Analysis of tumor stem cell recruitment and integration in microvascular architectural heterogeneity during oral mucosal carcinogenesis: an in vivo experimental study

Tumor stem cells (TSCs) play a central role in oral mucosal carcinogenesis by promoting tumor growth, invasion, and angiogenesis. Microvascular architectural phenotype heterogeneity (MAPH) and vasculogenic mimicry (VM) contribute to vascular complexity within tumors and resistance to conventional anti-angiogenic therapies. However, the mechanisms through which TSCs influence MAPH and VM formation during oral mucosal carcinogenesis remain poorly understood. In this study, we aimed to investigate the role of TSCs in mediating MAPH and VM throughout oral mucosal carcinogenesis. Animal models representing normal mucosa, precancerous lesions, and high-differentiated, moderate-differentiated, and low-differentiated squamous carcinomas were established. TSCs derived from HSC4 cells (isolated through laser capture microdissection (LCM) and validated using stemness markers and sphere-forming assays) were injected into the experimental models. Data were analyzed using one-way analysis of variance (ANOVA) and nonparametric tests, with multiple testing correction applied for proteomic analyses. Microvascular morphology analysis revealed stage-specific heterogeneity. In the TSC-treated groups, fused morphology predominated in the precancerous and highly differentiated stages, budding morphology appeared in the moderately differentiated stage, and finger-like morphology was characteristic of the poorly differentiated stage. LCM proteomics identified 2,215 differential proteins, with angiopoietin-like protein 2 (ANGPTL2), cytokeratin 15 (CK15), and eukaryotic translation elongation factor 1 epsilon 1 (EEF1E1) as key candidates. In the TSC-treated groups, CK15 was upregulated, whereas ANGPTL2 and EEF1E1 were downregulated. In the control groups, CK15 gradually increased across stages, ANGPTL2 peaked in highly differentiated carcinomas, and EEF1E1 peaked in moderately differentiated carcinomas. TSCs intervention shifted ANGPTL2 and EEF1E1 peaks to the precancerous and highly differentiated stages, respectively. These findings suggest that targeting these molecules may provide novel anti-angiogenic strategies for preventing or treating oral mucosal carcinogenesis.