Establishment and Authentication of Cell Lines

The human HNC cell lines HN4 and HN30 were kindly provided by the University of Maryland Dental School, USA. CAL 27, 293 T and MC-3 T3-E1 cells were purchased from the American Type Culture Collection (ATCC, USA). Human primary fibroblasts were isolated from tumors (CAFs) and adjacent normal tissues (NFs) from HNC patients by primary culture and were identified by the presence of CAF-specific markers (α-SMA, FSP1, FAP) [23 (link)]. All these cells except MC-3 T3-E1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified 5% CO₂ atmosphere, while MC-3 T3-E1 cells were maintained in MEM-α medium containing 10% FBS. All cell lines have been authenticated by short tandem repeat technology (STR) and tested for mycoplasma contamination.