Human bone marrow-derived MSCs (Catholic MASTER Cells) were obtained from the Catholic Institute of Cell Therapy (Seoul, Korea). Cells were thawed, and the culture process was performed according to the manufacturer's instructions. Human bone marrow aspirates were obtained from the iliac crest of healthy donors aged 20 to 55 years after approval by the Institutional Review Board of Seoul St. Mary's Hospital (approval numbers KIRB-00344-009 and KIRB-00362-006). Bone marrow aspirates from each consented donor were sent to the GMP-compliant facility of the Catholic Institute of Cell Therapy (Seoul, Korea, http://www.cic.re.kr) for the isolation, expansion, and quality control of human bone marrow-derived MSCs. Cells were plated in a culture dish and cultured with human bone marrow-derived MSC basal medium supplemented with MSC growth supplement at 37℃ with 5% CO₂. The MSC growth medium was used for all cell expansion procedures, unless otherwise mentioned. During cell expansion, cells were tested for bacterial sterility, mycoplasmal sterility, and the endotoxin level (<3 EU/mL).

The recombinant adenoviral vector encoding the gene for enhanced green fluorescent protein (Ad-GFP) and mouse IFN-β (Ad-IFN-β) was constructed and produced using the AdEasy vector system, following the manufacturer's instructions (Quantum Biotechnologies, Carlsbad, CA, USA)3). MSCs were infected with a multiplicity of infection of 50 for Ad-GFP or Ad-IFN-β. Enzyme-linked immunosorbent assays (ELISA) was done to confirm the transgene expression of MSC-GFP and MSC-IFN-β. The IFN-β proteins were checked in both supernatants (Fig. 1) and the supernatants at day 3 after infection were acquired for in vitro experiments.

The GBM cell line GL26 was obtained from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY, USA). All media was supplemented with 2 mmol/L L-glutamate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum purchased from Invitrogen. Cells were incubated at 37℃ in a humidified atmosphere containing 5% CO₂/95% air.

To analyze the antitumor effects of TMZ and IFN-β in the GL26 glioma tumor cell line, cells were washed twice with the medium and incubated with medium containing MSC-GFP (control), medium containing MSC-IFN-β, or medium containing MSC-IFN-β with 20 µM TMZ. After exposure to the various conditions, cells were detached by trypsinization and the viable cell population was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)1).

The established human glioma tumor cell line, GL26, was used in this experiment. C57BL/6 mice (6-8 weeks old; Harlan-Sprague-Dawley) were used in accordance with institutional guidelines under the approved protocols. For the intracranial implantation of syngeneic GL26 tumor cells in the brains of C57BL/6 mice, animals were stereotactically inoculated with 1×10⁵ GL26 cells [in 3 µL of phosphate-buffered saline (PBS)]into the right frontal lobe (2 mm lateral and 1 mm anterior to bregma, at a depth of 2.5 mm from the skull base) using a guide-screw system (Hamilton syringe, Hamilton Company, Reno, NV, USA). To increase the uniformity of the xenograft take and growth, cells were injected simultaneously using a multiport Microinfusion Syringe Pump (Harvard Apparatus, Holliston, MA, USA). Animals were anesthetized with xylazine/ketamine during the procedure.

For the survival experiment, intracranial glioma-bearing mice were randomly divided into four groups (n=5 in each groups) after tumor implantation and treated with intratumoral injections of saline (PBS), TMZ alone, MSCs infected with Ad-IFN-β (MSC-IFN-β), or a combination of TMZ with MSC-IFN-β. TMZ was injected intraperitoneally and MSC-IFN-β was injected intracranially, directly to the tumor. The therapeutic treatments were administered on the 14th day after tumor inoculation. The brains from the treated mice (n=5/groups) were serially sectioned (20 µm-thich sections, obtained every 200 µm into the tumor) and then stained with hematoxylin and eosin. Tumor size was determined as described previously4). The section with the maximum tumor area was calculated via a computer using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).

