Single-cell transcriptome analysis reveals DNMT1+ epithelial cells promote lymphatic metastasis via CXCL17-mediated TAM infiltration

Lymph node (LN) metastasis is the leading cause of unfavorable prognosis in bladder cancer (BCa), which involves a highly complex tumor microenvironment. The detailed molecular mechanisms that drive BCa LN metastasis, however, are not yet fully elucidated. In this study, single-cell transcriptomic profiling of 153,339 cells from seventeen BCa patients with or without LN metastases was analyzed. We integrated single-cell transcriptomic data and bulk RNA sequencing data, and employed multiple strategies for the analysis and identification of heterogeneity in epithelial and macrophage cells in BCa. Furthermore, mouse model and cell experiments were performed to validate and dissect the characteristics of the immunosuppressive microenvironment for LN metastases. This study found that DNMT1+ epithelial cells, characterized by a high potential for epithelial-mesenchymal transition, exhibited a propensity for LN metastasis. Notably, a specific subpopulation of tumor-associated macrophages (TAMs), marked by selective expression of SELENOP, has been identified. Intercellular crosstalk analysis observed the CXCL signal flow directed primarily from DNMT1+ epithelials to SELENOP+ TAMs. Through both in vitro and in vivo validation, it was suggested that DNMT1 promotes the recruitment and M2-type polarization of TAMs by enhancing CXCL17 expression and secretion. Additionally, CXCL17-mediated reprogramming of TAMs is associated with increased lymphangiogenesis and LN metastasis in BCa. Mechanistically, DNMT1 was found to bind the PTP1B promoter and enhance methylation enrichment at this region, which is consistent with reduced PTP1B expression and activation of JAK2/STAT3 signaling, thereby promoting the expression and secretion of CXCL17. These findings uncover key molecular mechanisms that shape the lymphatic metastatic niche and offer a foundation for novel strategies aimed at targeting pro-metastatic elements to suppress BCa progression and metastasis.