The samples used in the present study were obtained from 172 patients newly diagnosed with glioma that underwent sample resection without therapy between 2010 and 2015 at the Department of Neurological Surgery, the Affiliated Hospital of Nantong University. The collection of the glioma specimens was performed in accordance with the protocols approved by the Ethics Committee of the Affiliated Hospital of Nantong University. The clinical demographic details of the 172 patients are outlined in Table I. Specimens were snapped frozen in liquid nitrogen immediately after surgery and stored at −80°C until use.

Human GSCs used in the present study were isolated from glioblastoma patients with IDH1 mutation as previously described (23,24). In brief, a few minutes after tumor removal, tissues were dissociated into a cell suspension by 0.25% trypsin digestion. Cells that passed through a 100 µm strainer (Falcon, Oxnard, CA, USA) were cultured in the medium composed of Dulbecco's modified Eagle's medium (DMEM) with 2% B27, epidermal growth factor (EGF, 10 ng/ml) and basic fibroblast growth factor (bFGF, 10 ng/ml). Cells were incubated at 37°C with 95% air, 5% CO₂ and 100% humidity. Two weeks later, floating primary tumor spheres were collected. The sphere cells were harvested, dissociated into single cells and plated into a 24-well plate for the subsphere-forming. Subspheres from one mother cell were characterized by immunocytochemistry against CD133 and nestin.

To induce differentiation of GSCs, the cells (1×10⁵ cells/ml) were plated onto poly-D-lysine coated coverslips in 96-well plates containing DMEM with 10% fetal bovine serum (FBS).

Immunohistochemistry was performed using protocols described previously by Gurung et al (25). In brief, the TMA slides were deparaffinised, rehydrated, washed and endogenous peroxidase was blocked using Bond-III 'Dewax Protocol D' following the manufacturer's instructions (Leica Biosystems, Newcastle, UK). Epitope retrieval was achieved using Bond-III 'Protocol H1(30)' (Leica Biosystems). The slides were incubated with antibodies against anti-IDH1-R132H (1:400; Dianova GmbH, Hamburg, Germany), CD133 (1:500; Abcam, Cambridge, UK) or nestin (1:500; Abcam) at room temperature (RT) for 1 h. Antibody binding was detected using diaminobenzidine (DAB) with hematoxylin counterstaining following Bond-max and Bond-x 'IHC protocol F' (Leica Biosystems). The immunostaining results were interpreted independently by two expert pathologists who were blinded to the clinical data. The staining intensity was scored using the following scale of four grades: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. At least 5 areas of each core were viewed and the proportion of cells in each core staining positively was assigned a score (0-100%). A semi-quantitative histopathology (H) score was obtained by multiplying the staining intensity score with the percentage score (0–300). An H-score higher than the median was considered positive.

To over-express IDH1-wt, the coding sequence of IDH1 was amplified using the following primers: forward primer 5′-ATGTCCAAA AAAATCAGTGGCGG-3′ and reverse primer 5′-GTTTGGCC TGAGCTAGTTTG-3′. This fragment was then sub cloned into the lentiviral vector pLenti6.3-MCS-IRES2-EGFP vector (Invitrogen, Carlsbad, CA, USA) using the restriction sites for BamHI and AscI. To overexpress the R132H mutant form of IDH1, we procured commercial synthetic sequences of IDH1 mutated at Arg 132, and sub-cloned them into the pLenti6.3-MCS-IRES2-EGFP vector. An empty vector (EV) was used as the control. The vectors and the Packaging Mix were cotransfected into the 293FT cell line to generate lentiviral stocks, which were used to transfect the target cells.

For immunofluorescent staining, cells were first fixed in 4% PFA, followed by incubation in hydrogen peroxide, blocking of non-specific antibody binding sites, and overnight incubation with the following primary antibodieså: rat anti-β-catenin (1:2,000; Abcam), mouse monoclonal anti-nestin (1:600; Roche Diagnostics GmbH, Mannheim, Germany), rat anti-CD133 (1:600; Roche Diagnostics GmbH) and mouse monoclonal anti-MAP-2 (1:600; Roche Diagnostics GmbH). The following day, cells were labeled secondary antibody with Alexa Fluor 568-conjugated goat anti-rat (1:1,000; Molecular Probes, Eugene, OR, USA) or 488-conjugated goat anti-mouse (1:800; Invitrogen). Cell nuclei were counter-stained with Hoechst 33342 for 30 min at RT. Immunopositive cells were observed under a fluorescent microscope.

