Single-cell analyses reveal mechanisms of cancer stem cell maintenance and epithelial-mesenchymal transition in recurrent bladder cancer.

Purpose: Bladder cancer (BC) treatment remains a major clinical challenge due to therapy resistance and a high recurrence rate. Profiling intratumor heterogeneity can reveal the molecular mechanism of BC recurrence. Experimental Design: Here, we performed single-cell RNA-seq and ATAC-seq on tumors from 13 patients with low recurrence risk, high recurrence risk and recurrent BC. Results: Our study generated a comprehensive cancer cell atlas consisting of 54,971 single cells and identified distinct cell subpopulations. We found that the cancer stem cell subpopulation is enriched during BC recurrence with elevated expression of EZH2. We further defined a subpopulation-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the NCAM1 gene, thereby inactivating the cell invasive and stemness transcriptional program. Furthermore, taking advantage of this large single-cell dataset, we elucidated the spectrum of epithelial-mesenchymal transition (EMT) in clinical samples and revealed distinct EMT features associated with BC subtypes. We identified that TCF7 promotes EMT in corroboration with scATAC-seq analysis. Additionally, we constructed regulatory networks specific to recurrent BC. Conclusions: Our study and analytical approaches herein provide a rich resource for the further study of cancer stem cells and EMT in the BC research field.