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Bystander effect in glioma suicide gene therapy using bone marrow stromal cells
Stem Cell Research (2012) 9, 270–276
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www.elsevier.com/locate/scr
SHORT REPORT
Bystander effect in glioma suicide gene therapy using bone marrow stromal cells Shaoyi Li a, b , Chunyu Gu a, b , Yun Gao a , Shinji Amano a , Shinichiro Koizumi a , Tsutomu Tokuyama a , Hiroki Namba a,⁎ a
Department of Neurosurgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan b Department of Neurosurgery, Second Affiliated Hospital of China Medical University, Shenyang 110004, China
Received 29 February 2012; received in revised form 27 July 2012; accepted 8 August 2012 Available online 22 August 2012
Abstract An established rat intracranial glioma was successfully treated through the tumoricidal bystander effect generated by intratumoral injection of rat bone marrow stromal cells (BMSCs) transduced with the herpes simplex virus-thymidine kinase gene (BMSCtk cells) followed by systemic ganciclovir administration. In the present study, we tested the bystander effect of this treatment strategy when using human BMSCs as the vector cells. Human BMSCtk cells were mixed with various kinds of brain tumor cell lines (human and rat glioma cells) and examined in vitro and in vivo tumoricidal bystander effects, by coculture study and co-implantation study in the nude mouse, respectively. A significant in vitro bystander effect was observed between human BMSCtk cells and any of the tumor cells examined in the ganciclovir-containing medium. A potent in vivo bystander effect against human and rat glioma cells was also demonstrated when ganciclovir was administered. Migratory activity of the human BMSCs toward the tumor cells was enhanced by the conditioned media obtained from both human and rat glioma cells compared to the fresh media. The results of this study have demonstrated that the bystander effect generated by BMSCtk cells and ganciclovir is not cell type-specific, suggesting that the strategy would be quite feasible for clinical use. © 2012 Elsevier B.V. All rights reserved.
Introduction Glioblastomas, the most common and malignant primary brain tumor, have a dismal prognosis, despite modern refinement of diagnostic techniques and treatment strategy including surgery and radio/chemotherapy. Median survival of the patients with glioblastoma is generally about a year from the time of
⁎ Corresponding author. Fax: + 81 53 435 2282. E-mail address: [email protected] (H. Namba). 1873-5061/$ - see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scr.2012.08.002
diagnosis and this has not significantly improved for more than three decades (DeAngelis, 2001; Stewart, 2002; Stupp et al., 2005). As a novel treatment strategy, gene therapies using the herpes simplex virus-thymidine kinase (HSVtk) gene and ganciclovir (GCV) were clinically tested (Rainov, 2000; Ram et al., 1997). However, the results were unsatisfactory partly due to limited migratory activity of the vector-producing cells derived from fibroblasts, which could not cover all of the glioma cells that widely infiltrated into the surrounding brain tissues. To improve the migratory activity of the effector cells, use of the neural stem cells and bone marrow stromal cells (BMSCs), that display extensive tropism for brain lesions including gliomas, has been introduced (Aboody et al., 2000;
Bystander effect using bone marrow stromal cells Benedetti et al., 2000; Li et al., 2007). In the previous study, we demonstrated that established intracranial glioma in the rat brain was successfully treated by intratumoral injection of BMSCs transduced with HSVtk gene (BMSCtk cells) followed by intraperitoneal GCV administration (Amano et al., 2009). This is mainly due to a very potent “bystander effect”, where tumor cells that are not transduced with HSVtk gene are eliminated when mixed with HSVtk-expressing cells by GCV administration (Culver et al., 1992), as well as an active tumor tracking ability of BMSCs (Lee et al., 2003; Nakamizo et al., 2005). In fact, we have demonstrated a surprisingly potent bystander effect between BMSCtk and C6 rat glioma cells (Amano et al., 2009). These observations suggest that “BMSCtk therapy” is promising as a novel clinical treatment strategy for glioblastomas. Bystander effect between HSVtk-positive cells and HSVtknegative cells has been extensively studied especially from the aspect of cell-to-cell communication. Connexin43, a major molecule in the connexin family gene products, has been reported to be related to the bystander effect in suicide gene therapies (Dilber et al., 1997; Elshami et al., 1886; Mesnil et al., 1996; Vrionis et al., 1997; Ziu et al., 2006). We previously tested bystander effect between HSVtk-positive and HSVtknegative tumor cells and found that the bystander effect was very potent in 9L cells with high connexin43 expression compared with in C6 cells with low connexin43 expression (Namba et al., 2001). However, we consequently found that the bystander effects between BMSCtk and C6 cells were very potent. In the present study, we examined bystander effect generated by human and rat BMSCtk cells on various kinds of human and rat brain tumor cells in both in vitro and in vivo conditions. The results have demonstrated that tumoricidal bystander effect in the suicide gene therapy using BMSCtk cells and GCV is not cell type- and species-specific, suggesting a possibility of “BMSCtk therapy” using pre-established BMSCtk cell.
