Primary HNSCC Tumor Cell Lines

Primary tumor cell lines were obtained from patients treated at the OHSU Department of Otolaryngology for HNSCC from 2014 to 2019 (Dermatology Molecular Profiling Resource Repository, IRB #10071). Cell culture methods have been previously described.^{24 (link),34 (link)} Primary tumor samples were collected from the operating room in DMEM/F12 Media, supplemented with 2x antibiotic/antimitotic (Invitrogen, Carlsbad, CA). Tissues were washed 3 times in fresh DMEM/F12 Media and minced before plating. Cells were cultured in DMEM/F12 Media (Gibco, 11320082), supplemented with 5% supplemented BCS (Hyclone), 1x antibiotic/antimitotic (Invitrogen, Carlsbad, CA), 1.8 × 10–4 M adenine (Sigma, St. Louis, MO), 0.4 μg/mL hydrocortisone (Sigma), 1 × 10–10 M cholera enterotoxin (Sigma), 2 × 10–9 M triiodothyronine (Sigma), 5 μg/mL insulin (Sigma), and 10 μg/mL epidermal growth factor (Invitrogen). Fibroblasts were removed from primary cultures by differential trypsinization using Trypsin-EDTA (0.25%), phenol red (Gibco, 25200056). Frozen cultured cell lines were used within five passages of the original tumor.