HNSCC Tumor Sphere Culture Assay

HNSCC cell lines CAL27 and FaDu were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12, 10% fetal bovine serum (FBS), at 5% CO₂ and 37 °C humidified incubators with anti-vibration platform. For the tumor sphere culture assay, single-cell suspensions were resuspended in culture media containing 1% N2 supplement (Gibco), 2& B27supplement, 20 ng/mL basic fibroblast growth factor (bFGF-2, R&D), and 10 ng/mL epithelial growth factor (EGF, R&D) and plated in ultra-low attachment plates (Corning) at a density of 1 × 10³ cells per well as previously reported49 (link). Medium was replenished twice a week and spheres counted within 2 weeks. To evaluate the tumor sphere size, cells were plated in 96-well ultra-low attachment plates (Corning), at a density of 1 cell per well. The number and size of spheres formed were evaluated using an inverted microscope.