Single-cell profiling identifies rare normal mesenchymal-like urothelial cells associated with sarcomatoid urothelial carcinoma [bulk RNA-seq]

The bladder urothelium is a stratified epithelium, categorized as basal, intermediate, and umbrella cells. While epithelial–mesenchymal transition is known to occur during bladder injury, regeneration, and sarcomatoid tumor formation, it remains unclear whether normal urothelial cells intrinsically possess a mesenchymal-like epithelial state. We analyzed scRNA-seq data of EPCAM+ urothelial cells from both mouse and human urothelium. Vimentin-positive cells were evaluated by immunostaining to determine their distribution in normal urothelial layers and sarcomatoid tumors. Here, we identified and characterized a rare population of mesenchymal-like urothelial cells, termed M-cells, representing ~1–3% of urothelial cells. M-cells co-express epithelial markers with mesenchymal and stemness genes and engage stem-associated transcriptional regulators. Lineage tracing with the Upk2-Cre mT/mG reporter mice confirmed their urothelial origin, excluding a fibroblast lineage. M-cells are enriched within the basal layer but present across all urothelial compartments. M-cells exhibit high transcriptional plasticity between basal cells and intermediate cells. In addition, we identified a 16-gene transcriptional signature uniquely expressed in M-cells but absent in fibroblasts. RNA deconvolution of MB49 tumors demonstrated strong enrichment of this signature, suggesting that MB49 tumors are more likely to represent sarcomatoid urothelial carcinoma rather than fibroblast-derived sarcoma. The clinical relevance of M-cells and the M-cell state was validated in primary human sarcomatoid tumors and their corresponding patient-derived xenografts. Together, these findings establish transcriptionally unique and rare mesenchymal-like cells within the normal urothelium as a novel highly plastic population with translational implications for both mouse and human sarcomatoid urothelial carcinoma.