Culture of GBM TSs and reagents
GBM TSs were derived from two individual patients with GBM. These GBM TSs, denoted TS15-88 and TS13-64, were established from fresh GBM tissue specimens, as approved by the institutional review board of the Yonsei University College of Medicine. TSs were cultured according to standard methods [21–23]. Briefly, cells were cultured in TS complete medium, consisting of Dulbecco’s modified Eagle’s medium/F12 (Mediatech, Manassas, VA, USA), 1x B27 (Invitrogen, Carlsbad, CA, USA), 20 ng/mL basic fibroblast growth factor (bFGF; Novoprotein, Summit, NJ, USA) and 20 ng/mL epidermal growth factor (EGF; Novoprotein). All in vitro experiments were performed under the culture conditions for TSs. DFS, an anti-GBM lignan, was isolated from a chloroform soluble fraction of A. japonica extracts, as described [19].
Western blot
Lysates of GBM TSs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on Tris-glycine gels. Cytosolic and nuclear fractions were prepared using cytoplasmic and nuclear extraction reagents (Thermo Scientific, Rockford, IL, USA), respectively, according to the manufacturer’s instructions. Proteins were transferred to nitrocellulose membranes and probed with antibodies against cleaved capase-3, CD133, PDPN, β-catenin, CD44, snail, Cyclin D1 and cMYC (Cell Signaling Technology, Danvers, MA, USA); Sox2 (Merck Millipore, Darmstadt, Germany); Msi-1 (Abcam, Cambridge, UK); nestin (Novus Biologicals, Centennial, CO, USA); N-cadherin, Zeb1 (Sigma-Aldrich, St. Louis, MO, USA); Twist, Oct3/4, BAX, Bcl-2, phospho-β-catenin, PARP and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA); and FOXM1 (Bethyl Laboratories, Montgomery, TX, USA), along with Western Lightning Plus-enhanced chemiluminescence reagent (PerkinElmer, Lawrence, MA, USA). Images were captured using an ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Little Chalfont, UK).
Immunoprecipitation
To determine interactions between FOXM1 and β-catenin, GBM TSs were treated with DFS for 72 h, lysed with lysis buffer and incubated with anti-FOXM1 or anti-β-catenin antibody on a rotator overnight at 4ºC. Protein-antibody-protein A/G agarose complexes were prepared using protein A/G sepharose beads (Santa Cruz Biotechnology) for 4 h at 4 ºC. After extensive washing with RIPA lysis buffer, the immunoprecipitated complexes were resuspended in reducing sample buffer and boiled for 10 min. The precipitated proteins were subjected to immunoblotting with the indicated antibodies.
Luciferase reporter assay
Canonical TCF/LEF signaling activity was measured using TOPflash/FOPflash reporter assays. Briefly, 1 x 105 cells were seeded into each well of a 24-well plate for 1 day before transfection. The cells were transfected with 100 ng of firefly luciferase reporter plasmid and 10 ng of internal control plasmid pRL-TK (Promega, Madison, WI, USA) using Lipofectamine 3000 (Invitrogen) for 72 h according to the manufacturer's protocol. Samples were lysed with 1⋅ reporter lysis buffer for 15 min and centrifuged, and the supernatants were used for luciferase and β-galactosidase assays, according to the manufacturers' protocols. After confirming that the cells had been transfected using β-galactosidase assays, luciferase activity in 10 µg of protein lysate was quantified using the Dual Luciferase Reporter Assay System (Promega). Each sample was analyzed in triplicate.
Evaluation of cell viability
The effects of DFS on the survival of GBM TSs were determined using WST assays (Promega). Briefly, 1⋅104 single dissociated GBM TSs were seeded into each well of a 96-well plate. The plates were incubated at 37℃ for 24 h and treated with DFS for 72 h. WST reagent (10 µl/well) was added and absorbance was measured at 450 nm after incubating at 37°C for 1 h. Each experiment was repeated three times in triplicate, and the results are expressed as the percentage of viable cells relative to controls.
