Orthotopic Transplantation of EGFRvIII-Expressing Neurospheres

Tumors were also generated by orthotopic transplantation of EGFRvIII-expressing neurospheres, which were produced in vitro. Subventricular zones (SVZs) were dissected from P0-P2 Cdkn2a^−/−; EGFRvIII^fl-stop-fl;Pten^fl/fl mice and dissociated into single cells with Accutase (Biolegend, 423,201). The cells were cultured in serum free neural stem cell media containing 1× B27 supplement (Life Technologies, 17604044), 20 ng/ml b-FGF (ThermoFisher, PHG0261), 5 μg/ml heparin (Stem Cell Technologies, 7980), 25 ng/ml EGF (Lonza, cc-4017FF) and 1 X penicillin/streptomycin (Genesee, 25512) in DMEM: F12 medium (Gibco, 10565018) (Laks et al., 2009 (link)). The cells from individual animals were initially plated in 6-well plates to grow neurospheres over 1–2 weeks. 500,000 cells were then plated in 6-well plates and treated with concentrated adenovirus Cre (Vector Biolabs) at 50–100 MOI, and then cultured in 10 cm dishes for another 5–7 days before they were checked by western blot to confirm EGFRvIII expression and loss of PTEN protein expression. They were passaged for less than 8 passages before being injected into 6-week old C57BL/6 mice to form tumors. About 200,000 cells in 5 μl 1× DPBS were injected per animal using a 23-gauge Hamilton syringe at a position 2.5 mm to the right and 1 mm anterior to the bregma at a depth of 2.5–3 mm.