre and post enfortumab vedotin ± pembrolizumab: A single-cell spatial transcriptomic analysis.

818 Background: The current treatment landscape of metastatic urothelial cancer has recently expanded beyond platinum-based chemotherapy to include antibody-drug conjugates such as enfortumab vedotin and immune checkpoint inhibitors such as pembrolizumab. Nevertheless, treatment resistance and disease progression remain major challenges. Here, we apply single-cell spatial transcriptomics to characterize the locally advanced and metastatic urothelial carcinoma microenvironment and identify immune neighborhoods predictive of response to enfortumab vedotin ± pembrolizumab. Methods: Thirty-one formalin-fixed paraffin embedded (FFPE) bladder and metastatic site samples were obtained from ten patients with metastatic urothelial cancer and analyzed using the 10X Genomics Xenium In Situ Single-Cell Spatial Transcriptomics platform. The cohort included both primary bladder tumors and metastatic sites, totaling 27 tumor specimens and 4 matched benign samples. Patients received either singe-agent enfortumab vedotin (n = 9) or combination enfortumab vedotin plus pembrolizumab (n = 1). Three patients achieved a complete clinical response and contributed only pre-treatment samples. Five patients experienced disease progression and contributed both pre- and post-treatment samples. Two additional patients who progressed provided only two post-treatment samples. Data processing and downstream analysis were performed using Seurat v5.0. Results: In total, 419,644 single cells from ten patients passed quality control. In the pretreatment setting, CD4+ FOXP3+ regulatory T cells and CD8+ T cells were detected in significantly higher proportions among immune cells from patients who achieved a complete clinical response compared to nonresponders (p < 0.05). Spatial analysis revealed a distinct immune-enriched niche that was consistently present in responders (p < 0.05). This niche was predominantly composed of CD4+ T cells, CD4+ FOXP3+ regulatory T cells, CD8+ T cells, macrophages and dendritic cells. Ligand-receptor analysis demonstrated that this niche was highly enriched for antigen-presenting, costimulatory, and chemokine signaling pathways, indicative of active immune engagement. Cell-cell communication analysis further showed that CD8+ T cells served as the dominant signal senders, whereas dendritic cells and macrophages acted as the primary signal receivers, together defining a myeloid-T cell interactive microenvironment associated with therapeutic response. Conclusions: Spatial single cell-profiling revealed that patients responding to enfortumab vedotin ± pembrolizumab possess a pre-existing immune activated niche enriched in antigen presentation and T-cell co-stimulation.