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Keywords:

  • Umbilical cord blood-derived mesenchymal stem cells;
  • Irradiation;
  • TRAIL;
  • Glioma;
  • Gene therapy

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Irradiation is a standard therapy for gliomas and many other cancers. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for cancer gene therapy. Here, we show that tumor irradiation enhances the tumor tropism of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and the therapeutic effect of TRAIL delivered by UCB-MSCs. The sequential treatment with irradiation followed by TRAIL-secreting UCB-MSCs (MSC-TRAIL) synergistically enhanced apoptosis in either TRAIL-sensitive or TRAIL-resistant glioma cells by upregulating the death receptor 5 and by inducing caspase activation. Migration assays showed greater MSC migration toward irradiated glioma cells and the tumor site in glioma-bearing mice compared with unirradiated tumors. Irradiated glioma cells had increased expression of interleukin-8 (IL-8), which leads to the upregulation of the IL-8 receptor on MSCs. This upregulation, which is involved in the migratory capacity of UCB-MSCs, was confirmed by siRNA inhibition and an antibody-neutralizing assay. In vivo survival experiments in orthotopic xenografted mice showed that MSC-based TRAIL gene delivery to irradiated tumors had greater therapeutic efficacy than a single treatment. These results suggest that clinically relevant tumor irradiation increases the therapeutic efficacy of MSC-TRAIL by increasing tropism of MSCs and TRAIL-induced apoptosis, which may be a more useful strategy for cancer gene therapy. STEM CELLS 2010;28:2217–2228


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The prognosis of patients with glioblastoma multiforme (GBM), the most common malignant glioma, is extremely poor because glioma cells are resistant to conventional therapies [1–3] and complete surgical removal is difficult because of the invasiveness and infiltration into the adjacent functional brain parenchyma [4].

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have the capacity to differentiate into osteocytes, chondrocytes, and adipocytes [5–8]. MSCs have tumor-targeting properties, can be isolated easily and expanded to the numbers required for use, and they can be genetically manipulated with viral vectors, suggesting a potential clinical use for cancer gene therapy [9, 10]. MSC-mediated therapeutic gene delivery is a promising strategy for improving the efficacy and minimizing the toxicity of current gene therapy approaches in the treatment of glioma. We and others have demonstrated that MSCs can deliver multiple therapeutic genes, such as interleukin-2 (IL-2) [11], IL-12 [12], interferon-β [13], thymidine kinase [14], and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [15–17], selectively to tumor environments and that such delivery elicits a significant antitumor effect in animal models. Human umbilical cord blood-derived MSCs (UCB-MSCs) were proven recently to be advantageous in cell procurement, storage, and transplantation [18], and they do not trigger an immense immune reaction in unrelated donor transplantation [19]. These characteristics suggest that human UCB-MSCs have potential clinical use as effective delivery vehicles for therapeutic genes in the allogenic treatment of glioma.

The intrinsic homing property of MSCs to tumors suggests that this might be a promising strategy for delivering therapeutic agents into tumors. However, for clinical applications, it would be desirable if a sufficient quantity of engineered MSCs that localize within tumors is achieved in the MSC-mediated gene therapy. This requires the development of methods to improve the migratory capacity of MSCs to tumors, which would thereby increase the delivery of the therapeutic genes. Enhancing the MSC localization to tumors has been investigated by genetically manipulating MSCs to overexpress target receptors against epidermal growth factor-producing gliomas [20], and the overexpression of any receptor on MSCs related to tumor tropism may improve their migration capacity to specific tumor cells. Another potential method to enhance engraftment is based on the observation that MSCs migrate to damaged tissue after local tumor irradiation. Several studies have shown greater MSC migration to irradiated tumors compared with unirradiated tumors [21, 22]. The mechanism responsible for this increased migration and the factors related to the targeted tropism of MSCs remain to be elucidated; however, it is thought that irradiation increases inflammatory signaling, which involves secretion of chemokines or growth factors and may attract MSCs to the tumor microenvironment [21].

TRAIL is one of the most promising candidates for cancer therapy because of its selective induction of apoptosis in a wide variety of transformed cells without damaging normal cells and tissues [23]. Although TRAIL induces apoptosis preferentially, not all tumor cells are sensitive to TRAIL. In particular, most malignant glioma cells are resistant to TRAIL-induced cytotoxicity despite the expression of death-inducing TRAIL receptors on their surface [24], suggesting that TRAIL treatment by itself may be ineffective for cancer therapy and that new strategies are necessary to overcome TRAIL resistance. Recent reports show that chemotherapeutic agents or radiotherapy can enhance TRAIL sensitivity by increasing the expression of TRAIL receptor 1 (death receptor 4, DR4) and/or TRAIL receptor 2 (death receptor 5, DR5) in a range of tumors [25]; these findings suggest that a synergistic antitumor effect might be achieved using combination therapies. In the treatment of glioma, combination treatment with soluble recombinant TRAIL [26] or selective DR4 and DR5 agonistic antibodies [27] and irradiation synergistically enhances TRAIL-induced cell death.

In this study, we verified that tumor irradiation enhances the engraftment of TRAIL-secreting UCB-MSCs (MSC-TRAIL) into the tumors and the therapeutic potential of combined treatment with MSC-TRAIL.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

UCB-MSC Preparation and Cell Culture

Human UCB-MSCs were kindly provided by Medipost Co. (Seoul, Korea, http://www.medi-post.co.kr), which was isolated and expanded as reported previously [28]. UCB-MSCs were subcultured at a concentration of 5 × 104 cells/cm2 in minimum essential medium-α (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and used for experiments during passages 5–8. Human glioma cell lines were obtained from the American Type Culture Collection (Manassas, VA, http://www.atcc.org), and primary glioma cells were obtained from fresh specimens of glioma-bearing patients undergoing surgery after ethical approval and written informed consent. Glioma cells were maintained in Eagle's minimum essential medium or Dulbecco's modified Eagle's medium (DMEM; Invitrogen). Normal human astrocytes were obtained from the Applied Cell Biology Research Institute (Kirkland, WA, http://www.cellsystems.com) and cultured in DMEM. Enhanced green fluorescent protein (EGFP)-expressing U-87MG cells (U87-EGFP) were prepared as reported previously [15]. All cells were supplemented with antibiotics and 10% fetal bovine serum, and incubated at 37°C in a humidified atmosphere containing 5% CO2.

