J Korean Neurosurg Soc.  2015 May;57(5):323-328. 10.3340/jkns.2015.57.5.323.

Combination Therapy for Gliomas Using Temozolomide and Interferon-Beta Secreting Human Bone Marrow Derived Mesenchymal Stem Cells

• Park JH
• Ryu CH
• Kim MJ
• Jeun SS

• KMID: 1881728
• DOI: http://doi.org/10.3340/jkns.2015.57.5.323

Abstract

OBJECTIVE
Malignant gliomas are the most common primary tumors of the central nervous system and the prognosis of patients with gliomas is poor. The combination of interferon-bata (IFN-beta) and temozolomide (TMZ) has shown significant additive antitumor effects in human glioma xenograft models. Considering that the poor survival of patients with human malignant gliomas relates partly to the inability to deliver therapeutic agents to the tumor, the tropism of human bone marrow-derived mesenchymal stem cells (MSC) for malignant gliomas can be exploited to therapeutic advantages. We investigated the combination effects of TMZ and MSCs that secrete IFN-beta on gliomas.
METHODS
We engineered human MSCs to secret mouse IFN-beta (MSC-IFN-beta) via adenoviral transduction and confirmed their secretory capacity using enzyme-linked immunosorbent assays. In vitro and in vivo experiments were performed to determine the effects of the combined TMZ and MSC-IFN-beta treatment.
RESULTS
In vitro, the combination of MSC-IFN-beta and TMZ showed significantly enhanced antitumor effects in GL26 mouse glioma cells. In vivo, the combined MSC-IFN-beta and TMZ therapy significantly reduced the tumor size and improved the survival rates compared to each treatment alone.
CONCLUSION
These results suggest that MSCs can be used as an effective delivery vehicle so that the combination of MSC-IFN-beta and TMZ could be considered as a new option for the treatment of malignant gliomas.

Keyword

Temozolomide; Glioma; Mesenchymal stem cell; Interferon-beta

MeSH Terms

Animals
Bone Marrow*
Central Nervous System
Enzyme-Linked Immunosorbent Assay
Glioma*
Heterografts
Humans
Interferon-beta*
Mesenchymal Stromal Cells*
Mice
Prognosis
Survival Rate
Tropism
Interferon-beta

Figure

• 

  Fig. 1 In vitro transgene expression for MSC-IFN-β. IFN-β protein production was detected in the supernatants from IFN-β-transduced MSCs by ELISA. MSC-GFP was used as a control (data not shown). IFN-β : interferon-bata, MSC : mesenchymal stem cell, MSC-IFN-β : MSCs to secret mouse IFN-β, ELISA : enzyme-linked immunosorbent assay, GFP : green fluorescent protein.

• 

  Fig. 2 Sensitivity of the GL26 glioma cell line to TMZ and IFN-β. A : The viability of glioma cells was analyzed via MTT assay 24 h, 48 h, and 72 h after TMZ (0-500 µM) treatment. B : The number of viable cells was counted 72 h after the GL26 cell line was treated with MSC-IFN-β alone and in combination with TMZ (20 µM, which was selected as subtoxic dose). The results are representative of two independent experiments. All data are represented as the mean±SEM. *p<0.05, Student's t-test. Points, mean; bars, SEM. TMZ : temozolomide, IFN-β : interferon-bata, MSC-IFN-β : MSCs to secret mouse IFN-β, SEM : standard error of the mean, MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

• 

  Fig. 3 In vivo effects of TMZ and MSC-IFN-β on tumor growth and the survival of intracranial glioma-bearing mice. TMZ, MSC-IFN-β, or both were injected intratumorally to glioma-bearing mice at day 14 after GL26 (1×105 cells) inoculation. The PBS was used as a control. A : Representative photographs of H&E staining from each group show tumor growth. Magnification, ×1. B : The tumor sizes in each group were determined by histological analysis at day 35 after tumor inoculation. Columns, mean; bars, SEM. *p<0.05, **p<0.01; Student's t-tests. C : Survival curve of glioma-bearing mice. At day 14 after GL26 inoculation, TMZ, MSC-IFN-β, or both were injected intraperitoneally and intratumorally. Analyses of their survival were conducted by a log-rank test based on the Kaplan-Meier method. 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). The results are representative of two independent experiments (n=5 in each groups). TMZ : temozolomide, MSC-IFN-β : MSCs to secret mouse IFN-β, SEM : standard error of the mean, PBS : phosphate-buffered saline, H&E : hematoxylin and eosin.

• 

  Fig. 4 Apoptotic effects of TMZ, MSC-IFN-β, or both in combination. The PBS group was used as a control. A : Apoptotic cells were detected by TUNEL staining in cryosections. TUNEL-positive nuclei (red) were stained and counterstaining was conducted with DAPI (blue). Magnification, ×200. B : The TUNEL staining intensity was quantified by computerized image analysis. The results are representative of two independent experiments (n=5 in each groups). Columns, mean; bars, SEM. *p<0.05; Student's t-test. TMZ : temozolomide, MSC-IFN-β : MSCs to secret mouse IFN-β, PBS : phosphate-buffered saline, SEM : standard error of the mean, TUNEL : terminal deoxynucleotidyltransferased UTP nick end labeling, DAPI : 4,6-diamidino-2-phenylindole.

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