TCGA dataset analysis
For Fig. 1 datasets were obtained from the TCGA website (https://tcga-data.nci.nih.gov). The data were derived from 538 adult (18+ years) cases of GBM. This database includes survival information and tumor samples acquired from patients undergo genomic profiling and expression analysis. Tumor samples are required to contain at least 80 % tumor nuclei and no more than 50 % necrosis, and a secondary pathology assessment must confirm the samples represent GBM. For our analysis we used SurvExpress® which is a gene expression database and web-based tool (http://bioinformatica.mty.itesm.mx/SurvExpress) based on several datasets including the TCGA, to provide survival analysis and risk assessment using a biomarker gene list as input to a Cox proportional-hazards regression . Cox regression relates the time of death to a number of explanatory variables known as covariates, in this case genes. For our analysis STAT3 was the specific gene of interest. We analyzed STAT3 gene expression level with respect to GBM in 538 subjects, and survival analysis was censored by survival months, which means that even though patients may have been lost to follow-up, partial data was incorporated in the survival curves. Risk analysis was performed in which a predicted risk for a specific patient genetic profile was determined. The subjects were then partitioned into low risk and high risk groups . STAT3 expression is shown for each risk group (Fig. 1a) and survival was plotted according to the Kaplan–Meier estimator (Fig. 1b). Cox regression and Kaplan–Meier log rank analysis both indicated that the survival data were significant at the 95 % confidence level.
Reagents for cell based studies
The JAK-2 inhibitor SAR317461 was obtained from Targegen (now Sanofi-Aventis), Alamar Blue was purchased from AbD Serotech, and Anti-pY-STAT3 and anti-STAT3 were purchased from Cell Signaling Inc. Horseradish peroxidase-linked anti-rabbit or mouse IgG were acquired from Jackson ImmunoResearch (West Grove, PA, USA), and Odyssey Inc. supplied Odyssey® Blocking Buffer and IRDye 680.
Establishment of primary GSC tumorspheres from GBM patients
GBM tissue acquisition, processing, and culture of GSCs
We have published on these methods and our tumorsphere lines elsewhere [26, 27]. GBM (grade IV glioma) tumor samples were obtained from 7 adult human (>21 years) surgical patients without the exclusion of either sex or any ethnic/racial groups, under an approved UCSD MCC IRB protocol (IRB #100936), with written, informed patient consent. IRB ethical guidelines were strictly followed, and patient samples were de-identified. The tumors are obtained at surgery and multiple samples are taken covering all parts of the tumor. The tumor samples were immediately washed 2–3 times with 5–10 ml of PBS/NSC basal medium to remove blood and debris, and the tissue was minced for 1–3 min with a No. 10 scalpel blade. The minced tissue was combined and enzymatically dissociated by using 3–5 ml of pre-warmed Accutase® (Life Technologies) for 10–15 min in a 37 °C water bath. The solution was subsequently centrifuged and 10–15 ml of basal medium was added to the tube and filtered through a 40 micron cell strainer to remove clumps and debris. After further washing cells were plated in NSC medium supplemented with 20 ng/ml EGF, 10 ng/ml bFGF and heparin (2 ng/ml), antibiotics added, and the cultures incubated at 37 °C in 5 % CO2.
Passaging and expansion of patient GBM derived tumorspheres
We cultured five patient derived tumorsphere cell lines GBM4, GBM8, SK1035, SK987, SK892, SK429 and SK262. When the tumorspheres reached an average size of 150–200 μm in diameter, subculture was initiated. The content of each flask was removed and placed in an appropriately sized sterile tissue culture tube, and centrifuged at 190g for 6 min at room temperature. The supernatant was removed and the pellet dissociated to create a single cell suspension. The cell suspension was centrifuged, the supernatant was aspirated, and the cells resuspended in 1 ml of NSC medium, and incubated at 37 °C in 5 % CO2.
Culture of immortalized GBM lines
Human U87, U251 and A172 GBM cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in 5 % CO2–95 % air.
Cell viability assay
The cytotoxic effect of SAR317461 was determined in triplicate for all 10 GBM lines using the Cell Proliferation Reagent Alamar Blue assay (AbD sciences). Cells (2 × 103 cells/well, 100 µl) added to in 96-well flat-bottomed plates, incubated at 37 °C and 5 % CO2–95 % air overnight. After exposure to the JAK2 inhibitor (SAR317461) at concentrations between 0.1 and 40 µM, for 72 h, cell viability was determined by adding Alamar Blue to the cells and 6–12 h later measuring fluorescence using excitation and emission wavelengths of 560 and 590 nm, respectively. Results were expressed as percent viability = [F1(treated cells) − background/F(untreated cells) − background] × 100 %. Dose–response curves were plotted by using GraphPad® Prism software and EC50 values were calculated.
Untreated and SAR317461 treated human GBM tumorsphere cells were harvested by disassociation, washed with ice-cold PBS, and lysed in NP40 buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1 % SDS, 1 % NP-40, 0.1 % sodium deoxycholate, protease inhibitor (Complete Mini®, Roche Scientific) and Phostop® (phosphotase inhibitor, Roche Scientific). The loading control was GAPDH for all blots. Protein quantification was performed with a BCA protein assay kit from Pierce (Fisher Scientific). Protein was separated by 10 % SDS-PAGE and transferred to nitrocellulose membranes, then transferred to nitrocellulose membranes followed by blocking with Odyssey® Blocking Buffer. Subsequently the membranes were probed with anti-STAT3, antiphospho-STAT3, antiphospho-JAK-2, antiphospho-STAT5 and anti-PARP antibodies (Cell Signaling Inc.), and anti-Bcl-XL (Santa Cruz Biotechnology) and anti-GAPDH antibody (Genetex). After washing with distilled water the membranes were incubated in blocking buffer and IRDye 680 for 1 h under no light conditions, then scanned and read on an Odyssey infrared plate reader (Li-Cor Biosciences).
Cell cycle analysis
For cell cycle analysis, GBM4 and GBM8 glioblastoma cells were plated at 200,000 cells/well and incubated for 16 h with 2 µM of SAR317461 and DMSO. Cells were dissociated, fixed in ice cold methanol, then incubated at 22 °C in for 30 min PBS containing 50ug/ml RNAse A. Cellular DNA was stained with 250 μl of PI (10 μg/ml), followed by flow cytometric analysis (FACS Calibur® Flow Cytometer, BD Biosciences).
For the autophagy studies the U251 cell line was engineered to stably express GFP-LC3. 1 µM SAR317461 was added to the medium and then cell viability was measured using the MTT assay. A starvation control was utilized by culturing U251 cells in media without FBS. Also for each category, starvation control, starvation, and JAK2 inhibitor, a vehicle control was also used. In addition to the cell viability assay the cells were imaged on an Olympus fluorescence microscope to assess their morphology.
For the cell viability studies the overall mean average deviation in percent between replicates used to construct the IC50 curves for 10 separate cell lines was calculated to be approximately 8.49 %. The standard deviation of IC50 values for seven tumorsphere lines treated with SAR317461 IC50 value is approximately 8.1 with a mean of 4.88 (4.88 ± 8.1). Hence the IC50 of 25 for SK892, which does not express pSTAT3, is more than 2 standard deviations outside this interval. The IC50s for U87, A172 and U251 are 7–8 μM. Statistical differences between cell viability obtained in cells treated with SAR317461, with or without autophagy inhibitors, were determined using a 2-tailed, paired t test and the 95 % level of confidence. Thus test conditions that attained p values of less than 0.05 relative to control were declared to be statistically significant.