To determine the level of mRNA expression in cells, the total RNA was extracted from 1×10⁶ cells in each group using UNIQ-10 Spin Column RNA Purified kit (Sangon Biotech Co., Ltd., Shanghai, China). The first strand cDNA was synthesized using RevertAid™ First Strand cDNA Synthesized kit (Fermentas, Burlington, ON, Canada). First Strand cDNA was subsequently subjected to Corbett RG-6000 PCR system (Qiagen, Dusseldorf, German) using FastStart Universal SYBR-Green Master Mix (Roche, Basel, Switzerland). The reactions were optimized by varying the annealing temperatures from 50 to 55°C, the sense and anti-sense primers were synthesized as follows: GAPDH 5′-GCAA GTTCAACGGCACAG-3′ and 5′-GCCAGTAGACTCCACG ACAT-3′; CD133 5′-TTCTGCCTGTGTAACTTTGCA-3′ and R, 5′-TTGTTGTGCAACGTCTTCAAGTAT-3′; nestin 5′-AG CGTTGGAACAGAGGTTGGA-3′ and 5′-TGTTTCCTCCCA CCCTGTGTCT-3′; β-catenin 5′-ATTGAAGCTGAGGGAGC CAC-3′ and 5′-TCCTGGCCATATCCACCAGA-3′; LEF1 5′-AAATAAAGTGCCCGTGGTGC-3′ and 5′-CATGCCTTG TTTGGAGTTGACA-3′; TCF4 5′-CGGCGGTGGGGGGA TGAC-3′ and 5′-GGCCGCTTCTTCCAAACTTTCC-3′.

To determine the level of protein expression in cells, aliquots of total protein (50 µg per lane) were electrophoresed on a 12% SDS-polyacrylamide gradient gel and transferred to nitrocellulose membranes (Millipore, Darmstadt, Germany). Washed in rinse buffer at RT and incubated in blocking buffer (5% fat-free milk in rinse buffer) for 30 min, the membranes were incubated for 2 h at RT with the following primary antibodies: anti-IDH1 R132H (1:500; Abcam;), anti-IDH1 (1:500; Dianova GmbH), anti-β-catenin (1:4,000; Abcam), anti-LEF1 (1:500; Santa Cruz Biotechnolgy, Santa Cruz, CA, USA), anti-TCF4 (1:200; Santa Cruz Biotechnolgy), anti-nestin (1:600; Roche Diagnostics GmbH), anti-CD133 (1:600; Roche Diagnostics GmbH) and anti-MAP-2 (1:800; Invitrogen). The following day, they were incubated with the HRP-conjugated secondary antibodies and visualized using the enhanced chemiluminescence (ECL) system (Pierce Biotechnology Inc., Rockford, IL, USA). In addition, β-actin was used as a reference protein.

The cells of EV, IDH1-wt and IDH1-mu groups were cultured in the above mentioned medium. At day 7 in vitro (DIV), some cell spheres of three groups were dissociated into single-cell suspension mechanically by trypsin digestion. Collected cells were washed once in cold phosphate-buffered saline (PBS). Cells were analyzed using Cell Cycle and Apoptosis Analysis kit (Beyotime Institute of Biotechnology, Haimen, China) by flow cytometry (Epics XL; Beckman Coulter, Fullerton CA, USA), according to the manufacturer's protocol.

To measure changes in cell migration, we performed the Transwell migration assay using 24-well BD Matrigel invasion chambers (BD Biosciences, San Jose, CA, USA) as previously described (20). In brief, 5×10⁴ cells were seeded into the upper well in DMEM (without serum) and incubated for 24 h to allow the cells to migrate into the lower well through an 8-µm pore membrane. After 24 h, the latter were fixed and stained for easy visualization and quantification. The average number of cells per field was determined by counting the number of cells in six random fields per well. To determine the invasive properties, cells were plated into chambers coated with Matrigel, which were then inserted into a 24-well plate and incubated for 48 h in DMEM and 10% FBS. At the end of the assay, the cells that adhered to the lower well were fixed, stained and visualized.

Statistical analysis was performed using statistics package for social science 21.0 (SPSS 21.0). The group comparison was analyzed using the independent t-test or one-way ANOVA. Associations between positive rate of GSC markers and the clinicopathological characteristics were analyzed using the Pearson's chi-squared test and the correlation analysis was compared using the Pearson test. Five-year overall survival (OS) was estimated using the Kaplan-Meier method and the differences in survival among groups were compared using the log-rank test. P<0.05 was considered statistically significant.