Materials and methods Establishment of human BMSCtk cells The human BMSCs (from female of 19 years of age) and human BMSC growth medium were purchased from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). Thawing of cells and initiation of culture process were performed based on the manufacturer's instruction. Human BMSCs were plated in 10 cm tissue culture dishes in the concentration of 5 × 10 3 cells/cm 2 and cultured with human BMSC growth medium. Medium was changed every 2–3 days. The HSVtk retrovirus-producing cells (PA317; mouse fibroblast cell line with HSVtk gene) were kindly provided by Genetic Therapy Inc. (Gaithersburg, MD). The PA317 cells were cultured in the medium for 48 h and the supernatant was collected, filtered through 0.22 μm filter (Toyo Roshi Kaisha, Ltd) and the retrovirus containing supernatant was stored in −80 °C until following use. The method of infecting the BMSCs was described previously (Namba et al., 2000). Briefly, the BMSCs were plated at 5×10 3 cells/cm 2 in the human BMSC growth medium in a 25-cm 2 tissue culture flask and cultured for 24 h. Thereafter, the medium was replaced with 3 ml fresh medium and 1 ml of the supernatant of HSVtk retrovirus in the presence of 8 μg/ml Polybrene (Aldrich Chemical Company Inc., Milwaukee, WI) for 5 h. After washing,
271 the cells were maintained with fresh medium. The drug selection with 150 μg/ml G418 (Sigma-Aldrich Japan K.K., Tokyo, Japan) was performed for 1 week. The drug-resistant cells were collected and used for further experiments (BMSCtk cells).
Establishment of rat BMSCtk cells All the following experiments were performed according to the Rules of Animal Experimentation and the Guide for the Care and Use of Laboratory animals of the Hamamatsu University School of Medicine. BMSCs were obtained from 6-week Sprague–Dawley rats as previously described (Gu et al., 2010). Briefly, bone marrow from the femora and tibia bone was suspended in murine BMSC culture medium (Stem Cell Technologies Inc. Mesencult®) after aspiration by 5 ml syringe connected with 23 G needle. Depletion of macrophages was not performed. The total bone marrow cells were filtered through a 70 μm nylon cell strainer and plated in 10 cm tissue culture dished in the concentration of 5× 10 3 cells/cm 2. New medium was changed 24 h later and then every 2–3 days. BMSCs attach on the bottom of the dishes and form colonies. The HSVtk gene transfer methods and drug selection were the same as described above except using murine BMSC medium instead of human BMSC growth medium.
In vitro sensitivity of human BMSCtk cells to GCV To test the sensitivity to GCV, the obtained human BMSCtk cells and wild-type BMSCs were cultured in the medium containing various concentrations (0.01 to 1000 μg/ml) of GCV (Syntex Chemical Inc., Palo Alto, CA) in a 96-well plate (1 × 10 4 cells/well) and were incubated for 7 days. Viability of the cells was estimated using the tetrozolium-based colorimetric assay (MTT assay) (Mosmann, 1983).