Adenovirus Infection

The recombinant replication-deficient adenoviral vector encoding the gene for EGFP was constructed using the AdEasy Vector System (QBiogene, Carlsbad, CA, http://www.qbiogene.com). Adenovirus carrying the secretable trimeric form of the TRAIL gene was kindly provided by Dong-A Pharmaceutical Co. (Yongin, Korea, http://www.donga. co.kr), which was engineered as described previously [29]. Cell permeable peptides (CPPs) were provided by Prof. Young-Chul Sung (Pohang University of Science and Technology, Pohang, Korea). To transfect UCB-MSCs, adenoviruses at 50 multiplicity of infection were pretreated with 0.01 μM of CPPs in serum-free medium (SFM) for 30 minutes at room temperature, and the cells were then incubated with the premixed virus-CPPs complex for 30 minutes as described previously [15].

Irradiation

Cells were plated in either 35- or 100-mm dishes overnight and then exposed to γ-irradiation using a 137Cs Gammacell 3000 Elan source (MDS Nordion, Ottawa, ON, Canada, http://www.mds.nordion.com) at various doses as indicated. After irradiation, the cells or culture supernatants were used for subsequent assays. For the irradiation of murine tumors, stereotactic radiotherapy was performed with a 6-MV X-ray produced by a linear accelerator (Digital Mevatron MX2; Siemens Co., Concord, NH, http://www.siemens.com). The mice were anesthetized with ketamine/xylazine and positioned such that the apex of each tumor was at the center of a 6-mm aperture in the secondary collimator and irradiated at various doses as indicated.

Cell Viability Assay

Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cytotoxicity assay (Sigma, St Louis, MO, http://www.sigma-aldrich. com). Glioma cells were seeded in 96-well plates to measure TRAIL-induced cytotoxicity and in 35-mm dishes to confirm cell death by irradiation. Cells were treated with increasing doses of recombinant human TRAIL (rhTRAIL; R&D Systems, Minneapolis, MN, http://www.rndsystems.com) or irradiation and then analyzed after 48 hours. To assess the effect of combined treatment, tumor cells or irradiated tumor cells were seeded in 24-well plates and rhTRAIL was added. For the coculture experiments, MSC-TRAILs (2 × 104) were plated in the Transwell inserts containing 0.4-μm pores (Corning Inc., Corning, NY, http://www.corning.com), and then the tumor cells or irradiated tumor cells (5 × 104) were grown in the lower well of the Transwell plates. At 24 or 48 hours after treatment, cells were analyzed for viability. To confirm the cytotoxicity of UCB-MSCs and normal human astrocytes, irradiated, or unirradiated cells were seeded in the lower wells. For the inhibition studies, DR5/Fc chimera protein (R&D Systems) was added to the lower wells.

Flow Cytometry to Analyze TRAIL Death Receptors

Cells were analyzed for the surface expression of TRAIL death receptors with phycoerythrin-conjugated anti-human DR4 and DR5 (R&D Systems). Briefly, cells (2.5 × 105) were stained with each antibody on ice for 30 minutes. After washing with phosphate-buffered saline (PBS), the expression levels of these death receptors were analyzed by flow cytometry using a fluorescence-activated cell sorting (FACS) Vantage SE (Becton, Dickinson and Co., Franklin Lakes, NJ, http://www.bd.com).

Western Blotting

Cell lysates were prepared in 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (RIPA) buffer (Sigma) containing a protease inhibitor cocktail (Roche, Mannheim, Germany, http://www.roche.com) and subjected to Western blot analysis as described previously [15]. The membrane proteins were separated with Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit according to the manufacturer's protocol (Thermo Fisher Scientific Inc., Barrington, IL, http://www.thermofisher.com). Details of the antibodies are described in the Supporting Information.

Reverse Transcription Polymerase Chain Reaction

Total RNA was extracted from UCB-MSCs and tumor cells using TRIzol (Invitrogen). cDNA was synthesized with three micrograms of total RNA and oligo(dT) primer (Invitrogen) using an Omniscript RT kit (Qiagen, Valencia, CA, http://www.qiagen.com) for reverse transcription polymerase chain reaction (RT-PCR). Details of the primers are described in the Supporting Information. PCR was conducted as described previously [30].

Transfection of U-87MG Cells with Small Interfering RNA

IL-8 small interfering RNA (siRNA) was obtained from Bioneer (Daejeon, Korea, http://www.bioneer.co.kr). In brief, U-87MG cells were diluted with fresh medium without antibiotics and transferred to six-well plates with 2 × 105 cells per well. siRNA transfection was performed using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). The cells were exposed to IL-8 siRNA at a final concentration of 40 nM. Control treated cells were exposed to the scrambled siRNA (Santa Cruz Biotechnology, http://www.scbt.com).

Enzyme-Linked Immunosorbent Assay

Glioma cells in SFM were exposed to 0, 2, 5, 10, or 20 Gy using a γ-irradiator. Culture supernatants were harvested after 48 hours, and the enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentrations of IL-8, stromal cell-derived factor-1α (SDF-1α), and monocyte chemoattractant protein-1 (MCP-1) using Quantikine immunoassay kits from R&D Systems. The secretion level of TRAIL from transduced UCB-MSCs was measured as described previously [15].

