Unravelling the heterogeneity in oral cancer stem cells – A single-cell computational exploratory study

Cancer stem cells (CSCs) are key drivers of heterogeneity, recurrence, and therapy resistance in oral squamous cell carcinoma (OSCC). CD44, CD133, ALDH, and BMI1 have been highlighted as clinically relevant CSC markers, yet no single marker sufficiently defines CSC identity. This study aims to explore computationally CSC heterogeneity in Asian-derived ORL cell lines (ORL-48, ORL-115, ORL-174, ORL-214). Unsupervised clustering of single-cell qPCR datasets revealed enrichment of quadruple-positive (CD44⁺/CD133⁺/ALDH1A1⁺/BMI1⁺) subsets, alongside smaller heterogeneous fractions. PhenoGraph clustering and viSNE analyses of arcsinh-transformed flow cytometry datasets independently confirmed dominant CD44⁺ and BMI1⁺ fractions, smaller CD133⁺ and ALDH⁺ subsets, and rare CSC-enriched multi-marker clusters consistent with the single-cell qPCR analysis. In addition, sphere formation assays further validated the presence of self-renewing CSC-like populations across all lines. As this work was designed as an exploratory study, analyses were performed once per cell line without replicates; future studies will incorporate independent validations.