In vitro bystander effect between BMSCtk and various glioma cell lines Human glioblastoma (A-172, YKG-1, T98G), medulloblastoma (Daoy, TE671) cell lines and rat glioma cell lines (C6, 9L) were obtained from the American Type Culture Collection (Manassas, VA) and the Human Science Research Bank (Osaka, Japan). Tumor cells (5 ×10 3) were co-cultured with the same number of human or rat BMSCtk in complete DMEM medium with/without containing 1 μg/ml GCV in a 96-well tissue culture plate. As another controls, 5×10 3 tumor cells were cultured alone in the medium with/without GCV. The conditional medium was changed every 2 days and the number of living cells was determined by MTT assay on day 7. Viability of the cells was expressed as the percent absorbance of the respective tumor cells alone cultured without GCV.
In vivo bystander effect between human BMSCtk and glioma cells in nude mice We anesthetized 60 female BALB/c nude mice (19–21 g, 9 weeks old, Nippon SLC, Hamamatsu, Japan) with 0.4 ml/100 g Equithesin and placed in a stereotaxic apparatus
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Figure 1 In vitro bystander effect between human BMSCtk and various tumor cells (human and rat glioma, human medulloblastoma cell lines) tested with MTT assay (A). The proliferation of glioma cells co-cultured with BMSCtk cells was significantly inhibited when they were cultured in the presence of GCV (1 μg/ml, gray column, triplicate, mean ± standard error) compared to when cultured without GCV (open column, p b 0.01). In vitro bystander effect with rat BMSCtk cells showed similar results as human BMSCtk cells (B).
Figure 2 The tumor volume for each group (n = 5, mean± standard error) on day 21 (A). When treated with GCV for 10 days, no tumor was observed in the mice co-implanted with human BMSCtk and human glioma cells (A-172 and T98G) and only small tumors were observed in the mice co-implanted with human BMSCtk and rat C6 glioma cells (arrow). When treated with PBS, a large tumor (5–6 mm3) developed in the mice co-implanted with BMSCtk and glioma cells, which was not significantly different from the sizes of tumors in the mice implanted with tumor cells only regardless of GCV administration. Representative photomicrographs of coronal brain sections are shown (B, bar: 1 mm).
Bystander effect using bone marrow stromal cells
273 was analyzed by a log-rank test based on the Kaplan–Meier test using Statview 5.0 software. Another 10 mice were implanted with 2× 10 4 human BMSCtk alone to test tumorigenicity of BMSCtk cells. When the animals survived more than 100 days, they were euthanized and the brains were taken for histological analyses.
In vitro migration assay
Figure 3 Kaplan–Meier survival curves of the mice for each tumor cell line. The mice co-implanted with a mixture of human BMSCtk and human glioma cells (A-172 and T98G) and treated with GCV for 10 days survived more than 100 days (red lines), while those co-implanted with a mixture but treated with PBS (black lines) and those implanted with tumor cells only and treated with GCV (pink lines) or PBS (green lines) died in 3 weeks (there were no difference among those three groups). The mice co-implanted with a mixture of human BMSCtk and rat C6 glioma cells and treated with GCV for 10 days survived significantly longer than the other three groups and 2 out of 5 mice survived more than 100 days (red line).
