Clonal evolution and stromal crosstalk drive an invasive epithelial program in bladder cancer.

Tumor progression and metastasis in bladder cancer are driven by epithelial cell heterogeneity and dynamic interactions with the tumor microenvironment. To elucidate epithelial subpopulations associated with cancer progression, we performed single-cell transcriptomic profiling of bladder cancer tissues and identified a distinct epithelial subset, termed Meta-program 6 (MP6), that was significantly enriched in samples exhibiting lymphovascular invasion or lymph node metastasis. The MP6 gene signature correlated strongly with advanced tumor stage, lymph node involvement, and lymphovascular infiltration. CNV analysis revealed extensive chromosomal alterations, particularly chromosome 19 deletion, indicating genomic instability. Cell-cell communication analysis demonstrated active crosstalk between MP6 tumor cells and cancer-associated fibroblasts, including WNT and BMP signaling pathways, indicating that stromal crosstalk may contribute to the establishment of a pro-tumorigenic microenvironment. Transcription factor network inference further identified elevated activity of regulators such as TFCP2 and ELF1, implicating them in the acquisition of aggressive phenotypes. Immunohistochemical validation supported the clinical relevance of selected MP6-associated targets. Collectively, these findings define a clonally evolved epithelial subtype associated with lymphatic invasion and provide mechanistic insights into tumor cell plasticity and stromal-epithelial interactions underlying bladder cancer progression.