Synergistic inhibition of tumor growth by combination treatment with drugs against different subpopulations of glioblastoma cells

Mice were housed in animal rooms with controlled temperature (23 ± 2 °C) and humidity (55 ± 5%), exposed to a 12-h light-dark cycle, and allowed free access to water and food. All experimental procedures were in accordance with the National Institutes of Health guidelines and were approved by the National Cheng Kung University Medical Center Animal Care and Use Committee (project approval number #104064).

The human glioma cell line U87 was kindly provided by Dr. Michael Hsiao (Genomics Research Center, Academia Sinica, Taiwan) and rat glioma C6 cells was kindly provided by Dr. Shun-Fen Tzeng (National Cheng Kung University, Taiwan). The human glioma U87 cell line was cultured in Dulbecco’s modified Eagle medium (DMEM, Caisson) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 2 mM L-glutamine (Caisson), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Caisson). The rat glioma C6 cell line was cultured in DMEM/F12 (Caisson) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cultured cells were maintained in a humidified incubator at 37 °C in 5% CO₂/95% air. The cells were labeled with 1 ml CD133/L micromagnetic beads per million cells using a CD133 cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD133⁺ and CD133⁻ cells were plated onto 24-well culture dishes (5000 cells/well). CD133⁺ cells were plated in serum-free medium containing 10 μg/ml fibroblast growth factor 2 (FGF-2) and 10 μg/ml epidermal growth factor (EGF) and gave rise to non-adherent spheres on Ultra Low Attachment Multiple Well Plates (CORNING). CD133⁺ cells were allowed to form spheres/aggregates in a suspension culture, and were then dissociated and passaged using Accutase Cell Detachment Solution (BD Biosciences) at 37 °C for 30 min.

C6 and U87 glioblastoma parental cells were trypsinized and suspended with ice-cold phosphate-buffered saline (PBS), centrifuged at 800 g for 5 min, and then resuspended in 1 × PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA. Magnetically labeled anti-CD133 antibody from the Miltenyi Biotec CD133 cell isolation kit was used to isolate glioma CD133⁺ cells, as previously described [13]. CD133-PE conjugated antibody was applied for cell staining and evaluating the efficiency of magnetic separation via flow cytometry. The cell suspension was then placed within an autoMACS separator for magnetic separation. Labeled cells migrated toward the magnet; the unlabeled cells in suspension were drawn off. The remaining (labeled) cells were resuspended and then returned to the separator for further separation. The magnetic separation procedure was repeated twice to increase the efficiency of the magnetic separation. After the final elusion of the positive fraction of interest, the harvested cells suspended in culture medium were allowed for the downstream application. The separation purity was conducted via flow cytometry with a FACSCalibur machine (BD Biosciences).

Glioma cells (2 × 10³ cells per well) were seeded in 96-well plates. Culture medium containing vehicle or drugs was added to the medium of each well, and cells were incubated at 37 °C for the indicated time. Cytotoxicity assayed via 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H–tetrazolium monosodium salt (WST-1) reagent was used to measure cell viability. After aspirating drugs from wells, WST-1 was diluted in fresh culture medium (1:10) to a final volume of 100 μl and added into each well. The absorbance of soluble formazan was measured at 440 nm with a microplate reader. Cell viability is presented as the percentage of survivors relative to the vehicle-treated control culture. The absorbance of soluble formazan was measured at 440 nm with a microplate reader (Molecular Devices).

We used the response additivity approach [14]. In this approach, a positive drug combination effect occurs when the observed combination effect (E _AB ) is greater than the expected additive effect given by the sum of the individual effects (E _A  + E _B ). The combination index (CI) was calculated as: CI = (E_A + E_B)/E_(A + B).

Glioma cells were treated with medium containing minocycline (Mino), WP1066, or vehicle in a 10-cm dish at 37 °C. At the indicated time, cells were centrifuged at 4000 rpm and pellets were collected and stored at −80 °C. Cell pellets were lysed in a lysis buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and supplemented with protease (Roche) and phosphatase inhibitors (Roche). Lysates were shaken at 40 rpm on ice for 1 h and then centrifuged at 13,000 rpm for 30 min at 4 °C. Supernatants were collected and protein concentration was measured via the Bradford assay. The proteins were re-suspended in a 5X sample buffer (12.5 mM Tris, 25% glycerol, 4% SDS, 1.54% DTT, and 0.02% Bromophenol blue) and boiled in water for 10 min. Protein electrophoresis was conducted on 15%, 10%, or 9% SDS-polyacrylamide gel under 100 V. The separated proteins were transferred to a PVDF membrane (Immunobilon transfer membranes, Millipore) using a semi-dry transfer system (BIO-RAD) under 400 mA and 20 V for 2 h. The membrane was then immersed in 5% nonfat milk or 3% BSA) for 1 h at room temperature for non-specific blocking; it was reacted at 4 °C overnight with the following primary antibodies: CD133 (1:6000, Merck Millipore), NANOG (1:1000, ProSci Inc.), SOX-2 (1:1000, abcam), Caspase 3 (1:2000, Cell Signaling), Caspase 8 (1:2000, Cell Signaling), Caspase 9 (1:2000, Cell Signaling), and β-Actin (1:400,000, Millipore). HRP-conjugated secondary antibody (Jackson ImmunoResearch Lab., USA) was incubated at room temperature for 1 h. After three rinses with TBST for 10 min each, ECL-plus chemical reagents (PerkinElmer) were added to the membrane. The films were exposed and developed until an optimal image was obtained, but not saturated. The films were scanned and images were analyzed and quantified using ImageJ software (NIH) to evaluate the expression of proteins of interest. The protein levels in all groups are expressed as a percentage of those in controls.