Evaluation of ATP levels
Dispersed GBM TSs were plated in 96-well plates at a density of 1⋅104 cells per well. After incubating for 24 h, TSs were treated with DFS for 3 days, and then ATP levels were compared using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s protocol. Briefly, a volume of CellTiter-Glo Reagent equal to the volume of cell culture medium was added to each well, after which cells were incubated at room temperature for 10 min, and luminescence was measured.
Cell cycle and apoptosis analysis
To analyze the cell cycle, GBM TSs treated with DFS for 72 h were collected, fixed with 70% ethanol, dissociated with accutase (Sigma), and stained with 100 µg/ml of propidium iodide (PI, Sigma) at 4ºC for 15 min in the dark. To evaluate DFS-induced apoptosis in GBM TSs, GBM TSs were treated with the indicated concentration of DFS. Apoptotic cell death was detected by double staining with fluorescein-isothiocyanate (FITC)-conjugated annexin V (BioLegend, San Diego, CA, USA) and PI. The stained cells were analyzed using an LSR II flow cytometer (BD Biosciences, Bedford MA, USA).
Neurosphere formation assays
Ten single dissociated GBM TSs were seeded in each well of a 96-well plate and treated with DFS after 24h. The plates were incubated for 3 weeks in TS complete medium, with medium changed every week. The number of sphere-positive wells was counted, and the ratio of sphere-positive wells in the treated relative to the control group was calculated. The radius of spheres in each group was also measured. Images were captured and analyzed using ToupView software (ToupTek Photonics, Zhejiang, China).
Three-dimensional (3D) invasion assays
Each well of a 96-well plate was filled with mixed matrix composed of matrigel (Corning Incorporated, Tewsbury, MA, USA), collagen type I (Corning Incorporated) and TS complete medium. Single spheroids had been seeded inside the matrix prior to gelation, followed by the addition of TS complete medium over the gelled matrix to prevent drying. The invaded area was quantified as occupied area at (72 h - 0 h)/0 h.
Processing of transcriptome data
Total RNA was extracted from GBM TSs and used for RNA sequencing. The quality of the reads was checked using FastQC (v.0.10.1) and the sequencing adapters were removed using Trimmomatic (v. 0.38). Low quality reads were filtered according to the following criteria: reads containing more than 10% of skipped bases (marked as ‘N’s), reads containing more than 40% of bases with quality scores less than 20, and reads whose average quality scores of each read were less than 20. Filtered reads were mapped to the human reference genome using the aligner Tophat [24]. Gene expression levels were measured with Cufflinks v2.1.1 [25], using the gene annotation database of Ensembl release 72 [26]. Non-coding regions were removed using the –mask option. Using GENE-E software, the average linkage of hierarchical clustering was determined with Pearson’s correction as a distance metric and expression levels depicted as heat maps.
Mouse orthotopic xenograft model
Male athymic nude mice aged 4–8 weeks (Central Lab. Animal Inc.,Seoul, Korea) were housed in micro-isolator cages under sterile conditions and monitored for at least one week before study initiation to ensure proper health. Lighting, temperature, and humidity were controlled centrally. Dissociated Luc-TS13-64 cells were pretreated with DFS for 72 h using the pretreatment protocol [27, 28]. Cells were selected by trypan blue staining, and 5×105 viable cells were implanted into the right frontal lobe of each mouse at a depth of 4.5 mm using a guide-screw system [21–23, 29]. Mice showing a >15% reduction in body weight compared with the maximum were euthanized according to the approved protocol.
Statistical analysis
Levels of significance for comparisons among treatment groups were determined using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Survival was analyzed using the Kaplan-Meier method and compared by log-rank tests. All the graphs and statistical analyses were performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA) software, with P-values < 0.05 considered statistically significant.