(Narishige Scientific Instrument Lab., Tokyo, Japan). The method of cell implantation was the same as previously described (Li et al., 2007). Briefly, 2 ×10 4 tumor cells (A-172, T98G, or C6 cells) with/without 2 ×10 4 human BMSCtk were infused with a 10 μl microsyringe (Hamilton Company, Reno, NV) to the point of 3 mm ventral from the dura through the burr hole (co-ordinates with respect to bregma: 0.2 mm posterior, 2 mm right) for 5 min. Half of the animals (n = 5) for each group underwent intraperitoneal administration of 50 mg/kg of GCV twice daily (100 mg/kg/day) from day 0 for 10 days, and the other half (n = 5), phosphate-buffered saline (PBS). All the animals were killed on day 21 for histological analyses. Serial coronal sections (5 μm) were obtained and stained with hematoxylin and eosin. The tumor area of each section was measured using NIH Image software (rsbweb.nih.gov). The total volume of the tumor (mm 3) was calculated by summing up the cross-sectional areas. Another 60 mice were treated in the same way as above for the survival study. When the mice developed symptoms such as severe paresis and/or ataxia, or when their body weight decreased to less than 80%, they were euthanized. Survival
The in vitro migratory capacity of human BMSCtk cells, another important factor for the success of “BMSCtk therapy”, was then assessed using the 24-well Matrigel Invasion Chamber (BD Biosciences Discovery Labware, Bedford, MA), which contains an 8-μm pore size PET membrane that has been treated with Matrigel Basement Membrane Matrix in the insert (Li et al., 2007; Mohanam et al., 1993). First, 0.5 ml Dulbecco's Modified Eagle's Medium (DMEM) (without serum) was added to the interior of the inserts and bottom of wells and allowed to rehydrate for 2 h at 37 °C under 5% CO2. Then, the medium was carefully removed without disturbing the layer of Matrigel Matrix on the membrane. The BMSCtk cells were washed twice in BMSC growth medium and resuspended to 1× 10 5 cells/ml. 0.5 ml of the cell suspension (5 × 10 4 cells) was added to the upper insert. The lower chamber was filled with 0.75 ml conditioned medium obtained by incubating C6 cells for 24 h in DMEM (with or without 10% fetal bovine serum (FBS)) or unconditioned fresh DMEM (with or without 10% FBS). After incubating the Matrigel Invasion Chambers for 24 h at 37 °C under 5% CO2, the non-invading cells and/or Matrigel Matrix were removed from the upper surface of the membrane in the inserts with a cotton swab. The cells migrating to the lower surface of the membrane were stained with the DiffQuik kit (International Reagents), which was accomplished by sequentially transferring the inserts through three solutions and allowing the inserts to air dry. Ten random fields per membrane were counted using × 200 magnification. Each assay was performed in triplicate, and results were expressed as the mean number of cells migrating per field ± standard error. At the same time, 0.5 ml of the cell suspension (5 × 10 4 cells) of PA317 or C6 cells in DMEM (containing 0.1% BSA) was also tested for their migratory capacity using the same conditioned medium as described above.
Results In vitro sensitivity of human BMSCtk cells to GCV
Human BMSCtk cells were cultured in the medium containing various concentrations (0.01 to 1000 μg/ml) of GCV and the viability of the cells was tested with MTT assay (Mosmann, 1983). At a GCV concentration of 1 μg/ml, almost no BMSCtk cells survived, while no significant toxicity was observed on wild-type BMSCs. The 100% lethal dose of GCV for wild-type BMSCs was 300 μg/ml, 100 times higher than that of BMSCtk cells (Supplementary Fig. 1). We used a 1 μg/ml GCV in the following in vitro studies, because this concentration of GCV killed all BMSCtk cells but not wild-type BMSCs and tumor cells.
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Figure 4 In vitro migratory capacity of human BMSCtk cells when they were exposed to different media. 0.5 ml of the BMSCtk cell suspension was added to the upper insert of Matrigel Invasion Chamber and the lower chamber was filled with 0.75 ml of the conditioned DMEM obtained by incubating human glioma cells (A-172, T98G) or rat C6 cells for 24 h or fresh DMEM (with or without 10% FBS). BMSCtk cells showed a potent migratory capacity when the conditioned medium was used as attractant in the lower chamber, while only a few migrating BMSCtk cells could be observed when they were exposed to the fresh DMEM. The migration was further enhanced with 10% FBS. A: Mean number of migrating cells ± standard error (triplicate). B: Representative photomicrographs of the micropore membrane after cell migration.
In vitro bystander effect between BMSCtk and various tumor cell lines The proliferation of tumor cells, irrespective of human or rat cells, co-cultured with human BMSCtk cells in the presence of GCV (1 μg/ml) (Fig. 1A, closed column) was significantly inhibited as compared to that without GCV (Fig. 1A, open column), which was not different from the proliferation of tumor cells cultured alone with or without GCV (data not shown). This is due to a significant bystander effect between human BMSCtk and tumor cells. Relative viability of “tumor + BMSCtk” groups was around 100% (Fig. 1A, open column), suggesting that BMSCtk did not have significant stimulatory or inhibitory effects on tumor cell proliferation. When BMSCs (and not BMSCtk cells) were co-cultured with tumor cells in the GCV containing medium, no bystander effect was exerted (data not shown). The similar bystander effect was also observed between rat BMSCtk and various tumor cells (Fig. 1B). These observations suggested that the in vitro bystander effect generated by BMSCtk cells and GCV was not specific to tumor cell types and species.