For immunostaining of non-adherent spheres, cells were seeded on Ultra Low Attachment Multiple Well Plates for 7 days. Cells were then fixed with 4% paraformaldehyde (PFA) in PBS and stained with primary antibodies against CD133 (mouse monoclonal; Merck Millipore), Nestin (rabbit polyclonal; Proteintech), SSEA-1 (rabbit polyclonal; Bioss), and SOX-2 (rabbit polyclonal; Abaca). The secondary antibodies used were Alexa Fluor®594-conjugated Goat-anti-rabbit IgG (Jackson ImmunoResearch) and Alexa Fluor®488-conjugated Sheep-anti-mouse IgG (Jackson ImmunoResearch). DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich) dye was used to stain the nuclei.

To count the total number of neurospheres, CD133⁺ and CD133⁻ cells were suspended and seeded at 2.5 × 10⁴ cells/well in Ultra Low Attachment Multiple Well Plates in stem cell medium. After incubation for 14–21 days at 37 °C, floating neutrospheres were counted using inverted fluorescence microscopy (Olympus IX71). A cluster of more than five single cells was counted as a neurosphere.

The lentiviral vector pAS2.EGFP construct was obtained from the National RNAi Core Facility at Academia Sinica, Taipei, Taiwan. Firefly luciferase cDNA was put into pAS2.EGFP and a bi-cistronic lentivirus expression vector was constructed. Lentiviruses were produced by co-transfecting the GFP-Luc-expressing lentiviral vector, the envelope plasmid (pMD2.G), and the packaging plasmid (pCMV-dR8.91) into 293 T cells using calcium phosphate. The culture medium was changed on day 2, and the viral supernatants were harvested and titrated.

For tumorigenesis, 10- to 12-week-old male nude mice (BALB/cAnN-Foxnlnu/CrlNarl mice, National Laboratory Animal Center) were subcutaneously injected with 1 × 10⁴ CD133⁺ or CD133⁻ cells. For the intracranial tumor model, U87 and C6 glioma cells were transduced with a lentiviral vector expressing GFP and firefly Luc. Luciferase-expressing CD133⁺ cells (5 × 10³ cells in Fig. 3b, 1 × 10⁵ cells in Fig. 7) were injected intracranially into the 10- to 12-week-old male nude mice. The sorted cells were immediately implanted into the nude mice without further culture/amplification. Nude mice were anesthetized with chlorohydrate and placed on a stereotaxic device. Subsequently, a Hamilton syringe with a 30-gauge needle was mounted on the stereotaxic device. U87 luciferase-expressing glioma cells were injected 1.5 mm caudal and lateral to the bregma, and at a depth of 3.5–4 mm into the left side of the brain. Ten days after tumor implantation, Mino (50 mg/kg in saline with 10% DMSO), WP1066 (20 mg/kg in DMSO and polyethylene glycol), or their combination was injected intraperitoneally once per day for 10 days into the mice. Tumors were monitored via longitudinal bioluminescence imaging, for which mice were injected with 100 μg of luciferin (Caliper), simultaneously anesthetized with isoflurane, and subsequently imaged with a cooled charge-coupled device camera (IVIS-200, Xenogen). Tumor light output was quantitated using the Living Image 2.5 software package (Xenogen).

The method used to calculate combination drug interaction was based on previous reports [15, 16]. Fractional tumor volume (FTV) was calculated as tumor volume experimental/mean tumor volume control. Expected FTV was calculated as FTV of Minocycline × FTV of WP1066. A synergistic effect is suggested when the ratio of expected FTV/observed FTV is more than 1. A ratio of <1 indicates a less than additive effect.

Experiments were performed at least in triplicate. All results are presented as mean ± standard error of the mean (SEM). Independent experiments were analyzed using the unpaired t test. One-way analysis of variance (ANOVA) was used to analyze differences in neurosphere numbers, various signaling inhibitors, and cell viability. Bonferroni multiple comparison tests were used as post hoc comparisons. Data were considered significant at the p < 0.05 level.