Single-cell profiling uncovers a rare mesenchymal-like urothelial state associated with sarcomatoid bladder cancer

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