In Vitro Migration Assay

The migratory ability of UCB-MSCs was determined using Transwell plates that were 6.5 mm in diameter with 8-μm-pore filters (Corning Inc.). Cells that had been irradiated or unirradiated tumor cells (1 × 106) were incubated in SFM for 48 hours and the resulting conditioned medium (CM) was used as the chemoattractant. MSCs or MSC-TRAIL (2 × 104 cells) were seeded into the upper well, and 600 μl of CM was placed in the lower well of the Transwell plate. For the siRNA inhibition assay, U-87MG cells were transfected with IL-8 siRNA and CM was prepared from the culture supernatant of unirradiated or irradiated cells. For the antibody-neutralizing assay, antibodies (R&D Systems) against chemokine (C-X-C motif) receptor 1 (CXCR1) (0.2 μg/ml) and IL-8 (0.2 μg/ml) were pretreated in the lower well. The filter was stained with the Three-Step Stain Set (Diff-Quik, Sysmex, Kobe, Japan, http://www.sysmex.com), and the cells that migrated to the lower side of the filter were counted under a light microscope (Axio Imager A1; Carl Zeiss, Jena, Germany, http://www.zeiss.com) for five high-power fields (×400).

Animals and Tumor Model

Male athymic nude mice (6- to 8-weeks old; Charles River Laboratories, Wilmington, MA, http://www.criver.com) were used in accordance with approved protocols of our institutional guidelines. To evaluate tumor growth, mice were injected subcutaneously with 1 × 106 U-87MG cells (in 100 μl PBS) on the left or right flank region. For the intracranial xenografts of human glioma, mice were anesthetized intraperitoneally with ketamine/xylazine, and then inoculated stereotactically with 1 × 105 U-87MG cells (in 3 μl PBS) into the right frontal lobe (2-mm lateral and 1-mm anterior to the bregma at a depth of 2.5 mm from the skull base) or into both hemispheres via a Hamilton syringe (Hamilton Company, Reno, NV, http://www.hamiltoncompany.com) using a microinfusion pump (Harvard Apparatus, Holliston, MA, http://www.harvardapparatus.com).

In Vivo Migration of UCB-MSCs

Seven days after tumor cell inoculation into both hemispheres, the tumors were irradiated only in the right frontal lobe. On the next day, EGFP-expressing MSCs (MSC-EGFP) or MSC-TRAILs (1 × 106 cells in 100 μl PBS) were injected intravenously through the tail vein. Migration toward the tumors was assessed at 5 days after MSC inoculation by direct visualization using a fluorescence microscope or confocal microscope (LSM 510 Meta; Carl Zeiss) and by ELISA assay for TRAIL.

Therapeutic Efficacy Study

To assess the inhibition of tumor growth by the combined treatment, U-87MG cells were inoculated subcutaneously as described earlier. When the diameter of the tumors reached the expected range, mice were randomized into four groups (n = 7/group), and the tumors were injected with PBS or MSC-TRAIL (5 × 105 cells in 10 μl PBS) intratumorally. The tumors were irradiated 24 hours before MSC-TRAIL injection. The tumor volume was calculated using the formula A × B2 × 0.5, where A and B represent the larger and smaller diameter of the tumor, respectively. To evaluate the survival of the treated animals, U-87MG cells were inoculated orthotopically as described earlier. After 7 days, mice were randomized (n = 5/group) and exposed to a single intratumoral irradiation. On the next day, unirradiated or irradiated tumors were injected with MSC-TRAIL (2 × 105 cells in 5 μl PBS) intratumorally. Survival was followed for a maximum of 90 days.

Immunohistochemistry

Mouse brains were perfused with PBS followed by 4% paraformaldehyde under deep anesthesia. The excised brains were fixed, embedded, snap frozen in liquid nitrogen, and stored at −70°C until use. Tissues were cryosectioned (14 μm) and then stained with IL-8 (R&D Systems). The primary antibodies were detected with fluorescein-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). To detect apoptotic activity, brain tissues were stained using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (Roche) developed using Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories). In some sections, brains were stained with H&E to visualize the tumor mass, and nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma) for counterstaining.

Statistical Analysis

All data are expressed as mean ± standard error of the mean. The significance of differences between test conditions was assessed using Student's t test. Probability values less than .05 were considered as significant. Statistical analysis of survival was performed by a log-rank test.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Synergistic Enhancement of TRAIL-Mediated Cytotoxicity by Irradiation

We first determined the effects of TRAIL or irradiation separately in human glioma cells. The treatment with various doses of rhTRAIL-induced cell death with varying sensitivity. LN-18, T98G, and A172 cells were sensitive to TRAIL, U-373MG and U-251MG cells were resistant, and U-87MG cells had intermediate sensitivity to TRAIL (Supporting Information Fig. S1A). Interestingly, most of the primary glioma cells were resistant to the treatment with rhTRAIL. A single irradiation at various doses also affected cell viability, although the inhibition of cell growth did not exceed 20% except in one cell line (U-251MG; Supporting Information Fig. S1B). Next, we examined whether TRAIL treatment combined with irradiation enhances the therapeutic potential in human gliomas. Concurrent treatment of U-87MG, U-373MG, and primary glioma cells (GBM2) with irradiation plus rhTRAIL caused substantial and synergistic death compared with the treatment with a single agent alone, p < .01 (Fig. 1A). We also tested the therapeutic effects of sequential treatment with irradiation followed by TRAIL or TRAIL followed by irradiation. Interestingly, pretreatment with irradiation followed by TRAIL induced significantly (p < .05) more apoptosis than did concurrent treatment in both TRAIL-responsive U-87MG and TRAIL-resistant U-373MG and GBM2 cells. However, the reverse treatment induced less apoptosis than the sequential treatment of irradiation followed by TRAIL or the concurrent treatment. In addition, sequential treatment with rhTRAIL (10–100 ng/ml) combined with irradiation caused death of the human glioma cell lines (U-87MG and U-373MG) and primary glioma cells (GBM2 and GBM5) in a dose-dependent, synergistic manner (Supporting Information Fig. S2). These results suggest that sequential treatment with irradiation followed by TRAIL enhances the therapeutic potential in TRAIL-sensitive or TRAIL-resistant glioma cells.