In vivo bystander effect between human BMSCtk and glioma cell lines Since a potent in vitro bystander effect was observed between human BMSCtk and various tumor cells, we then tested in vivo bystander effect by an intracranial co-implantation study of human BMSCtk and glioma cells in the nude mouse. When
tumor cells alone were implanted, a large tumor (5-6 mm 3) developed regardless of GCV administration (Fig. 2A). When a mixture of BMSCtk and human glioma cells (A-172 and T98G) was co-implanted, no tumor was observed in the GCV group, while tumors of similar sizes as those of the tumoralone groups developed in the PBS group (Fig. 2A). Representative mouse brain sections of each group were shown in Fig. 2B (A-172, T98G). The survival times of the mice implanted with a mixtures of BMSCtk and human glioma cells and treated with GCV (Fig. 3, A-172 and T98G, red lines) were more than 100 days, while those treated with PBS were about 3 weeks (black lines), which were similar to those of the tumor-alone groups treated with GCV (pink lines) or PBS (green lines). All the mice implanted with BMSCtk alone survived more than 100 days and no tumor formation was observed (Fig. 2A). Surprisingly, a similar in vivo bystander effect was observed between BMSCtk and rat C6 glioma cells (Fig. 2, C6). When human BMSCtk cells were mixed with rat C6 cells and coimplanted into the mice brain, small tumors developed in some mice of the GCV group (Fig. 2B, arrow). The survival was also significantly prolonged in the GCV group and 2 out of 5 mice survived more than 100 days (Fig. 3, C6).
In vitro migration of human BMSCtk cells to the conditioned medium Migratory activity of human BMSCtk cells was evaluated in Matrigel invasion assay (Li et al., 2007; Mohanam et al.,
Bystander effect using bone marrow stromal cells 1993). BMSCtk cells actively migrated through the Matrigel Matrix layer and micropore when the lower chamber was filled with the conditioned medium from the tumor cells, while they hardly migrated when the lower chamber was filled with the fresh DMEM (p b 0.01, Fig. 4). The migratory capacity of BMSCtk cells to the tumor conditioned medium was further enhanced when 10% FBS was added in the lower chamber compared with respective controls (p b 0.01, Fig. 4).
Discussion We used commercially-available human BMSCs as the vector cells in the present study. Thus, a limitation of the present study is that the commercially-available BMSCs do not represent general BMSCs that are obtained from the patients. They may not behave as “stem cells” that have multipotent differentiation capacity, and therefore, more studies are obviously needed to establish ideal vector cells for clinical use. Induced pluripotent stem cells, recently established and vigorously examined in the field of reconstructive neurosciences, could be also suitable vehicles for gene therapy (Takahashi et al., 2007; Yu et al., 2007). We have recently confirmed that induced pluripotent stem cells have a potent migratory activity toward the conditioned medium of brain tumors and this activity is also not tumor cell type-specific, or dependent on the species as shown in the present migration assay (Koizumi et al., 2011). In the present study, we used the same number of BMSCtk cells as that of tumor cells (BMSCtk/tumor cell ratio of 1:1). The previous in vitro study testing the potency of the bystander effect, using rat BMSCtk or HSVtk-transduced neural stem cells and C6 cells at various ratios (BMSCtk/tumor cell ratio of 1:1 to 1:128) showed a very potent bystander effect up to BMSCtk/ tumor cell ratio of 1:16 (Amano et al., 2009; Li et al., 2005b). Therefore, a dose response study at different BMSCtk/tumor cell ratios should be also performed between different BMSCtk/tumor cell combinations. In the present study, we only performed a co-implantation study, because the aim of the study was to test cell type- and species-specificity. We obviously