Single-cell stemness analysis highlights Midkine-LRP1 pathway and identifies new bladder cancer subtypes.

Bladder cancer (BC) is among the ten most common malignant tumors worldwide. Tumor stem cells contribute significantly to postoperative recurrence and disease progression. Understanding stem cell interactions with other tissue cells and developing a prognostic model may improve BC management. Through single-cell RNA sequencing, we identified tumor stem cells in BC tissue. We identified 91 genes specifically upregulated in the stem cell cluster versus other clusters. Of these, 67 whose high expression correlated with poorer patient survival were defined as high-risk stemness genes (HRSGs). Further analysis showed that the MDK-LRP1 axis is the primary communication pathway between stem cells and other cell types, involving 31 of these HRSGs. Based on these 31 HRSGs, patients were stratified into two stemness clusters (ST cluster A and B), with ST cluster B associated with poorer prognosis. We further selected prognosis-related genes from differentially expressed genes between the clusters to construct a risk model. Patients in ST cluster B exhibited higher risk scores, aligning with clinical outcomes. Among the HRSGs, ACTN1 emerged as a key gene, showing elevated expression in patients with poor survival and advanced disease stages. Immunohistochemistry confirmed significantly increased ACTN1 protein levels in BC tissues. Additionally, protein interface analysis indicated that the Cys104 residue of Midkine potentially interacts with both LRP1 and ACTN1 within a 5 Å distance, suggesting a critical interaction site. These findings provide novel insights into stem cell-mediated BC progression and offer potential prognostic and therapeutic targets.