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Figure 1. Cytotoxic effects of rhTRAIL and therapeutic potential of MSC-TRAIL combined with irradiation in glioma cells. (A): For the effect of combined treatment, tumor cells or irradiated tumor cells were seeded in 24-well plates. Cells were cultured for 24 hours after exposure to irradiation (10 Gy), followed by treatment with or without rhTRAIL (10 ng/ml) for another 24 hours. In addition, cells were treated with rhTRAIL for 24 hours, followed by treatment with or without irradiation for 24 hours. As a concurrent treatment, irradiation plus rhTRAIL were exposed for 48 hours. #, p < .01 compared with irradiation or TRAIL; *, p < .05. (B): The effect of MSC-TRAIL in combined treatment was analyzed by coculture experiment. MSC-TRAIL in Transwell inserts (0.4-μm pores) and irradiated glioma cells in lower wells of 24-well plates were cocultured for 24 hours and the viability was determined by MTT assay. Glioma cells were exposed to irradiation (10 Gy) 1 day before coculture experiment. The viability of glioma cells treated with irradiation or MSC-TRAIL alone was analyzed after 48 hours. (C): MSCs and NHA cells were exposed to irradiation (10 Gy) for 24 hours, followed by treatment with rhTRAIL (10 ng/ml) or MSC-TRAIL in Transwell inserts for another 24 hours. The viability of cells treated with irradiation, rhTRAIL, or MSC-TRAIL alone was analyzed after 48 hours. Abbreviations: GBM, glioblastoma multiforme; IR, irradiation; MSCs, mesenchymal stem cells; MSC-TRAIL, TRAIL-secreting mesenchymal stem cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHA, normal human astrocytes; rhTRAIL, recombinant human TRAIL; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

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Therapeutic Potential of TRAIL-Secreting UCB-MSCs Combined with Irradiation

MSC-TRAIL was engineered efficiently with adenovirus-mediated transduction as described in “Materials and Methods” section. To determine whether the growth inhibition was caused specifically by the release of soluble TRAIL, we used Transwell plates containing semiporous membranes to separate the cells. Coculture experiments showed that MSC-TRAIL had greater therapeutic efficacy compared with rhTRAIL in sequential treatment after irradiation (Fig. 1B), indicating that MSC-TRAIL secreted biologically relevant quantities of TRAIL protein continuously. We also examined the effects of the combined treatments on MSCs and normal human astrocytes. Treatment with irradiation, rhTRAIL, or MSC-TRAIL alone or combination with irradiation followed by rhTRAIL or MSC-TRAIL showed marginal cytotoxicity in these cells (Fig. 1C), indicating that UCB-MSCs or normal human astrocytes are resistant to both the combination and single treatment. These results indicate that MSC-based TRAIL gene therapy combined with irradiation is better than the treatment with rhTRAIL.

Enhanced TRAIL-Induced Apoptosis via Upregulation of DR5 by Irradiation

To explore the underlying mechanisms by which irradiation enhances TRAIL-induced apoptosis, we determined whether irradiation could increase the expression of TRAIL receptors. Flow cytometry showed that the membrane expression level of DR5 was increased in a dose-dependent manner in glioma cell lines and primary glioma cells except in U-251MG cells (Fig. 2A). Western blot analysis also showed a dose-dependent increase in DR5 protein expression after irradiation (Fig. 2B). Flow cytometry and Western blot analysis showed that the expression of another death receptor, DR4, was not affected by irradiation (data not shown). DR5 expression increased by as much as 90% in U-87MG and GBM2 cells, and TRAIL-induced cytotoxicity was increased more by irradiation in these cells than in U-373MG cells. In contrast, 10 Gy of irradiation did not increase DR5 protein expression appreciably in U-251MG cells, which had not responded to the combination treatment. To confirm that the induction of apoptosis by the combination therapy is mediated through DR5, we used DR5/Fc chimera proteins to block the interaction of DR5 and TRAIL. DR5/Fc efficiently blocked apoptosis induced by combined treatment of irradiation followed by rhTRAIL or MSC-TRAIL (Fig. 2C). We next investigated whether the irradiation-enhanced TRAIL-induced apoptosis is mediated through caspase activation (Fig. 2D). The combination treatment induced cleavage of caspase-8, an initiator caspase. The intrinsic pathway also became activated, as shown by cleavage of caspase-9 and Bid. Ultimately, the combination treatment caused significant cleavage of caspase-3, a major effector caspase, indicating that irradiation and TRAIL induce apoptosis by activating multiple caspases. The cleavage of caspase-8 and caspase-9, Bid, and caspase-3 was inhibited in the presence of DR5/Fc, suggesting that the synergistic enhancement of apoptosis caused by the combined treatment is mediated through the specific interaction between TRAIL and DR5.

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Figure 2. Enhanced TRAIL-induced apoptosis by upregulation of DR5 and caspase activation after irradiation. (A): Glioma cells were treated with increasing doses of irradiation (0–10 Gy) for 24 hours and DR5 expression was determined by flow cytometry analysis. (B): Cells were exposed to irradiation (0–10 Gy) for 24 hours, and then total cell extracts were analyzed by Western blot with anti-DR5 antibody. β-actin expression was used as a loading control. (C): U-87MG, U-373MG, and GBM2 cells were treated with irradiation (10 Gy), followed by rhTRAIL (3–100 ng/ml) and MSC-TRAIL with or without DR5/Fc chimera protein (100 ng/ml), and then the viability of glioma cells was determined by MTT assay. (D): U-87MG cells were treated with irradiation (10 Gy), MSC-TRAIL, or irradiation followed by MSC-TRAIL with or without DR5/Fc chimera protein (100 ng/ml). After 48 hours, total cell extracts were analyzed by Western blot with antibodies against caspase-8, Bid, caspase-9, and caspase-3. β-actin expression was used as a loading control. Abbreviations: DR5, death receptor 5; GBM, glioblastoma multiforme; IR, irradiation; MSC-TRAIL, tumor necrosis factor-related apoptosis-inducing ligand-secreting mesenchymal stem cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; rhTRAIL, recombinant human TRAIL.

