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

Qian et al. Cancer Cell International (2025) 25:376 https://doi.org/10.1186/s12935-025-04009-0 Cancer Cell International Open Access RESEARCH Single-cell stemness analysis highlights Midkine-LRP1 pathway and identifies new bladder cancer subtypes Cheng Qian4†, Jin Qin3, Zixian Lin1, Shenggan Wang1, Anmei Deng4* and Tengda Li1,2*† Abstract 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. Keywords Bladder cancer, Stemness, Single-cell sequencing, Cell chat, Protein interface analysis, ACTN1, Midkine, LRP1 † Cheng Qian and Tengda Li have contributed equally to this work. *Correspondence: Anmei Deng Tengda Li 1 School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China 2 Department of Chemistry, Fudan University, Shanghai 200438, China 3 Fudan University, Shanghai 200438, China 4 Institute of Integrated Chinese and Western Medicine, PuDong Traditional Chinese Medicine Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201200, China © The Author(s) 2025. 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To view a copy of this licence, visit http://creati vecommons.org/licenses/by-nc-nd/4.0/. Qian et al. Cancer Cell International (2025) 25:376 Introduction Bladder cancer (BC) poses a substantial global health burden, owing to its consistently high incidence and associated mortality [1, 2]. According to the World Health Organization (WHO), there were 610,000 new cases of BC and 220,000 deaths globally in 2022 [3]. The traditional classification of BC based on histopathology revealed that urothelial carcinoma is the most common subtype [4]. Among all patients with BC, 25% have muscle-invasive BC and 75% have non-muscle-invasive BC [5, 6]. They were also classified by the Tumor Node Metastasis (TNM) system [7]. BC is staged using the TNM system: the T category ranges from Tis (carcinoma in situ) and Ta (non-invasive papillary carcinoma) through T1 (invasion of the lamina propria), T2 (invasion of the muscularis propria), T3 (extension into perivesical tissue), to T4 (invasion of adjacent organs or pelvic/abdominal wall); the N category classifies regional lymph node involvement as N0 (no metastasis), and N1–N3 denote progressively increasing degrees of regional lymph node involvement and the M category distinguishes M0 (no distant metastasis) from M1 (distant metastasis present) [8–10]. Although the clinical features and classification of BC have been previously described, patients are often treated with uniform therapeutic approaches, disregarding tumor heterogeneity [4–7]. This lack of individualized treatment frequently contributes to poor clinical outcomes. Moreover, the key molecular drivers underlying BC development and progression remain insufficiently characterized [11]. Therefore, establishing a more refined classification system for BC is essential to identify potential therapeutic targets and to enable clinicians to design more precise and effective treatment strategies. BC stem cells are the main causes of tumor complex biological behavior and are involved in tumor occurrence, recurrence, and metastasis [12]. Reportedly, BC stem cells maintain the tumor cells’ self-renewal and proliferation and can migrate to distant organs to promote metastasis [12]. Normal stem cells with fibroblast growth factor receptor 3 mutations can transform into BC stem cells and promote the formation of non-muscle invasive BC, while mutations in p53 or PTEN may promote the development of muscle-invasive BC[12]. The signatures in the crosstalk between mesenchym al stem cells and BC stem cells can predict the patients’ prognosis or immune response [13]. Single-cell sequencing (scSeq) is a powerful technique for amplifying and sequencing transcriptome or genome at a single-cell level [14]. It enables the identification of novel tumor cell subsets and the construction of comprehensive single-cell gene expression profiles [15–17]. Prior to the advent of scSeq, conventional highthroughput sequencing approaches often overlooked genetic material derived from cancer stem cells and other Page 2 of 18 low-abundance yet functionally critical cell populations [14, 18]. The development of single-cell sequencing has effectively addressed this limitation [14, 18]. For instance, in breast cancer, scSeq has been employed to elucidate the dynamic metabolic evolution of early disseminated lesions [19]. More recently, scSeq has also been utilized to invest (...truncated)