Supporting Information Ortiz et al. 10.1073/pnas.1401952111 SI Materials and Methods monoclonal, 5 μg/mL) was added followed by Tyramide-Alexa Magnetic Resonance Imaging. Brains of injected mice were scanned Fluor 488 (Invitrogen, cat. no. T20922). at 8-, 11-, 16-, and 20-wk postinjection with a 200 MHz Bruker 4.7T Biospec MRI scanner equipped with a 560 mT/m ID 12-cm Immunostaining Image Analysis. Whole slides were scanned with gradient (Bruker Biospin MRI; Resonance Research). For mouse Pannoramic Flash Scanner (3DHistech). Image analysis of tumor brain imaging, brain coronal T2-weighted images using fast spin- areas was performed with Metamorph software (Molecular echo RARE sequence (Rapid Acquisition with Relaxation En- Devices). For analysis of immunohistochemistry images, color hancement) was acquired with TR 1.5 s, TE 50 ms, RARE factor thresholds were set for brown positive staining and for total area + of 8, slice thickness of 0.7 mm, field of view 30 × 20mm, in-plane (brown staining blue nuclei). Percent of brown staining to total resolution of 117 × 125 μM, and 24 averages. Tumor volume was area was calculated. For analysis of dual immunofluorescence calculated by contouring, measuring the tumor areas, and cal- images, a grayscale threshold and standard area was set for green culating the sum of the areas multiplied by the distance between (Iba1), red (p-Stat3), and blue (DAPI). For each sample, the the centers of two adjacent slices. number of DAPI-positive nuclei within each stained area, and the number of DAPI within colocalized areas was calculated. Histology and Immunostaining. Brains from mice were collected and fixed with 10% (vol/vol) neutral buffered formalin (Sigma). Human Tumor Collection, Tissue Lysates, and Immunoblotting. Fresh Tissues were embedded in paraffin and 5-μm sections were used human glioblastoma multiforme (GBM) tissue samples were for analysis. Sections were stained with H&E or used for im- obtained from patients who consented under an Institutional munohistochemical analysis. Review Board-approved protocol. Tumor lysates were lysed in Immunohistochemistry was performed at the Molecular Cytol- CelLytic MT Mammalian Tissue Lysis/Extraction Reagent (Sigma) ogy Core Facility of Memorial Sloan-Kettering Cancer Center supplemented with Complete Mini EDTA-free (Roche) and using Discovery XT processor (Ventana Medical Systems). Tissue PhosSTOP (Roche) protease inhibitor mixes. Protein lysates sections were stained with the following antibodies: p-Stat3 Tyr-705 were run in SDS/PAGE gels and transferred to nitrocellulose (Cell Signaling, cat. no. 9145; rabbit monoclonal, 0.5 μg/mL), Stat3 for immunoblotting. The following antibodies were used: p-STAT3 (Cell Signaling, cat. no. 9132; rabbit polyclonal, 0.16 μg/mL), Iba1 Tyr-705 (Cell Signaling, cat. no. 9145; rabbit monoclonal, 1:2,000) (Wako Chemicals, cat. no. 019–19741; rabbit polyclonal, 0.5 μg/mL), and STAT3 (Cell Signaling, cat. no. 9132; rabbit polyclonal, 1:1,000). Gfap (Dako, cat. no. Z033429-2; rabbit polyclonal, 1 μg/mL), Olig2 Quantification of Western blot by densitometry analysis was per- formed using ImageJ software. (Millipore, cat. no. AB9610; rabbit polyclonal, 2 μg/mL), and Nestin (BD Pharmigen, cat. no. 556309; mouse monoclonal, μ Flow Sorting of RCAS PDGF-GFP Tumors. Tumors were dissected 5 g/mL). For rabbit antibodies, tissue sections were blocked in from mice injected with replication-competent avian sarcoma- 10% (vol/vol) goat serum with 2% (wt/vol) BSA in PBS. For the leukosis virus long terminal repeat with splice acceptor retrovirus mouse antibody, tissues were blocked with Biotinylated Mouse on (RCAS) PDGF-GFP and enzymatically and mechanically dis- Mouse (M.O.M.) anti-Mouse Ig Reagent (Vector Labs, cat. no. sociated into single-cell suspensions by treatment with papain and MKB-2225). Primary antibody incubation was done for 5 h, ovomucoid as previously described (1). Single-cell suspensions + − followed by a 60-min incubation with biotinylated goat anti- were made in PBS with 10% (vol/vol) FBS, and GFP DAPI rabbit IgG (Vector Labs, cat. no. PK6101; 1:200 dilution) or cells were sorted on a MoFlo Cell Sorter (Dako Cytomation). biotinylated anti-mouse IgG (Vector Labs, cat. no. BMK-2202) ’ according to the manufacturer s instructions. Detection was per- Human Genetic Analysis and Microarray Analysis of Mouse Tumor formed with Blocker D, Streptavidin-HRP and DAB kit (Ventana Cells. The Cancer Genome Atlas data used was previously de- ’ Medical Systems) according to the manufacturer s instructions. scribed (2, 3). The GBM oncoprint was generated using the cBio Slides were counterstained with hematoxylin and cover-slipped Portal as previously described (4) + − with Permount (Fisher Scientific). Sorted GFP DAPI tumor cells from mice injected with CD34 staining was performed on the Leica Bond RX (Leica RCAS-PDGF-GFP were stored at −80 °C in TRIzol LS Reagent Biosystems) using the Bond Polymer Refine Detection Kit (Leica (Ambion). Samples