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Enhanced Migration of UCB-MSCs Toward Irradiated Tumors

Next, we tested whether tumor irradiation would affect the tropism of UCB-MSCs. In the Transwell migration assay, the migration of MSC-TRAIL was increased by CM from irradiated glioma cells in a dose-dependent manner (Fig. 3A). Migration of MSC-TRAIL was two to three times greater to glioma cells irradiated with 10 Gy than to the control (untreated) cells (Fig. 3B). We also confirmed that unmodified MSCs migrated in a similar pattern to MSC-TRAIL (data not shown), indicating that the migratory ability of UCB-MSCs was not affected by adenoviral transduction. In addition, we investigated whether implanted MSCs could migrate toward intracranial gliomas treated with in vivo irradiation (Fig. 3C). Although EGFP-fluorescent MSCs were observed in both irradiated and untreated tumors, more MSCs were found in irradiated tumor tissues. Tumor size regression in response to irradiation was also observed by H&E staining. After normalization to the ratio of irradiated and untreated tumor volume, the number of MSCs that migrated to irradiated tumors was higher than those that migrated to untreated tumors. Moreover, we showed that increased expression of TRAIL delivered by MSCs in irradiated tumors compared with untreated tumors quantitatively (Fig. 3D). Similar pattern of increased TRAIL expression was also observed in mice treated with four daily fraction of 2.5 Gy, a more clinically relevant model, which was also confirmed in vitro migration assay (Supporting Information Fig. S3).

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Figure 3. Increased migration ability of umbilical cord blood-derived MSCs toward irradiated tumors and irradiation-induced increase of IL-8 expression in glioma. (A): The migratory ability of MSC-TRAIL in response to conditioned medium from untreated or irradiated (2 and 10 Gy) tumors was determined using a Transwell plate (8-μm pores). Representative photomicrographs of stained filters show migrated cells. Magnification, ×200. (B): Cell migration was compared and evaluated after staining by taking photographs and counting cells that had migrated under a light microscope. *, p < .001 compared with control; **, p < .05 compared with control. (C): Seven days after U-87MG inoculation into both hemispheres, tumor-bearing mice were exposed to irradiation (10 Gy) only in the right frontal lobe. MSC-EGFP (1 × 106 cells in 100 μl PBS) were injected i.v. through the tail vein at 24 hours after irradiation. Migration was assessed at 5 days after MSC-EGFP inoculation by direct visualization using fluorescence microscope. Sections were shown at original magnification (×200). Dotted line, tumor edge. (D): Tumor-bearing mice in both hemispheres were exposed to either single irradiation (10 Gy) or daily fractionated radiation (2.5 Gy × 4) only in the right frontal lobe. MSC-TRAILs (1 × 106 cells in 100 μl PBS) were injected i.v. through the tail vein at 24 hours after the last treatment of irradiation. For the quantification of TRAIL delivered by MSCs in tumors, brain tissues containing tumors were homogenized at 5 days after MSC-TRAIL inoculation and then assessed by enzyme-linked immunosorbent assay (n = 5 per group). Tumor tissues from the mouse without irradiation were used as a control. (E): Seven days after tumor irradiation (10 Gy), irradiated or unirradiated (control) mice were sacrificed and then stained with IL-8 antibody (green). Magnification, ×200. Dotted line, tumor edge. (F): IL-8 mRNA levels were determined in the tissues from untreated and treated with irradiation in normal or tumor-bearing mice. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBM, glioblastoma multiforme; IL-8, interleukin-8; IR, irradiation; MSC-EGFP, enhanced green fluorescent protein-expressing mesenchymal stem cells; N, normal; T, tumor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

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To verify whether the appearance of factors secreted by irradiated tumor cells correlate with enhancement of the MSC migration, we examined the expression level of cytokines such as IL-8, SDF-1α, and MCP-1, which are implicated in the attraction of MSCs to tumors [31, 32]. Expression levels of IL-8 and MCP-1, but not SDF-1α, increased in a dose-dependent manner (Supporting Information Fig. S4A). In our previous report [30], the increase in IL-8 enhanced the migration of UCB-MSCs, whereas the treatment with MCP-1 had no dose-dependent effect on UCB-MSC migration. Thus, IL-8 was selected for further analysis among the cytokines listed earlier. We used immunohistochemical staining to investigate the expression level of IL-8 in tumors irradiated in vivo (Fig. 3E). IL-8 expression increased markedly in irradiated tumors compared with untreated tumors. Measurements of IL-8 mRNA levels using RT-PCR confirmed that U-87MG, U-373MG, and GBM2 cells and U-87MG-xenografted tumor tissues expressed more IL-8 in irradiated cells (Supporting Information Fig. S4B) or tissues (Fig. 3F) compared with their unirradiated controls. IL-8 expression was undetectable and was not induced by treatment in normal human astrocytes or normal mouse brain.

Role of Irradiation-Induced Expression of IL-8 in Enhanced MSC Migration

We next used an inhibition assay with siRNA and specific antibodies to investigate whether the upregulation of IL-8 is required for the enhanced migration of MSCs to irradiated tumors. Expression of IL-8 was suppressed successfully by IL-8 siRNA in U-87MG cells, which secreted less IL-8 into the culture medium (Fig. 4A). In the Transwell migration assay, migration of MSC-TRAIL to the irradiated cells transfected with the control siRNA was greater than in the unirradiated controls (p < .01). By contrast, the migration activity of MSC-TRAIL toward CM was inhibited significantly (p < .001) in both irradiated and unirradiated tumor cells transfected with IL-8 siRNA (Fig. 4B). Interestingly, exposure of MSCs to the irradiated glioma cells by coculturing in the Transwell plate increased the expression of CXCR1, the specific IL-8 receptor (Fig. 4C). This observation could be explained by the increased secretion of IL-8 by irradiated tumor cells and was confirmed by treatment with recombinant IL-8 to the MSC culture medium and Western blot analysis using the lysate from the membrane fraction of MSCs (Fig. 4D). In addition, treatment with specific antibodies against CXCR1 or IL-8 inhibited MSC migration toward the CM from both irradiated and unirradiated U-87MG cells (Fig. 4E). These results suggest that the IL-8/CXCR1 axis plays a critical role in the irradiation-induced increase in the migratory activity of MSCs.

