Human samples and cell culture
GBM tissue samples were obtained from patients accepting surgical resection of tumors at the Department Neurosurgery, Xijing Hospital, Fourth Military Medical University. The diagnosis was confirmed by pathology. Informed consent was obtained from each subject involved in this study. The use of human tissues was approved by the Ethics Committee, Xijing Hospital.
To culture primary GBM cells, fresh tumor tissues were dissociated into single cell suspensions by mechanical grinding. Cells derived from two patients (named as FMXJ-1 and FMXJ-2, respectively) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (1:1) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. Cells were passaged routinely with the same medium as stocks of primary GBM cells. To culture GSCs, the primary GBM cells were cultured under the neurosphere condition in DMEM/F12 containing 20 ng/ml epidermal growth factor (EGF, Peprotech, Rocky Hill, NJ), 20 ng/ml basic fibroblast growth factor (bFGF, Peprotech), B27 (1:50, Invitrogen) and N2 (1:100, Invitrogen) for 7 days to obtain tumor spheres (with > 50 cells)39. For re-plating, spheres were mechanically dispersed, counted, and cultured as above for 7 days. For differentiation, spheres were dissociated into single cells and cultured in DMEM/F12 median containing 10% FBS for 5 days. In some cases, cells were infected by ADV for 8 h before plating. In some experiments, primary GBM cells were cultured in the presence of CpG oligodeoxynucleotides (2 mM, InvivoGen, San Diego, CA) for 24 h, HMGB1 (1 mg/mL, GenScript, Piscataway, NJ) for 48 h, or Stattic, a STAT3 inhibitor (Calbiochem, San Diego, CA) for 4 h40, respectively. Cells were then transferred to GSC medium and cultured for 7 days, and tumor spheres were counted.
A172 and T98G glioma cell lines were purchased from ATCC (Manassas, VA), and cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin solution. All cells were cultured in a standard culture incubator with 5% CO2 in air and 100% relative humidity at 37 ℃.
Viral infection and transfection of cells
ADV particles, which were generated by co-transfecting HEK293A cells with pAdTrack-CMV and pHBAd-BHG using the Adeasy adenovirus system, were purchased from HANBIO Biotechnology (Shanghai, China) with viral titers of 1.26 ╳ 1010 PFU/ml. For infection, primary GBM cells were incubated with viral particles at 400 multiplicity of infection (MOI), and the medium was changed 8 h later. siRNA against different genes and negative control siRNA (NC) were purchased from RiboBio (Guangzhou, China). Primary GBM cells were transfected with 10 nM of siRNA using Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocol. Forty-eight hours after the transfection, cells were re-plated for tumor sphere assay, or total RNA or protein was extracted from the transfected cells for further experiments.
Tumor growth in vivo
BABL/c-A nude mice at 4-6 weeks of age were used for intracranial xenograft tumor inoculation according to a published protocol41. Briefly, tumor cells were labeled with a luciferase fusion reporter by lentivirus-mediated transfection. Different number of cells (500, 5,000, 10,000) in 3 ml of DMEM/F12 medium were injected into the brain hemisphere of mice under the navigation of a murine brain stereotaxic apparatus (RWD68000, RWD Life Sciences Co., Ltd, Shenzhen, China). Tumor growth was monitored by intracranial bioluminescence using an IVIS Kinetic Imager (PerkinElmer, Waltham, MA). Tumors were dissected from the mouse brain and fixed in 10% formalin. Samples were embedded in paraffin, and sections were made for immunohistochemistry or hematoxylin and eosin (H&E) staining. All animal experiments were approved by the Animal Experiment Administration Committee of the Fourth Military Medical University.
