We used the human glioblastoma cell line, T98G (provided by JCRB, Setagaya, Tokyo), in the present study. T98G cells were cultured in α-minimum essential medium (α-MEM) supplemented with 20 mM 4-(2-hydroxyethyl) piperazine ethane sulfonic acid, 8 mM NaHCO₃, 50 µg/ml streptomycin, 50 U/ml penicillin, and 10% fetal calf serum and maintained in a humidified incubator at 37°C containing a mixture of 98% air and 2% CO₂.

T98G cells were seeded onto non-adherent U-shaped bottom 96-well plates (PrimeSurface 96U, MS-9096U, Sumitomo Bakelite Co., Ltd.). The seeded cells were cultured in α-MEM at a density of 5,000 or 10,000 cells/well for 3 days at 37°C under conditions of 98% air and 2% CO₂. The seeded cells aggregated and formed a cell mass at the bottom of the nonadherent U-shaped wells on the plates. The spheroids formed were transferred to non-adherent 100-mm dishes (PrimeSurface 100Φ, MS-9090×, Sumitomo Bakelite Co., Ltd.) at a density of 96 spheroids/dish after 3 days of culture. The transferred spheroids were then successively cultured for 7–10 days until they reached a diameter of 300–500 µm.

After the spheroids were fixed with a solution containing 10% formalin and 10% sucrose for 1 h, the spheroids were rinsed with phosphate-buffered saline (PBS) (-). The spheroids were then embedded in Tissue-Tek O.C.T. Compound (Sakura Finetechnical Co., Ltd.) and sliced into frozen sections 10–20 µm in thickness using a cryostat.

Cryostat sections on glass slides were incubated for double immunofluorescent staining using anti-CD133^AC133 monoclonal antibody (Miltenyi Biotechnology; 1:10 dilution), anti-HIF-1α polyclonal antibody (Novus Biologicals; 1:100 dilution), or anti-nestin polyclonal antibody (Santa Cruz Biotechnology; 1:50 dilution) for 1 h at room temperature. Subsequently, the sections were washed thrice using PBS (-) and then incubated with Alexa Fluor 488 anti-rabbit and Alexa Fluor 546 anti-mouse secondary antibodies (Nacalai Tesque; 1:1,000 dilution) for 1 h at 37°C. After washing with PBS (-), the sections were embedded with SlowFade Gold antifade reagent containing 4,6-diamidino-2-phenylindole (Invitrogen; Thermo Fisher Scientific, Inc.), and covered with a glass coverslip. For immunofluorescent staining of monolayer cultured cells, cells were seeded onto a glass slide and cultured for 1 day at 37°C with a mixture of 98% air and 2% CO₂. After 1 day, the cells were washed with PBS (-), fixed with 10% formalin for 1 h, and then incubated for double immunofluorescent staining using the same method used for cryostat section staining.

After culturing the spheroids for 7–10 days, the spheroids were rinsed once with PBS (-) and then treated with 0.025% trypsin for 5 min at 37°C. Trypsin-treated spheroids were dispersed into single cells via pipetting. The single cells were centrifuged at 1,200 × g for 5 min, rinsed thrice using PBS (-), and treated for 10 min with a PE-labeled anti-CD133 monoclonal antibody (Miltenyi Biotechnology 1:10 dilution) and 7-aminoactinomycin D (7-AAD; 1:10 dilution) for dead cell-labeling. PBS containing 1% bovine serum albumin (BSA) was used for dilution. Subsequently, the single cells were centrifuged, rinsed thrice with PBS (-), and suspended in PBS containing 1% BSA. BD FACSJazz cell sorter (BD Biosciences) was used for flow cytometry analysis. Finally, CD133-positive and 7-AAD-negative cells or CD133-negative and 7-AAD-negative cells were sorted as SCPCs and SCNCs, respectively, according to the manufacturer's protocol (BD Biosciences). The clonogenic cell survival assay was performed within 1 h of sorting the cells.

The proportion of surviving cells was measured using a colony formation assay. Exponentially growing cells were plated at a density of 1.0×10³ or 3.0×10³ cells in three replicate 6-well plates. These cells were then incubated at 37°C for 3 h in a CO₂ incubator. Subsequently, the cells were irradiated in α-MEM with 150 kV X-rays emitted by an irradiator (Softex, M-150ME) at a dose rate of 1.1 Gy/min under normoxia at room temperature. Non-irradiated cells were treated with 0, 10, and 50 nM of NVP-AUY922 (Novartis) or dimethyl sulfoxide (DMSO) for 24 h, following which the medium containing NVP-AUY922 or DMSO was replaced with a NVP-AUY922-free or DMSO-free medium. We used doses of 10 and 50 nM of NVP-AUY922 on the basis of pre-experiments. Under combined treatment with X-rays and NVP-AUY922, the cells were irradiated in α-MEM containing NVP-AUY922 or DMSO. α-MEM containing NVP-AUY922 or DMSO was replaced with a NVP-AUY922- or DMSO-free medium 24 h after irradiation. The cells were subsequently incubated at 37°C for 10–14 days in a CO₂ incubator. The colonies formed were stained with crystal violet and dissolved in 20% methanol. Colonies containing more than 50 cells were counted as survivors. Three independent experiments were performed.