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Figure 4. Role of IL-8 in irradiation-induced umbilical cord blood-derived-MSC migration. (A): U-87MG cells were transfected with either control or IL-8 siRNA using lipofectamine reagent. Downregulation of IL-8 mRNA levels was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and IL-8 secretion was analyzed by enzyme-linked immunosorbent assay. (B): Effect of IL-8 knockdown in U-87MG cells on the migration of MSC-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was examined using a Transwell migration assay. Representative photomicrographs of stained filters show migrated cells (left). Number of migrated MSC-TRAIL in response to conditioned medium from U-87MG cells transfected with control or IL-8 siRNA after irradiation was analyzed (right). Magnification, ×200. *, p < .01; **, p < .001 compared with control siRNA-transfected cells. (C): CXCR1 expression in UCB-MSCs induced by irradiated glioma cells was determined by coculturing in the Transwell plates (0.4-μm pores). CXCR1 mRNA levels were analyzed by RT-PCR after 48 hours. (D): Upregulation of CXCR1 expression was also confirmed by treatment with recombinant IL-8 to the MSC culture medium and Western blot analysis using the lysate from the membrane fraction of MSCs. Coomassie stain was used as a loading control. (E): Effect of the treatment with CXCR1 and IL-8 blocking antibodies on the migration of MSC-TRAIL was examined using a Transwell migration assay. Representative photomicrographs of stained filters show migrated cells. Magnification, ×200. Abbreviations: Ab, antibody; Ct, control; CXCR1, chemokine (C-X-C motif) receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-8, interleukin-8; IR, irradiation; MSCs, mesenchymal stem cells; WB, Western blot.

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Therapeutic Potential of Combined Therapy with Irradiation and MSC-TRAIL in a Xenograft Glioma Mice Model

Because the combined treatment enhanced cytotoxicity synergistically in vitro, we evaluated the therapeutic efficacy of this treatment in subcutaneous and intracranial xenograft mouse models after inoculation with U-87MG cells. The average tumor volume was smaller in mice treated with irradiation or MSC-TRAIL alone than in the control animals; however, the tumor volumes did not differ between animals treated with irradiation and MSC-TRAIL (Fig. 5A, 5B). Interestingly, sequential treatment with MSC-TRAIL after irradiation synergistically inhibited tumor growth. In addition, we assessed the survival of intracranial glioma-bearing mice treated with irradiation, MSC-TRAIL, or irradiation followed by MSC-TRAIL. Survival was dose-dependently greater in mice exposed to irradiation compared with unexposed control mice (Fig. 5C). We also confirmed that exposure to brain irradiation did not affect the survival of normal mice even at high doses of irradiation. More importantly, treatment of tumor-bearing mice with MSC-TRAIL after irradiation prolonged survival compared with mice administrated with MSC-TRAIL alone (Fig. 5D). Irradiation also had a dose-dependent therapeutic effect. To evaluate the ability to induce apoptosis and tumor size regression, TUNEL and H&E staining were performed (Fig. 5E). Although irradiation and MSC-TRAIL alone induced apoptosis in tumor tissues, sequential treatment with irradiation followed by MSC-TRAIL induced greater apoptosis compared with the single treatment alone. H&E staining showed nearly completely regressed tumor growth in the mice given the combined treatment.

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Figure 5. Effect of combined therapy with irradiation followed by MSC-TRAIL on tumor growth and survival. (A): Combined effect of irradiation with MSC-TRAIL on tumor growth was determined in a subcutaneous glioma mice model. When tumor reached 3–4 mm in diameter (the average tumor volume at the start of treatment, 16.7 ± 1.95 mm3), mice were exposed to irradiation (10 Gy), injected with MSC-TRAIL, and exposed to irradiation followed by MSC-TRAIL administration (24 hours after irradiation). Tumor volume was measured at an interval of 3 days. (B): Representative photograph shows growing tumor mass (arrow) of each treatment group. (C, D): The survival of orthotopic xenograft mice model was analyzed by a log-rank test based on the Kaplan–Meier method. (C): Tumor-bearing mice were exposed to irradiation dose-dependently (5, 7.5, and 10 Gy). Normal mice were also treated with irradiation (5 and 10 Gy) as a control. (D): Tumor-bearing mice were treated with PBS, MSC-TRAIL alone, or irradiation followed by MSC-TRAIL in a dose-dependent manner (24 hours after irradiation). (E): Apoptosis in the treated groups was analyzed by TUNEL staining. Magnification, ×200. Dotted line, tumor edge. Representative photographs of H&E staining from each treatment group shows tumor size regression induced by apoptosis. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; IR, irradiation; MSC-TRAIL, tumor necrosis factor-related apoptosis-inducing ligand-secreting mesenchymal stem cells; N, normal brain; T, tumor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

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Furthermore, to evaluate tumor-specific apoptosis-inducing ability of TRAIL secreted from UCB-MSCs in irradiated gliomas, TUNEL staining was done. MSC-TRAIL-treated tumors were almost completely apoptotic, and apoptosis was confined to the glioma cells and not adjacent normal nonglioblastoma cells (Fig. 6A–6G). Apoptotic activity detected in MSC-TRAIL-treated normal brain tissues was negligible (Fig. 6H–6K). Accordingly, apoptotic cells were detected only in the tumor mass and were not seen in normal brain parenchyma, indicating that MSC-TRAIL migrated toward tumor cells and induced tumor cell death specifically, and thus this combined treatment had no adverse toxic side effects. In addition, we quantitatively measured the levels of TRAIL protein and its longevity in vivo to determine whether the stable expression of TRAIL transduced with adenovirus is another toxicity concern. TRAIL protein expressed in tumor tissues began to decrease after day 7 and persisted by 3 weeks (Fig. 6L).

