Glutamine

The human oral squamous cell carcinoma (OSCC) cell lines (SAS and SCC9) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). SCC9 cells were cultured in Dulbecco’s modified Eagle’s medium-F12 supplemented with 10% fetal bovine serum (FBS), 1% NEAA, 1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4), hydrocrostine (0.4 mg/L), 1 mM sodium pyruvate and 2 mM glutamine (Sigma, St. Louis, Mo, USA). SAS cells were cultured in Dulbecco’s modified Eagle’s medium-F12 supplemented with 10% FBS, 1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4) and 1 mM sodium pyruvate. The cells culture was maintained at 37 °C in a humidified atmosphere of 5% CO₂.

Human fibroblasts were seeded onto T75 flasks previously coated with 0.1% gelatin (Sigma) at a concentration of 7.8 × 10⁴ cells/cm². Then, 24 h after plating, cells were exposed to the epigenetic eraser 5-aza-CR (Sigma) at a 1µM concentration for 18 h [30 (link),31 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 (link)]. At the end of 5-aza-CR exposure, 7 × 10⁶ epigenetically erased human dermal fibroblasts (hEpiE) were resuspended in 300 μL of DMEM supplemented with 10% FBS, 2 mM glutamine (Sigma, Milan, Italy), and 1% antibiotic/antimycotic solution (Sigma, Milan, Italy) and seeded onto scaffold fragments to repopulate the decellularized ovarian ECM-based scaffolds. Cell repopulation was performed in 5% CO₂ at 37 °C for 7 days. Half of the medium volume was refreshed every other day. Cultures were arrested after 24 and 48 h and at Day 7. Samples were subjected to histological evaluations, DNA quantification, the TUNEL assay, and gene expression analysis. hEpiE plated in standard plastic 4-well multidishes (Nunc, Milan, Italy) and cultured in DMEM supplemented with 10% FBS, 2 mM glutamine (Sigma, Milan, Italy), and 1% antibiotic/antimycotic solution (Sigma, Milan, Italy) were used as the control.

The GSC lines GB7 and G166 were obtained from human GBM biopsies [9 (link),46 (link),47 (link)]. The cells were cultured on laminin-coated dishes (1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) or as neurospheres (on uncoated plastic) and maintained in serum free medium consisting of DMEM/F12 (1:1; v:v) (Corning, New York, NY, USA) supplemented with 1% streptomycin, 50 IU/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA), 1% glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% N2 supplement (Invitrogen, Monza, Italy), 2% B27 (Invitrogen, Monza, Italy), 20 ng/mL EGF (Recombinant Human Epidermal growth factor, Peprotec, London, UK), and 20 ng/mL FGF (Recombinant Human FGF-basic, ABM, Richmond, Canada). The cultures were maintained at 37 °C in an atmosphere of 5% CO₂/95% air. Human glioblastoma U251MG cell line was cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA) plus 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 50 µg/mL streptomycin, 50 IU/mL penicillin, 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% non-essential amino acids (Sigma-Aldrich, St. Louis, MO, USA), and maintained at 37 °C in a 10% CO₂ atmosphere.

Monocytic Raw264.7 (ATCC^®, Manassas, VI, USA, US-TIB-71™) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, Waltham, MS, USA) in the presence of 10% fetal bovine serum (FBS), 2 mM glutamine (Sigma Aldrich, Darmstadt, Germany) and antibiotic solution (Sigma Aldrich, Darmstadt, Germany). Immature dendritic JAWSII cells (ATCC^® CRL-11904™) were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) (Thermo Fisher Scientific, Waltham, MA, USA) in the presence of 20% FBS 4 mM glutamine, 1 mM sodium pyruvate and antibiotic solution (Sigma Aldrich). Pancreatic islet endothelial MS1 cells (MILE SVEN 1) (ATCC^® CRL-2279™) were cultured in DMEM with 5% FBS and antibiotic solution.

For glutamine supplementation experiments, 4 % glutamine (Sigma) in sterile tap water was made fresh daily and offered as the sole source of drinking water for 5 consecutive days (Monday through Friday). Control mice were fed with only sterile tap water. To avoid undue stress from elevated ammonia concentrations, all mice drank glutamine-free water 2 days each week (Saturday and Sunday).

Patient-derived BT168 GSCs [50 (link),86 (link)] were cultured in serum-free medium consisting of DMEM/F12 (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 20 ng/mL EGF, 20 ng/mL bFGF, 2% B27 w/o vitamin A (Thermo Fisher Scientific, Waltham, MA, USA), 1% glutamine and 1% antibiotics (Sigma-Aldrich) in an incubator under standard culture conditions. GSC proliferation generated floating rounded spheres with well-defined borders, which were kept until they were suitably sized to ensure cell health, and well spaced from each other to avoid sticking. Twice a week, subconfluent spheres were centrifuged at 400 rpm for 20 min and then incubated with accutase (Thermo Fisher Scientific) for 15 min at 37 °C. Single GSCs were centrifuged at 800 rpm for 5 min and seeded in fresh medium. U87MG cells (from the American Type Culture Collection, Manassas, VA, USA) were routinely grown in DMEM supplemented with 10% FBS, 1% glutamine, and 1% antibiotics (Sigma-Aldrich). DETA/NO, TMZ and DMSO were from Sigma-Aldrich. DETA/NO spontaneously releases NO in aqueous media with a half-life of 20 h. Therefore, it was dissolved in sterile water and supplied to the cells every 48 h. TMZ was dissolved in DMSO and supplied every 72–96 h. Parallel to TMZ treatment, control cells were always supplied with DMSO, diluted as TMZ.