were processed by the Memorial Sloan- Biosystems, DS980). Anti-CD34 antibody (Abcam, cat. no. Kettering Cancer Center Genomics Core. Briefly, RNA was ex- ab8158; rat monoclonal, 16 μg/mL) was added for 30 min fol- tracted and quality checked using a bioanalyzer. RNA was analyzed lowed by a 30-min incubation of biotinylated anti-rat (Vector using the Affymetrix MOE 430A 2.0 chip following the manu- Labs, cat. no. BA-4001; 1:100 dilution). TUNEL staining was facturer’s instructions. Differentially expressed genes were deter- performed with the following reaction mixture: 0.1 M sodium mined using ANOVA. Principal component analysis and hierar- cacodylate pH 7, 0.1 mM DTT, 0.05 mg/mL bovine serum al- chical analysis were performed using the Partek Software Suite. bumin, 2 U/μl terminal transferase, 0.2 nm biotin-16-dUTP, and Genes were considered to be differentially expressed if their fold- 2.5 mM cobalt chloride for 1 h at 37°. The reaction was termi- change > 1.8 and P value < 0.05. Enriched pathways were iden- nated with 300 mM sodium chloride and 30 mM sodium citrate tified using Ingenuity Pathway Analysis (Ingenuity Systems, at room temperature for 15 min, incubated in avidin-biotin for Qiagen). Pathways with Benjami–Hochberg multiple testing cor- 30 min, and developed with 3,3′-diaminobenzidine for 3 min. rection P value < 0.05 and Bias-corrected z-score > 2werecon- Dual immunofluorescence of p-Stat3 and Iba1 or Gfap were sidered to be significant. performed using Discovery XT processor (Ventana Medical Systems). Staining with p-Stat3 Tyr-705 (Cell Signaling, cat. no. Side Population Assay. Tumors were dissected from mice injected 9145; rabbit monoclonal, 0.5 μg/mL) followed by Tyramide Alexa with RCAS PDGF-GFP and dissociated into single-cell sus- Fluor 568 (Invitrogen, cat. no T20914) was performed first. Next, pensions, as previously described (1). Single-cell suspensions Iba1 (Wako Chemicals, cat. no. 019–19741; rabbit polyclonal, were made in unsupplemented basal neural stem cell media 0.5 μg/mL) or Gfap (BD Pharmigen, cat. no. 561483; mouse (Lonza) and counted using a hemocytometer. Side population Ortiz et al. www.pnas.org/cgi/content/short/1401952111 1of10 analysis was performed by Hoechst 33342 exclusion as previously at room temperature. Cells were washed two times with HBSS:2% described (5). Cells were incubated with or without verapamil (vol/vol) FBS and suspended in PBS:10% (vol/vol) FBS for analysis (Sigma) and fumitremorgin c (Sigma) ABC inhibitors to set side with a FACSCalibur (BD Biosciences). Analysis was performed population gates (5). Flow cytometry was performed with a MoFlo using FloJo software (Treestar Inc.). Cell Sorter (Dako Cytomation) and analysis was performed using Flojo software (Treestar Inc.). Flow Cytometry for Nestin. Single-cell suspensions of neonatal mouse days 1–3 brains were made using papain digestion as done K i67 and GFP Flow Cytometry. Tumors were dissected from mice previously. Staining was performed as described previously using injected with RCAS PDGF-GFP and dissociated into single-cell Nestin antibody (BD Pharmigen, mouse monoclonal, cat. no. suspensions as previously described (1). Single-cell suspensions μ were made in PBS and counted using a hemocytometer. The 556309; 1.7 g/mL) and goat anti-mouse Alexa 568 (Life Tech- μ following protocol was performed to minimize quenching of native nologies, cat. no. A21124; 33 g/mL) (1). Flow cytometry was GFP in the tumor cells. One million cells were fixed with 1% performed on the FACSCalibur (BD Biosciences), and analysis paraformaldehyde in a buffer made of HBSS and 2% (vol/vol) was performed using FloJo software (Treestar Inc.). fetal bovine calf serum overnight at 4 °C. Cells were permeabilized using 0.5% TritonX-100 in HBSS and 2% (vol/vol) FBS for 10 min Statistical Analysis. Unless noted, Student t test was performed for at room temperature. Ki67 antibody conjugated to Alexa 647 (BD all statistical analysis. Log-rank statistical analysis was performed Pharmigen, cat no. 561126; mouse monoclonal, 1/60 dilution) for Kaplan–Meier curves and Fisher’s exact test was performed was made in HBSS:2% (vol/vol) FBS and applied to cells for 1 h for the tumor grade analysis. 1. Ciznadija D, Liu Y, Pyonteck SM, Holland EC, Koff A (2011) Cyclin D1 and cdk4 mediate 4. Cerami E, et al. (2012) The cBio cancer genomics portal: an open platform for exploring development of neurologically destructive oligodendroglioma. Cancer Res 71(19): multidimensional cancer genomics data. Cancer Discov 2(5):401–404. 6174–6183. 5. Bleau AM, et al. (2009) PTEN/PI3K/Akt pathway regulates the side population phenotype 2. Cancer Genome Atlas Research Network (2008) Comprehensive genomic characteriza- and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell 4(3):226–235. tion defines human glioblastoma genes and core pathways. Nature 455(7216):1061–1068. 3. Verhaak RG, et al.; Cancer Genome Atlas Research Network (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.