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Figure 6. Glioma-specific cytotoxicity not in the normal brain tissues of combined therapy with irradiation followed by mesenchymal stem cell (MSC)-TRAIL. Seven days after U87-EGFP inoculation into the right hemisphere, tumor-bearing mice were exposed to irradiation (10 Gy). MSC-TRAIL (2 × 105 cells in 5 μl PBS) were injected intratumorally at 24 hours after irradiation. Normal mice were also treated with irradiation followed by MSC-TRAIL in the right hemisphere as a control. Apoptosis was assessed at 7 days after MSC-TRAIL inoculation by TUNEL staining. (A, H): Representative photographs of H&E staining from each treatment group shows growing tumor mass. (B, I): Merged images of the treated site in the tumor-bearing brain or normal brain. Magnification, ×50. (B): MSC-TRAIL-treated brains stained with TUNEL (red) show the specific staining of apoptosis in the tumor (green) and the lack of staining in adjacent normal tissue. Dotted line, tumor edge. (I): A section from MSC-TRAIL-treated normal brains shows negligible TUNEL-positive cells. Dotted line, injection site. (C–E): Apoptotic cells were detected in the main tumor mass not in the normal tissues from rectangular sections in (B). Magnification, ×100. Dotted line, tumor edge. (F, G): A higher magnification (×400) of boxed area in (C) and (E) shows tumor-specific apoptosis. (J, K): A high-power view (×400) from the area indicated with (a) and (b) in (I). Nuclei were stained with DAPI (blue) for counterstaining. (L): For the quantification of longevity of TRAIL expression in tumor tissues, brain tissues were homogenized at 1, 4, 7, 10, 14, and 21 days (n = 3 per group) after treatment of MSC-TRAIL and then assessed by enzyme-linked immunosorbent assay. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; N, normal brain; T, tumor mass; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; U87-EGFP, enhanced green fluorescent protein-expressing U-87MG glioma cells.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Our results show that MSC-based TRAIL gene therapy combined with irradiation is more advantageous against glioma than is treatment with rhTRAIL and irradiation. We also found that the improved migratory ability of MSCs by irradiation enhanced the delivery of the therapeutic gene. This enhanced MSC tropism seems to be mediated, at least in part, by the axis of increased IL-8 secretion from irradiated tumors and upregulation of its receptor, CXCR1, on MSCs in response to irradiation of the tumor cells. These results suggest that clinically relevant tumor irradiation increases the antitumor effect of MSC-based TRAIL therapy by increasing tropism of MSCs and TRAIL-induced apoptosis.

Radiation is associated with the release of several cytokines from exposed tissue [33]. The production of cytokines after irradiation might be responsible for the attraction of MSCs. Inflammatory-related cytokines, such as tumor necrosis factor-α, platelet-derived growth factor, and SDF-1α, have been shown to be upregulated by irradiation [34, 35]. In addition, recent studies showed that IL-8, MCP-1, transforming growth factor-β1, neurotrophin-3, and vascular endothelial growth factor-A mediate the recruitment to gliomas of bone marrow-derived MSCs (BM-MSCs) [31, 32]. We have also shown that IL-8 can induce UCB-MSC migration and that the IL-8-mediated migration capacity of UCB-MSCs is superior to that of BM-MSCs because of greater expression of IL-8 receptors in UCB-MSCs than in BM-MSCs [30]. Therefore, we hypothesized that irradiation would induce the release of cytokines related to tumor tropism and would enhance the engraftment of UCB-MSCs into the irradiated tumors. We found that release of IL-8 was induced by irradiation and enhanced MSC migration; this finding was confirmed by siRNA and antibody inhibition assays. These findings indicate that MSC migration to irradiated tumors is affected by factors secreted from tumor cells, which induce specific receptor upregulation on MSCs, and thereby increase the migration toward the cytokine-secreting tumor. In this context, we found that CXCR1 expression on MSCs was upregulated in response to IL-8 secretion from irradiated tumors and that anti-CXCR1 treatment inhibited MSC migration markedly. Taken together, our results suggest that UCB-MSCs are more suitable than BM-MSCs when used in combination with irradiation as an antitumor gene-delivery vehicle for targeting gliomas.

TRAIL-based cancer therapies against glioma involving treatment with rhTRAIL and delivery of TRAIL using a herpes simplex virus [36] or an adenovirus [37] have been reported. However, clinical application is limited because of systemic toxicity, the short biological half-life, insufficient delivery into outgrowing glioma cells infiltrating the brain parenchyma, and a virus-mediated immune reaction [38, 39]. In a previous report, we engineered human UCB-MSCs to produce the secretable form of trimeric TRAIL [29] and showed that engineered MSC (MSC-TRAIL) can migrate toward gliomas and deliver TRAIL to infiltrating tumor cells [15]. We found that treatment with these cells has a significant antitumor effect and increases the survival of mice bearing gliomas compared with adenovirus-mediated gene therapy. Our findings suggest that MSC-based therapy has the advantages of offering continuous and concentrated local delivery of secretable therapeutic molecules such as TRAIL and a greater efficiency for a longer time period compared with the systemic delivery of therapeutic agents. However, we did not achieve complete tumor regression using MSC-TRAIL alone.