Apoptotic cells were visualized using a terminal deoxynucleotidyltransferased UTP nick end labeling (TUNEL) assay kit (Roche, Basel, Switzerland) developed using Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Endogenous peroxidase activity was blocked by 3% H₂O₂ for 10 min at room temperature. After washing with PBS, 50 µL of TUNEL reaction mixture was pipetted onto the sections, which were then incubated in a humidified chamber at 37℃ for 1 h. The reaction was stopped by adding wash buffer. In all sections, nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) for counterstaining. Apoptotic cells were also measured using a computer with the MetaMorphsoftware (Molecular Devices, Silicon Valley, CA, USA).

All data are expressed as the mean±the standard error of the mean. All statistical comparisons between the groups were determined using one-way analyses of variance and Student's t-tests. Survival analyses were conducted by a log-rank test based on the Kaplan-Meier method. Probability values of <0.05 were considered statistically significant.

To confirm the transgene expression of MSC-IFN-β, the IFN-β protein was checked in supernatants from MSC-IFN-β by enzyme-linked immunosorbent assays (Fig. 1). IFN-β protein production was well detected in the supernatants from IFN-β-transduced MSCs. To examine the involvement of MGMT in the GL26 cell line, the expression of the MGMT was analyzed by western blot and reverse transcription (RT)-polymerase chain reaction (PCR). As a result, MGMT was not detected in both examinations (data not shown). As GL26 cells were sensitive to temozolomide, to determine the combination effect with MSC-IFN-β clearly, a temozolomide concentration of 20 µM was chosen as a subtoxic dose for the following in vitro combination experiments (Fig. 2A).

To assess the combined antitumor effects of MSC-IFN-β and TMZ in the GL26 cell line, cells were incubated in culture medium containing 0-90 µL MSC-IFN-β supernatant and/or 20 µM TMZ for 72 h. Subsequently, the number of viable cells was analyzed via MTT assay. As shown in Fig. 2B, a significantly enhanced antitumor effect was observed for the combined treatment of MSC-IFN-β and TMZ (p<0.005, combined treatment vs. each single treatment). This result suggests that the combination of MSC-IFN-β and TMZ has combined antitumor effects in the glioma cell line.

To determine whether the MSC-IFN-β treatment alone and combined with TMZ showed antitumor effects on gliomas in vivo, glioma-bearing mice were treated with MSC-IFN-β, TMZ, or both. Tumor sizes were verified by histological staining (Fig. 3A). The average tumor area in each treatment group was decreased compared with PBS-treated mice. Additionally, as shown in Fig. 3A, the tumor sizes were mostly decreased in the combined treatment group. Histological analyses were performed for each treatment group, and the combined treatment was significantly better than MSC-IFN-β or TMZ alone (p<0.05, MSC-IFN-β vs. combination; p<0.01, TMZ vs. combination) (Fig. 3B). These results show that MSC-IFN-β and TMZ alone reduced the rate of tumor growth in glioma-bearing mice, while the combination of MSC-IFN-β and TMZ, had significantly better antitumor effects. Next, survival analyses were conducted, as shown in the survival curve of glioma-bearing mice (Fig. 3C). The survival of mice in the combined treatment group was significantly prolonged compared with the survival in each of the monotherapy groups (p<0.05).

To figure out the mechanism of the combined treatment, we focused on apoptosis. To determine whether apoptotic cell death was involved in the antitumor activity of transplanted MSC-IFN-β and TMZ, TUNEL staining and counterstaining with DAPI were conducted. TUNEL staining demonstrated a significant increase in the number of apoptotic cells in the combined treatment group compared to the number in each of the monotherapy groups (p<0.05, TMZ vs. combination and MSC-IFN-β vs. combination) (Fig. 4). These results demonstrate that the antitumor activity of transplanted MSC-IFN-β and TMZ is mediated by apoptosis.