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DOI

10.1016/j.jds.2025.05.032

First Page

2283

Last Page

2291

Abstract

Abstract Background/purpose Oral squamous cell carcinoma (OSCC) often recurs and has poor clinical outcomes, partly attributable to subpopulations that develop resistance to 5 fluorouracil (5FU). Elucidating how these resistant clones emerge and drive tumour aggressiveness is essential for improving OSCC treatment approaches. Materials and methods To establish 5FU-resistant cells, SCC25 cells were repeatedly exposed to 5FU, and single-cell clones were subsequently isolated using a microfluidic system. Three subclones-Holoclone, Meroclone, and Paraclone-were evaluated for their 5FU responses, expression of drug-efflux pumps (ABCB1, ABCG2), and resistance in three-dimensional (3D) cultures. Their levels of cancer stem cell (CSC) markers (OCT4, SOX2, CD44, CD133) and epithelial–mesenchymal transition (EMT) markers (E-cadherin, Vimentin, Twist) were also examined. In addition, Transwell assays were performed to assess migration and invasion. Results Compared with parental SCC25 cells, the three subclones exhibited markedly higher resistance to 5FU under 3D spheroid conditions, concurrent with upregulated ABCB1 and ABCG2 expression. All three subclones showed enhanced sphere-forming capacity and increased OCT4 and SOX2 levels, consistent with higher proportions of CD44+/CD133+ cells. Moreover, Holoclone, Meroclone, and Paraclone each displayed reduced E-cadherin alongside elevated Vimentin, and Twist, characteristic of EMT. Transwell assays confirmed increased migration and invasion, with Holoclone and Paraclone exhibiting particularly pronounced effects. Conclusion Extended 5FU treatment in OSCC selects for distinct subclones that exhibit CSC-like traits and EMT-related motility, promoting robust chemoresistance and heightened malignancy. These findings emphasise the importance of developing comprehensive therapeutic strategies that simultaneously target drug-efflux mechanisms, CSC markers, and EMT pathways to more effectively control OSCC progression.

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