Immunofluorescence
Cells cultured on coverslips were fixed in 4% paraformaldehyde (PFA) for 10 min and rinsed with PBS for three times, followed by permeabilization with 0.2% Triton X-100 for 10 min. Samples were blocked with 1% bovine serum albumin (BSA) for 30 min and incubated with primary antibodies overnight at 4 °C. Cells were then incubated with Cy2-conjugated secondary antibodies for 1 h. Washing with PBS was performed between each staining step. Nuclei were counter-stained with Hoechst for 5 min. The antibodies used included mouse anti-mitogen associated protein 2 (MAP2, 1:1000, Sigma, St. Louis, MO), rabbit anti-gial fibrilling acidic protein (GFAP, 1:500, Sigma), mouse anti-O4 (1:100, Sigma), Cy2-conjugated donkey anti-mouse (1:500) and Cy2-conjugated donkey anti-rabbit (1:500, Jackson ImmunoResearch, West Grove, PA). Samples were examined under a fluorescence microscope (FV-100, Olympus, Japan).
Flow cytometry
Single cell suspensions were prepared and incubated with a rabbit anti-CD133 (1:50, Proteintech) for 30 min at 4 ℃ in dark. Cells were washed and then stained with FITC-conjugated goat anti-rabbit secondary antibody, followed by FACS analysis using a FACS CaliburTM flow cytometer (BD Immunocytometry Systems, Franklin Lakes, NJ). Dead cells were excluded by propidium iodide (PI) staining. The acquired data were analyzed with FlowJo vX.0.6 software (Tree Star Inc., Ashland, OR).
Quantitative reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using the Trizol reagent (Invitrogen) and was reverse-transcribed into cDNA with a kit (Takara, Dalian, China). Quantitative (q)PCR was performed on an ABI PRISM 7500 Real-time PCR system (Life Technologies, Waltham, MA) using a SYBR Premix Ex Taq Kit (Takara), with β-actin as a reference control. Primers are listed in supplementary Table S1. All RT-qPCR experiments were performed in triplicates for at least 3 times.
Western blotting
Cells were lysed and soluble proteins were extracted using the radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail and sodium orthovanadate (Santa Cruz Biotechnology, Dallas, TX). Protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Protein samples were then run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% polyacrylamide, and electro-transferred onto polyvinyl difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were probed with specific primary and secondary antibodies, and developed with chemiluminescence (ECL, Thermo Fisher) using the ChemiDoc Touch Imaging System (BioRad). Quantification of bands was achieved by a densitometry, with β-actin as a reference control. Specific primary antibodies against the following proteins were used: c-MYC (SAB, 1:1000), SOX2 (R&D Systems, 1:1000), OCT4 (Cell Signaling, 1:1000), NANOG (SAB, 1:1000), STAT3 (SAB, 1:1000), pSTAT3 (SAB, 1:1000), MYD88 (R&D Systems, 1:1000), β-actin (Santa Cruz Biotechnology, 1:2000). Species-specific horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Jackson ImmunoResearch) and HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch) were used as secondary antibodies.
Bioinformatics
mRNA expression datasets and the associated clinical information were downloaded from TCGA (http://xena.ucsc.edu/getting-started/) and CGGA (http://www.cgga.org.cn). Gene set enrichment analysis (GSEA) was performed with the GSEA v2.0 software (Broad Institute of MIT, Massachusetts Institute of Technology). Probed gene sets were taken without further from the indicated publications, and downloaded from the KEGG pathway database (gseaftp.broadinstitute.org://pub/gsea/gene_sets_final/c2.cp.kegg.v6.2.symbols.gmt). The normalized enrichment scores (NES) with P values < 0.05 and false discovery rates (FDR) < 0.25 were considered statistically significant.
Statistical analysis
Statistical analysis was performed with the GraphPad Prism 6 software. All the results were presented as the mean ± standard error of mean (SEM). Comparisons between groups were performed using unpaired, two-tailed, Student’s t-test and Analysis of Variance (ANOVA) with 95% confidence interval. Survival analysis was calculated using Kaplan-Meier curves (log rank test). P < 0.05 was considered statistically significant.