Modulating cancer stemness in commercial OCSCC spheroid cultures through microenvironmental stress

Oral cavity squamous cell cancer (OCSCC) recurrence is linked to cancer stem cells (CSCs), which persist despite treatment. To enable reproducible CSC studies, we established a scalable system using the commercially available ATCC SCC-9 cell line subjected to physical and metabolic stress gradients-standard monolayer, AggreWell spheroids (moderate stress), and ultra-low attachment (ULA) spheroids (maximal stress)-in both nutrient-rich (RMk media) and nutrient-deficient (CSC media) conditions. SCC-9 cells were expanded in standard tissue culture flasks and seeded at defined concentrations into five experimental conditions: (1) standard attachment monolayer, (2) 24-well AggreWell 400 plates in RMk media, (3) 24-well AggreWell 400 plates in CSC media, (4) ULA plates in RMk media, and (5) ULA plates in CSC media. After four days, spheroids were imaged, dissociated with Accutase, and analyzed for CSC enrichment by flow cytometry. CD44 expression was quantified by median fluorescence intensity (MFI), and increased aldehyde dehydrogenase activity was detected using the Aldefluor assay. MFI for CD44 rose progressively with microenvironmental stress, from 18,340 (monolayer) to 42,924 (ULA plate in CSC media), representing a 2.3-fold increase. The double-positive CSC-like population (CD44high/Aldefluor+) was nearly absent in low-stress monolayer and moderate-stress AggreWell cultures, but increased to 0.12% in ULA RMk and tripled to 0.42% in ULA CSC media. These results demonstrate that both CSC prevalence and marker intensity can be systematically tuned by adjusting physical and metabolic stress. This protocol allows researchers to enhance CSC-like enrichment in a stepwise manner using a widely available OCSCC cell line. The methodology supports applications in drug testing, differentiation studies, and immune interaction assays, leveraging commercial plates and reagents for reproducibility across laboratories. The dynamic range of CSC induction provides a practical tool for studying OCSCC recurrence and therapeutic resistance.