Evaluation of glioma growth in vivo

For the TS516 xenograft model, 4–6 weeks old female SCID mice were injected subcutaneously with 10⁶ glioma cells, which were suspended in 100 μl of a 50:50 mixture of growth media and Matrigel (BD #356237). Mice were then randomly assigned to treatment groups (Dox or control). Ntv-a mice, and procedures for RCAS-mediated gliomagenesis have been described previously⁵⁴. Ntv-a pups were injected with a total of 200,000 DF1 cells transfected with various constructs: 100,000 RCAS-PDGFB plus 100,000 RCAS-shRanBP6 or RCAS-shLuc. After injection of the DF1 cells during the newborn period, mice were aged until they developed symptoms of disease (lethargy, poor grooming, weight loss, macrocephaly). Samples in panel ​panelii of Fig. ​Fig.66 are derived from tumors generated in a Ntv-a; Ink4a/Arf null background. RCAS-shRanBP6 and RCAS-shLuc constructs express a EGFP reporter that allowed to isolate the tumor cells by FACS. For the derivation of primary cells for FACS analysis, tumors were digested to a single-cell suspension by 10 min of incubation at 37 °C with 5 ml of papain digestion solution (0.94 mg/ml papain (Worthington), 0.48 mM EDTA, 0.18 mg/ml N-acetyl-l-cysteine (Sigma) and 0.06 mg/ml DNase I (Sigma) diluted in Earl’s Balanced Salt Solution (EBSS). After digestion, the enzyme was inactivated by the addition of 2 ml of 0.71 mg/ml ovomucoid (Worthington). The cell suspension was then passed through a 40-μm mesh filter to remove undigested tissue and centrifuged at a low speed (750 r.p.m.) to remove debris and obtain the cell pellet⁵⁴. Cells were then resuspended in 500 μl of PBS to be sorted. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center and CNIO-ISCIII Ethics Committee for Research and Animal Welfare (CEIyBA) and they were performed in accordance with the guidelines stated in the International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS).