GSC culture and hypoxic treatment
Two human GSC lines, GSL-1 and GSL-2, were used in the present study. These cell lines were previously established from the primary cell culture of tissues surgically obtained from the tumor periphery of GBM patients [6]. The stemness of these GSC lines (previously designated SFC-1 and SFC-2) was confirmed by evaluating their sphere-forming ability, and the lines were then renamed GSL-1 and GSL-2, respectively.
Cells were cultured in serum-free DMEM/Ham’s F-12 medium (Wako) containing 10 μg/ml insulin (Wako), 10 nmol/l recombinant human basic fibroblast growth factor, 10 nmol/l recombinant human epidermal growth factor, 5 μmol/l heparin, N2 supplement (Wako), GlutaMAX supplement (GIBCO), and penicillin/streptomycin/amphotericin B mixture (neural stem cell medium: NSCM). Growth factors were purchased from PeproTech (London, UK). The cells were maintained at 37°C under humidified 5% CO2/95% air atmosphere as the normoxic condition (21% O2). For severe hypoxia treatment, the cells were incubated for 3 h in an atmosphere of 1% O2, 5% CO2, and 94% N2 in a multi-gas incubator (APM-50D, ASTEC). For moderate hypoxia treatment, the cells were incubated for 6 h in an atmosphere of 5% O2, 5% CO2, and 90% N2.
For sphere formation assay, 1x106 cells were seeded onto 10cm culture dish and incubated for 7 days in the NSCM at normoxia. Then, the cells were sprit in six-well suspension culture plates at a density of 1000 cells/well in 2ml NSCM. After 7 days, formation of spheres was confirmed and the number was counted.
Treatment of cells with small interfering RNA (siRNA)
The sequences of siRNAs for HIF-1α, HIF-2α, CD44, and OPN are listed in Table S1. As a control for each siRNA, we used a corresponding random siRNA sequence (5’-GCGCGCUUUGUAGGAUUCG dTdT-3’). GSCs were transfected with each siRNA using Lipofectamine 3000 reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. After a 24-h incubation of GSCs transfected with each siRNA, the culture medium was changed to remove the Lipofectamine, and subsequent experimentation was performed.
Establishment of stable CD44-knockdown cells
Lentiviral particles were generated using the shRNA expression vector pLKO.1-puro, which carries a shRNA sequence against CD44 (CD44 MISSION shRNA, SHCLNG-NM_000610, Sigma Aldrich) together with the MISSION Lentiviral packaging mix (SHP001, Sigma Aldrich), according to the manufacturer’s instructions. Briefly, HEK 293T cells were co-transfected with the two products described above using Lipofectamine 3000 reagent (Invitrogen). The supernatant containing virus particles was harvested 48 h after transfection and used to infect GSCs. After 48 h of incubation, infected cells were selected using puromycin (0.5 µg/ml; Invitrogen).
RNA isolation and quantitative real-time–polymerase chain reaction (qRT-PCR)
Total RNA was extracted from GSCs and tissue of each tumor sample (core and periphery) using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with a gDNA remover kit (Toyobo). qPCR analysis was performed using Fast Start Universal SYBR Green Master Mix (Roche Diagnostic Japan) with an MJ mini instrument (BioRad, Hercules, CA, USA). All gene-specific mRNA expression values were normalized relative to the expression level of the housekeeping (reference) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Quantification of gene expression was performed using ΔCt values, wherein ΔCt is defined as the difference between the target and reference gene Ct values. All primer sequences are listed in Table S2.
Western blot analysis
Cells grown on poly-L-lysine–coated dishes were lysed using RIPA buffer solution. The lysates were electrophoresed, transferred onto nitrocellulose membranes, and immunoblotted with antibodies to β-actin (1:1000; mouse monoclonal; Sigma), HIF-1α (1:250; mouse monoclonal; BD Biosciences), HIF-2α (1:250; rabbit polyclonal; abcam), OPN (1/200, rabbit polyclonal, abcam) or CD44 (1:250; mouse monoclonal; Cell Signaling Technology). Following incubation with alkaline phosphatase–conjugated secondary antibody (Promega), immunoreactions were developed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Immunohistochemical analysis
Freshly dissected tissue blocks surgically obtained from the tumor periphery of GBM patients were frozen and sliced into 5-µm-thick sections. These sections were stored at −80°C for later use. The sections were permeabilized and blocked with Tris-buffered saline containing 0.1% Tween 20 (TBSt) and 1 mg/ml bovine serum albumin (BSA-TBSt) for 30 min. For double-labeling immunofluorescence, sections were incubated in a humidified chamber overnight at 4°C with a mixture of two primary antibodies diluted in BSA-TBSt, one antibody to OPN (1/200, rabbit polyclonal, abcam; 1/100, mouse monoclonal, R&D) and the other to Nestin (1/200, mouse monoclonal, Chemicon International). After washing with TBSt, sections were treated with DyLight 488-labeled anti-mouse and Cy3-conjugated anti-rabbit IgG secondary antibodies (1:1000, Jackson ImmunoResearch, West Grove, PA, USA). Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. The immunostained specimens were observed under a conventional microscope (BX52; Olympus, Tokyo, Japan).
Cells were fixed with 4% paraformaldehyde (PFA), permeabilized, and blocked with BSA-TBSt for 30 min. The cells were then incubated in a humidified chamber overnight at 4°C with a mixture of two primary antibodies diluted in BSA-TBSt: antibodies to CD44 (1:250, mouse monoclonal, Cell Signaling Technology) and HIF-1α (1/250, rabbit polyclonal, Novus) or HIF-2α (1/100, rabbit polyclonal, abcam) and OPN (1/250, mouse monoclonal, abcam). The cells were then treated according to the steps described above.
Sections of mouse brain were deparaffinized in Histo-Clear (Cosmo Bio), hydrated in a graded alcohol series and subjected to heat-activated antigen retrieval. After blocking endogenous peroxidase activity, the sections were incubated in a humidified chamber overnight at 4°C with monoclonal antibodies to CD44 (1:200, Cell Signaling Technology), Ki-67 (1:200, Dako), Nestin (1/200, Chemicon International), or Sox2 (1/200, R&D Systems) diluted in BSA-TBSt. Subsequently, the sections were washed with TBSt and incubated with biotinylated secondary antibody for 1 h at room temperature. The reaction complexes were stained with diaminobenzidine and counterstained with hematoxylin.
Measurement of OPN secreted out of GSCs
When GSCs reached confluence, the culture medium was removed, and the cells were washed with PBS. After addition of serum-free, low-glucose DMEM, the cells were cultured for 24 h. The cells were then centrifuged, and the supernatant was used as conditioned medium. The concentration of OPN in the conditioned medium was determined using an OPN ELISA kit (Sigma-Aldrich) according to the manufacturer’s protocol.
Cell invasion and migration assays
The invasiveness of cultured GSCs was assessed with an in vitro assay using Falcon cell culture inserts (Becton Dickinson Biosciences, CA, USA) and a reconstituted basement membrane, Matrigel (Becton Dickinson Biosciences), as previously described [24,25]. Briefly, GSCs were suspended in DMEM containing 0.1% BSA and seeded onto the insert filters at a density of 5×104 cells/insert. The insert was placed in the lower wells of the Falcon 24-well plate containing 500 μl of DMEM with 1% FBS and incubated for 24 h at 37°C under normoxic conditions or 1% O2 hypoxic conditions. GSC migration was assayed using the modified Boyden chamber method with 48-well microchemotaxis chambers (Nucleopore, Pleasanton, CA, USA), as previously described [26,27]. GSCs in DMEM containing 0.1% BSA (at a density of 1×104 cells/ml) were placed in the upper well, and DMEM containing 1% FBS was placed in the lower well. A polyvinylpyrolidone-free polycarbonate membrane with 8-μm pores (EMD Millipore, Bedford, MA, USA) was used. To examine the effect of OPN on CD44-induced migration and invasion, 1 μg/ml of OPN (Sigma Aldrich) was added to the upper well. To examine the effect of OPN addition on HA (Wako, Japan)–stimulated migration and invasion of GSCs, HA was added to the lower well at a concentration of 0.5 mg/ml. The chamber was incubated for 6 h at 37°C under normoxic conditions or 1% or 5% O2 hypoxic conditions. In both assays, cells on the upper membrane surface were mechanically removed. Cells that had invaded or migrated to the lower side of the membrane were fixed, stained with 0.1% crystal violet, and examined under a microscope (×400) to determine the number of cells in three random fields.
In vivo xenograft experiments
Control and CD44-knockdown cells (1×106) were suspended in 5 µl of Matrigel and injected into the brain of 6-week-old male NOD/SCID mice purchased from CLEA Japan, Inc. (n=6 mice per both group) that had been anesthetized intraperitoneally with a mixture of medetomidine (0.75 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg). MRI was performed to confirm tumorigenesis before the mice were euthanized by decapitation. The brain of each mouse was dissected and fixed in 4% PFA at 4°C overnight. After fixation, the brains were embedded in paraffin, sliced into 5-µm-thick sections, and stained with hematoxylin and eosin. The duration from cell injection to decapitation was evaluated as survival time.
Statistical analysis
Values are expressed as the mean ± standard deviation (SD), and the data were compared using the Student’s t-test (unpaired). Kaplan-Meier plots were generated to estimate unadjusted time-to-event variables. The log-rank test was performed to assess the statistical significance of differences between groups. Significance was set at P<0.05.