L glutamine

Human NSCLC cell lines (A549 and H1975) were purchased from the Shanghai Institute of Country Cell Bank. The cells were cultured in DMEM (Hyclone, Logan, UT, USA) containing 2 mM L-glutamine (Sangon Biotech, Shanghai, China) and 10% FBS (Gibco, Grand Island, NY, USA) in a 37ºC humidified atmosphere containing 5% CO₂. Digestion with trypsin-EDTA (Hyclone, Logan, Utah, USA) was performed when the cells were grown in culture flasks. The cells were subcultured (1:3) or used for experiments when the cell fusion proportion reached 90%.

The HUVECs were cultured in DMEM (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) enriched with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), bovine brain extract (12 µg/ml), L-glutamine (2 mmol/l) and sodium pyruvate (1 mmol/l) in the presence of penicillin (100 U/ml) and streptomycin (100 µg/ml; Sangon Biotech Co., Ltd).

Tumor tissue and adjacent healthy tissue samples were obtained from 52 patients with OSCC who were treated at Nanjing Stomatological Hospital (Nanjing, China) between July 2014 and July 2018. The inclusion criteria were as follows: i) Patients were diagnosed with OSCC by pathological biopsies; ii) patients with complete medical records; iii) patients with no history of another type of malignancy; and iv) patients provided written informed consent. The exclusion criteria were as follows: i) Patients who had been diagnosed with multiple diseases; and ii) patients who had received treatment prior to admission at Nanjing Stomatological Hospital. In total, the present study included 29 males and 23 females, with an age range of 33–65 years and a mean age of 45.3±4.4 years. The current study was approved by the Ethics Committee of Nanjing Stomatological Hospital (Nanjing, China).
The OSCC cell lines SCC090 and SCC25 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in Eagle's Minimum Essential Medium (ATCC) containing 2 mM L-glutamine (Sangon Biotech Co., Ltd., Shanghai, China) and 10% fetal bovine serum (FBS, Sangon Biotech Co., Ltd.) at 37°C with 5% CO₂.

Human embryonic kidney 293T cells, human HCC HepG2 cells were obtained from American Type Culture Collection (ATCC). Human HCC Huh7 cells were purchased from Sangon Biotech (#E680517, Shanghai, China). All three cell lines were cultured in DMEM medium, high glucose (#E600003, BBI Life Science, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (#10099141C, Gibco, Waltham, MA, USA), 2 mM l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Sangon Biotech).

Two human OsC cell lines, U-2OS and Soas-2, were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Human lung fibroblasts (MRC-5 cell line) were provided by Prof Shengquan Zhang (Department of Biochemistry and Molecular Biology, Anhui Medical University) as a gift. Cells were maintained in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL) (Sangon Biotech, Shanghai, China). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.