Orthotopic Glioma Transplantation Model

All mice developed tumors with retrovirus injection. Following IACUC guidelines, animals were sacrificed at the first sign of morbidity. Ex-vivo gross total resection of the tumor was performed and tumor cells were isolated using enzymatic digestion [10 (link), 11 (link)]. The isolated cells were cultured for up to 15 passages in a 2:1 ratio of basal media (DMEM, N2, T3, 0.5% FBS, and penicillin/streptomycin/amphotericin) in B104 conditioned media [5 (link)]. This media was further supplemented with PDGF-AA (Sigma-Aldrich; St. Louis, MO) and FGFb (Gibco; Grand Island, NY) to a concentration of 10 ng/ml. Intracranial injection of the freshly prepared tumor cells was performed as previously described for the retrovirus. 1.0×10⁴, 2.0×10⁴, and 1.0×10⁵ cells were injected into 3 naïve adult Pten^lox/lox/p53^lox/lox/luciferase-^stop-lox transgenic mice over 3 generations.