Radiotherapy has been found recently to restore or enhance TRAIL sensitivity by inducing DR4 and/or DR5 expression in a range of tumors including prostate, leukemia, breast, lung, colon, and head and neck cancers [40–42] and that this can provide an additive or synergistic tumoricidal effect by activating two independent stress pathways or death programs [25]. Thus, we sought to examine whether MSC-TRAIL treatment combined with irradiation enhances the therapeutic potential in glioma cells and in an orthotopic human glioblastoma xenograft mice model. Here, we showed for the first time that the combined therapy of irradiation followed by MSC-TRAIL significantly increased cytotoxicity in human glioma cells through TRAIL-induced apoptosis by upregulating DR5. Our in vivo therapeutic efficacy results also showed significant inhibition of tumor growth for the combined treatment compared with the treatment with a single agent alone. We achieved complete tumor regression and tumor-free animals after combined therapy at a dose of 7.5 Gy (one survivor of five animals) and 10 Gy (four survivors of five animals) at the end of this study. In addition, the viability of UCB-MSCs did not decrease significantly after exposure to rhTRAIL protein or secreted TRAIL from engineered MSCs, even after the combined treatment with irradiation. These findings confirmed that MSC-TRAIL secreted substantial quantities of TRAIL protein, and that the therapeutic gene was active continuously, and that the effect was enhanced by irradiation and selective only for transformed cells without damaging normal cells such as MSCs or normal astrocytes.

Many therapeutic approaches have been developed based on the use of MSCs as delivery vehicles of antitumor agents in the treatment of gliomas [43]. Targeted migration of MSCs to tumors makes them a very promising strategy for antitumor therapy; however, achieving a sufficient number of tumor-targeted MSCs may be a limitation [21]. Our findings indicate that radiation therapy might provide a way to overcome this issue. Because radiotherapy is a standard modality in cancer therapy, enhanced targeting of radiation-damaged tumors by MSCs and the specific antitumor effect of radiotherapy should allow maximal efficacy of MSC-based gene therapy within the tumor. This is also a particularly attractive strategy that might be relevant to MSCs transduced with other therapeutic genes besides TRAIL, as described here. In addition, because MSCs have been reported to be radioresistant [44], as are UCB-MSCs as shown in this study, continued radiotherapy after the administration of MSCs to tumors might provide prolonged benefit during the treatment course. An alternative approach might be to use local irradiation followed by the therapeutic gene delivery of MSCs overexpressing a cytokine receptor such as CXCR1. The increased expression of receptors critical to the migratory capacity of MSCs may increase the engraftment of MSCs in the target tumors and thus increasing the migratory efficiency should improve the therapeutic potential of MSC-based cancer gene therapy.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In summary, our results suggest that intracranial MSC-TRAIL delivery during surgical removal of the tumors followed by chemotherapy and radiotherapy are a potential strategy in the therapeutic treatment of intractable, and even recurrent, malignant gliomas.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We thank Prof. Young-Chul Sung (Pohang University of Science and Technology, Pohang, Korea) for experimental contributions and Dr. Yoon-Sun Yang (MEDIPOST Co., Ltd., Seoul, Korea) for providing the human UCB-MSCs. We also thank Dong-A Pharmaceutical Co., Ltd. (Yongin, Korea) for providing us with Ad-stTRAIL used in the study. This study was supported by a grant from the National R&D Program for Cancer Control (0820040), by a grant of the Korea Healthcare Technology R&D Project (A091309), Ministry for Health, Welfare and Family Affairs, Republic of Korea, and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0021527).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information
FilenameFormatSizeDescription
STEM_543_sm_suppinfofigure1.tif8121KFigure S1. Cytotoxicity of treatment with irradiation or rhTRAIL in human glioma cells. (A): Glioma cell lines (left panel) and primary glioma cells (right panel) were seeded in 96−well plates. Increasing doses of rhTRAIL (0–300 ng/ml) were treated to the wells. Cells were assayed for viability after 48 h by MTT assay. (B): Glioma cells were grown in 35 mm dishes overnight and then treated with increasing doses of irradiation (0–10 Gy) and analyzed after 48 h. Abbreviation: GBM, glioblastoma multiforme; rhTRAIL, recombinant human tumor necrosis factor−related apoptosis−inducing ligand.
STEM_543_sm_suppinfofigure2.tif2724KFigure S2. Synergistic effects of combined treatment with irradiation and rhTRAIL in glioma cells. Glioma cells were grown overnight and then treated with increasing doses of irradiation (0–10 Gy). For the effect of combined treatment, tumor cells or irradiated tumor cells were seeded in 24−well plates. Cells were cultured for 24 h after exposure to irradiation, followed by treatment with or without rhTRAIL of 10 ng/ml for U−87MG and GBM2 cells (A and B) or 100 ng/ml for U−373MG, U−251MG, GBM4, and GBM5 cells (C–F) for another 24 h. Cells were assayed for the viability using MTT assay. Abbreviation: GBM, glioblastoma multiforme; rhTRAIL, recombinant human tumor necrosis factor−related apoptosis−inducing ligand.
STEM_543_sm_suppinfofigure3.tif8121KFigure S3. Effect of fractionated radiation of glioma cells on the migration of MSC−TRAIL. (A): The migratory ability of MSC−TRAIL in response to CM from untreated and irradiated U−87MG glioma cells with single−fraction (2.5 and 10 Gy) or multiple fractions (2.5 Gy × 4) was determined using a Transwell plate (8 μm pores). Representative photomicrographs of stained filters show migrated cells. Magnification, ×200. (B): Cell migration was compared and evaluated after staining by taking photographs and counting cells that had migrated under a light microscope. *, P < .001 compared with untreated control.
STEM_543_sm_suppinfofigure4.tif2722KFigure S4. Expression levels of cytokines and upregulation of IL−8 mRNA in glioma cells after irradiation. (A): Glioma cells were irradiated with various doses and the expression levels of IL−8, SDF−1α, and MCP−1 in the culture supernatants were quantified by ELISA after 48 h. (B): IL−8 mRNA levels were determined in the cells from untreated or treated with irradiation. Abbreviation: IL−8, interleukin−8; SDF−1α, stromal cell−derived factor−1α; MCP−1, monocyte chemoattractant protein−1; GBM, glioblastoma multiforme; NHA, normal human astrocytes; GAPDH, glyceraldehyde−3−phosphate dehydrogenase.
STEM_543_sm_suppinfo.doc26KSupporting Information

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