The results of TMA-IHC showed that there were 48 patients with IDH1-mu and 124 patients with IDH1-wt. GSCs in patients with IDH1-mu or IDH1-wt were identified by CD133 and nestin TMA-IHC. Many CD133⁺ and nestin⁺ cells were found in the patients with IDH1-wt while a few CD133⁺ and nestin⁺ cells in the patients with IDH1-mu. The positive rate of GSCs in the patients with IDH1-wt was more than that in the patients with IDH1-mu. The difference among them was statistically significant (P<0.05) (Fig. 1A). The correlation between positive rate of GSC markers and a series of clinicopathological characteristics was conducted. However, the relationship between the expression patterns of GSC markers and the histologic grade in glioma was not analyzed in the present study because of the limited samples of high histologic grade glioma. In the following study, we will collect enough samples to carry out this analysis. As shown in Table I, positive rate of GSC marker was significantly correlated with tumor stage (r=0.274) and IDH1 mutation (r=0.180). Five-year OS of patients was estimated using the Kaplan-Meier. The median 5-year OS of patients with GSC marker negative was 41 months (95% CI, 36.64–44.39 months). The median 5-year OS of patients with GSCs marker positive was 26 months (95% CI, 19.28–31.89 months). The 5-year OS rate was significantly lower for GSC marker positive patients than for GSCs marker negative patients, the difference was statistically significant (P<0.05) (Fig. 1B).

GSCs were identified by CD133 and nestin markers in vitro. The cells were cultured in serum-free medium, many cells formed floating primary tumor spheres. Subspheres from one mother cell were CD133 and nestin double positive (Fig. 2A). The expression of IDH1-R132H in GSCs was detected by western blot analysis. IDH1-R132H protein level in IDH1-R132H modified GSCs was more than that in the EV and IDH1-wt modified GSCs. The difference among them was statistically significant (P<0.05) (Fig. 2B). The expression of CD33 or nestin mRNA and protein in the transfected GSCs were detected by RT-PCR and western blot analysis. CD33 or nestin mRNA and protein level in IDH1-R132H modified GSCs was lower than that in the EV and IDH1-wt modified GSCs (P<0.05) (Fig. 2C and D).

The β-catenin expression level was detected by RT-PCR, western blot analysis and immunofluorescence. Compared with IDH1-wt and EV groups, the mRNA and protein expression of β-catenin in IDH1-mu group were significantly decreased. The difference among them was statistically significant (P<0.05) (Fig. 3A). Many intense β-catenin⁺ cells were found in the EV and IDH1-wt groups, and a few weak β-catenin⁺ cells were found in the IDH1-mu group. The difference among them was statistically significant (P<0.05) (Fig. 3B). To further analyze the level of Wnt/β-catenin signaling in the GSCs, the concentration of the β-catenin co-actors and Wnt signaling mediators TCF4 and LEF1 was measured by RT-PCR and western blot analysis. The mRNA and protein expression levels of TCF4 and LEF1 were downregulated in IDH1-mu group, compared with IDH1-wt and EV groups. The difference among them was statistically significant (P<0.05) (Fig. 3C).

The proliferation ability of GSCs in 3 groups was detected by flow cytometry. The result showed the proliferation index (PI) of IDH1-mu group was 30.05±9.01, while the PI of EV and IDH1-wt groups were 43.86±9.85 and 42.64±10.43, respectively. The PI of IDH1-mu group was lower than that of other two groups (P<0.05) (Fig. 4A). The differentiation ability of GSCs in 3 groups was detected by immunofluorescence and western blot analysis. Scarce MAP-2⁺ neurons and many CD133⁺ cells were found in the 3 groups. Compared with IDH1-wt and EV group, the percentage of MAP-2⁺ neurons in IDH1-mu group was more than that of the other two groups, but the percentage of CD133⁺ cells in IDH1-mu group was less than that of the other two groups (P<0.05) (Fig. 4B). Compared with IDH1-wt and EV groups, the CD133 protein level was decreased and MAP-2 protein level was increased in the IDH1-mu group (P<0.05) (Fig. 4C). The apoptotic level of GSCs in 3 groups was measured by a flow cytometry assay. The percentage of apoptotic cells was significantly higher in IDH1-mu group than that in IDH1-wt and EV groups (P<0.05) (Fig. 4D). To investigate the migration and invasion ability of GSCs, we assayed the rate of cellular migration and invasion using Transwell assay system. The migrated and invasive cells in IDH1-mu group obviously decreased compared with other two groups (P<0.05) (Fig. 4E).