Establishing Head and Neck Cancer Cell Lines

As previously described 29 (link), the human HNC cell lines HN4, HN6 and HN30 were kindly provided by the University of Maryland Dental School, USA. SCC-4, SCC-9, SCC-25 and CAL-27 cells were purchased from the American Type Culture Collection (ATCC, USA). The human HB cell line was derived from HIOEC by treatment with benzo[a]pyrene 30 (link) and the Rca-T cell line was purified from tongue squamous cell carcinoma tissues induced by adding 4-nitroquinoline-1-oxide into Sprague-Dawley rats' drinking water 31 (link). CAFs and normal fibroblasts (NFs) were isolated from tumor and adjacent normal tissues of HNC patients by primary culture and were identified by the presence of CAF-specific markers (α-SMA, Vimentin). All these cells except SCC-4, SCC-9 and SCC-25 were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL, USA) supplemented with 10% heat-inactivated FBS (GIBCO-BRL), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37°C in a humidified 5% CO₂ atmosphere, while SCC-4, SCC-9 and SCC-25 cells were maintained in DMEM/F12 medium containing 10% FBS. In addition, normal primary head and neck epithelial cells were cultured in keratinocyte serum-free medium (KSF; GIBCO-BRL, USA) with 0.2 ng/mL recombinant epidermal growth factor (rEGF; Invitrogen, USA).