To analyze a significant interaction effect, two-way ANOVA followed by a post hoc Tukey's HSD test was conducted. Paired Student's t-test was also used for confirmation of the difference between CD133-positive and CD133-negative cells. P<0.01 was considered to indicate a statistically significant difference.

Fig. 2A-F shows anti-CD133^AC133 and anti-HIF-1α antibody immunofluorescence double-stained images. In monolayer cultured cells (Fig. 2A-C), only a few CD133-or HIF-1α-positive cells were observed occasionally. In contrast, in the cryostat sections from spheroids (Fig. 2D-F), CD133 positive or HIF-1α positive cells were observed. CD133 positive cells were also positive for HIF-1α in the hypoxic region, as suggested by HIF-1α positivity (Fig. 2F).

Fig. 2G-L shows CD133^AC133 and nestin (a marker for undifferentiated neural cell) antibodies double-stained immunofluorescence images. In monolayer cultured cells (Fig. 2G-I), only a few CD133- and nestin-positive cells were observed occasionally. In contrast, CD133 positive or nestin-positive cells were observed in the cryostat sections from spheroids (Fig. 2J-L). CD133 positive cells were also positive for nestin and were observed in the central region of spheroids (Fig. 2L).

Single cells that were dispersed from spheroids and stained immunofluorescently with anti-CD133AC133 antibodies were analyzed. CD133-positive and 7-AAD-negative cells (red-colored gating) represented ~0.8% of all measured single cells in monolayer cultured cells (negative control; Fig. 3A and C) and ~15% in the cells from spheroids (Fig. 3B and D). Sorted CD133-positive and 7-AAD-negative cells or CD133-negative and 7-AAD-negative cells (blue-colored gating) from spheroids were used for the cell viability assay.

SCPCs and SCNCs sensitivity to X-rays (6 Gy), NVP-AUY922 (10 and 50 nM), or X-rays combined with NVP-AUY922 was examined using a colony-formation assay. As shown in Fig. 4, the survival fractions among SCPCs groups (X-ray 6 Gy (0.41±0.021) vs. AUY 10 nM (0.53±0.025) or 50 nM (0.57±0.035), X-ray 6 Gy vs. X-ray 6 Gy + AUY 10 nM (0.26±0.015) or 50 nM (0.15±0.040), AUY 10 or 50 nM vs. X-ray 6 Gy + AUY 10 or 50 nM) were significantly different (P<0.01, Tukey's HSD) except for the survival fraction between groups of AUY 10 nM and AUY 50 nM with no significant difference (P>0.05). In the case of SCNCs, the survival fractions among groups (X-ray 6 Gy (0.24±0.006) vs. X-ray 6 Gy + AUY 10 nM (0.12±0.006) or 50 nM (0.11±0.040), AUY 10 nM (0.27±0.025) or 50 nM (0.28±0.010) vs. X-ray 6 Gy + AUY 10 or 50 nM) were significantly different (P<0.01) but those among groups (X-ray 6 Gy vs. AUY 10 or 50 nM, AUY 10 nM vs. AUY 50 nM, X-ray 6 Gy + AUY10 nM vs. X-ray 6 Gy + AUY 50 nM) were not significantly different (P>0.05). The difference in the proportion of surviving SCPCs between X-ray 6 Gy and X-ray 6 Gy + AUY 50 nM (Cohen's d=6.22, effect size) was larger compared with that of surviving SCNCs between X-ray 6 Gy and X-ray 6 Gy + AUY 50 nM (d=4.50). In addition, the difference in the proportion of surviving SCPCs between AUY 10 nM (d=8.79) or 50 nM (d=8.54) and X-ray 6 Gy + AUY 50 nM was larger compared with that of surviving SCNCs between AUY 10 nM (d=4.85) or 50 nM (d=5.66) and X-ray 6 Gy + AUY 50 nM. The larger Cohen's d values in SCPCs indicated that the sensitivity of SCPCs to X-rays or NVP-AUY was more strongly enhanced by combined treatment of X-rays and NVP-AUY than that of SCNCs.

Since the interaction was significant, we also confirmed the difference between CD133-positive and CD133-negative cells. The survival fractions of SCPCs exposed to X-ray 6 Gy, AUY 10 nM, and AUY 50 nM were significantly larger (P<0.01, Student's t-test) than those of SCNCs exposed to X-ray 6 Gy, AUY 10 nM, and AUY 50 nM, respectively, under X-ray irradiation or NVP-AUY922 treatment conditions, indicating a lower sensitivity of SCPCs to X-rays and NVP-AUY922. Under treatment with X-ray 6 Gy irradiation combined with a low concentration of AUY (10 nM), a significant difference in the proportion of surviving SCPCs and SCNCs was observed (P<0.01) but that of surviving SCPCs and SCNCs was not observed (P>0.05) under treatment with X-ray 6 Gy irradiation combined with a high concentration of AUY (50 nM). We did not use other HSP90 inhibitors because it was reported that other inhibitors have high cell toxicity (15–17).