Single-Cell Analyses Reveal Mechanisms of Cancer Stem Cell Maintenance and Epithelial-Mesenchymal Transition in Recurrent Bladder Cancer

Purpose: Bladder cancer treatment remains a major clinical challenge due to therapy resistance and a high recurrence rate. Profiling intratumor heterogeneity can reveal the molecular mechanism of bladder cancer recurrence. Experimental Design: Here, we performed single-cell RNA sequencing and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on tumors from 13 patients with low recurrence risk, high recurrence risk, and recurrent bladder cancer. 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 bladder cancer 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 bladder cancer subtypes. We identified that TCF7 promotes EMT in corroboration with single-cell ATAC with high-throughput sequencing (scATAC-seq) analysis. Additionally, we constructed regulatory networks specific to recurrent bladder cancer. Conclusions: Our study and analytic approaches herein provide a rich resource for the further study of cancer stem cells and EMT in the bladder cancer research field.

Automated summary

Bladder cancer treatment is challenging due to therapy resistance and high recurrence rates. Profiling intratumor heterogeneity can uncover molecular mechanisms of recurrence. This study used single-cell RNA sequencing and ATAC-seq to analyze tumors from different risk groups and recurrent cases, creating a comprehensive cancer-cell atlas. The research identified a cancer stem-cell subpopulation enriched during recurrence, highlighting the role of EZH2 in maintaining repression of specific genes related to invasion and stemness. Additionally, the study investigated epithelial-mesenchymal transition in clinical samples and identified regulatory networks specific to recurrent bladder cancer.