Dissecting the spatial architecture of plasmacytoid urothelial carcinoma to inform therapeutic strategies

Plasmacytoid urothelial carcinoma (PUC) is an aggressive histologic subtype of bladder cancer that frequently co-exists with conventional urothelial carcinoma (CUC). Patients with PUC have worse response to cisplatin-based neoadjuvant chemotherapy and worse outcomes than patients with CUC, emphasizing the importance of therapeutic development. Elucidating the biology of plasmacytoid urothelial carcinoma (PUC) may reveal therapeutic vulnerabilities specific to this variant. PUC is defined by loss of E-cadherin protein expression, and evidence from other cancer types suggests a complex relationship between E-cadherin signaling, epithelial-to-mesenchymal transition (EMT), tumor microenvironment (TME) remodeling, and responsiveness to immune checkpoint blockade. This study seeks to understand the impact of E-cadherin loss on TME composition in PUC and the crosstalk between cell types. Archival formalin-fixed paraffin embedded tissue samples from patients with urothelial carcinoma who underwent surgical intervention was obtained. Hematoxylin and eosin stains were performed and reviewed by a GU pathologist to identify areas of histology consistent with conventional or plasmacytoid UC. Immunohistochemistry for E-cadherin confirmed loss of E-cadherin in histologic areas of PUC. The 10X Genomics Xenium In Situ assay was utilized to perform spatial transcriptomics. The expression of 477 genes was profiled in situ using a combination of the 10X Genomics Human Multi-Tissue and Cancer panel and a custom 100-gene panel, designed to evaluate the expression of bladder cancer specific genes. Xenium data analysis was performed using a combination of the 10X Genomics Xenium Explorer and R-based packages, including Seurat and HoodScanR. Analysis focused on a mixed histology tumor with histologic areas of PUC and CUC. Within areas of PUC histology, CDH1 (gene encoding E-cadherin) RNA expression was absent, and genes associated with EMT signaling (Snail, vimentin) were upregulated. Relative proportions of immune cells were characterized and PUC-specific cellular neighborhoods were identified. In the area of PUC histology, increased infiltration of macrophages was identified, with different macrophage phenotypes identified between PUC and CUC areas. Using spatial transcriptomics at single cell resolution, this study delineates PUC architecture and demonstrates the interplay between E-cadherin loss, EMT, and macrophage activation. Further characterization of PUC-specific expression and TME components will identify potential therapeutic vulnerabilities.