Murine Glioma Cell Line Generation

Murine glioma cell lines were generated from retrovirus induced orthotopic murine glioma models as previously described^{21 (link)}. Briefly, C57Bl/6 neonatal (p4) mice harboring floxed p53 and stop-flox mCherry-luciferase were anesthetized using hypothermia and orthotopically injected with a PDGFA–internal ribosomal entry site (IRES)–cyclization recombination (Cre) retrovirus (stereotaxic coordinates relative to bregma: 1 mm anterior, 1 mm lateral, 1 mm deep), resulting in tumor cells that overexpress PDGFA and mCherry-Luciferase, and have deleted p53^{36 (link)}. End-stage tumors were harvested and tumor cells isolated and cultured in basal media (BFP), containing DMEM (Gibco™ 11965092) with 0.5% FBS (Gibco™ 16000044), antibiotic-antimycotic (Thermo Scientific 15240096), N2 supplement (Thermo Fisher Scientific, 17502-048), and 10 ng/ml each of recombinant human PDGF-AA (Peprotech, 100-13 A) and FGFb (Peprotech, 10018B50UG). Three biological replicates of PDGFA driven cells made from three independent tumors with the same genetic background were used for this study. A Pten^−/− P53^−/− PDGFB⁺ cell line was also used^{21 (link)}. All cells were grown at 37 °C with 5% CO2. For all murine glioma cell lines cysteine methionine deprived media was made from basal DMEM without cysteine, methionine and glutamine (Thermo Fisher Scientific, 21013024) that was supplemented with L-glutamine to a final concentration of 4 mM. All other components of the media were the same for control and CMD media. Human glioma cells were cultured as previously described^{37 (link),38 (link)}. TS543 cell neurosphere cell lines were cultured in Neurocult media as previously described^{37 (link)}, but were dissociated and plated in a single cell monolayer in 96 well plates in either BFP or cysteine methionine deprived BFP for dose response experiments. KNS42 cell lines were cultured in DMEM + 10%FBS or cysteine methionine deprived DMEM + 10%FBS. Thus, for all experiments the only difference between CMD media and control media used for each cell line was the concentration of cysteine and methionine.