have to test the effect on an established intracranial tumor as in our previous treatment studies (Amano et al., 2009; Li et al., 2005a). In vivo bystander effect between human BMSCtk and rat glioma cells was a little weaker than that between human BMSCtk and human glioma cells (Figs. 2 and 3), though the difference was not very obvious in in vitro conditions (Fig. 1). Precise mechanism is unknown but in vivo cell-to-cell communication, which is important for generation of bystander effect, may be different between human and rat cells. Bystander effect is still potent between cells of different species, but use of human BMSCs is recommended for treatment of human gliomas. In conclusion, we have demonstrated that the bystander effect by the human BMSCtk cells and GCV exerts against various kinds of human brain tumor cells, and also against rat glioma cells. Even after we changed the vector cells from neural stem cells to BMSCs for convenience for clinical use, it is still time-consuming to obtain sufficient number of BMSCtk cells from the patients when considering the duration for life-expectancy of glioblastoma patients. If pre-established
275 allogeneic or heterogeneic vector cells can be used, the strategy becomes much more feasible because those cells can be used at the time of the initial operation of glioblastoma. The results suggest that several established BMSCtk cell lines prepared in advance can be used for the BMSC-based HSVtk/ GCV gene therapy of glioma patients provided that safety issues due to immunological reactions are properly solved. Then “BMSCtk therapy” would become easier and more feasible as a treatment strategy for malignant gliomas that is inevitably fatal at the present time. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scr.2012.08.002.
Conflict of interest The authors have no conflict of interest.
References Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., Snyder, E.Y., 2000. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U. S. A. 97, 12846–12851. Amano, S., Li, S., Gu, C., Gao, Y., Koizumi, S., Yamamoto, S., Terakawa, S., Namba, H., 2009. Use of genetically engineered bone marrow-derived mesenchymal stem cells for glioma gene therapy. Int. J. Oncol. 35, 1265–1270. Benedetti, S., Pirola, B., Pollo, B., Magrassi, L., Bruzzone, M.G., Rigamonti, D., Galli, R., Selleri, S., DiMeco, F., DeFraja, C., Vescovi, A., Cattaneo, E., Finocchiaro, G., 2000. Gene therapy of experimental brain tumors using neural progenitor cells. Nat. Med. 6, 447–450. Culver, K.W., Ram, Z., Walbridge, S., Ishii, H., Oldfield, E.H., Blaese, R.M., 1992. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256, 1550–1552. DeAngelis, L.M., 2001. Brain tumors. N. Engl. J. Med. 344, 114–123. Dilber, M.S., Abedi, M.R., Christensson, B., Björkstrand, B., Kidder, G.M., Naus, C.C., Gahrton, G., Smith, C.I., 1997. Gap junctions promote the bystander effect of herpes simplex virus thymidine kinase in vivo. Cancer Res. 57, 1523–1528. Elshami, A.A., Saavedra, A., Zhang, H., Kucharczuk, J.C., Spray, D.C., Fishman, G.I., Amin, K.M., Kaiser, L.R., Albelda, S.M., 1886. Gap junctions play a role in the ‘bystander effect’ of the herpes simplex virus thymidine kinase/ganciclovir system in vitro. Gene Ther. 3, 85–92. Gu, C., Li, S., Tokuyama, T., Yokota, N., Namba, H., 2010. Therapeutic effect of genetically engineered mesenchymal stem cells in rat experimental leptomeningeal glioma model. Cancer Lett. 291, 256–262. Koizumi, S., Gu, C., Amano, S., Yamamoto, S., Ihara, H., Tokuyama, T., Namba, H., 2011. Migration of mouse-induced pluripotent stem cells to glioma-conditioned medium is mediated by tumorassociated specific growth factors. Oncol. Lett. 2, 283–288. Lee, J., Elkahloun, A.G., Messina, S.A., Ferrari, N., Xi, D., Smith, C.L., Cooper, R., Albert, P.S., Fine, H.A., 2003. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: a potential antiglioma cellular vector. Cancer Res. 63, 8877–8889. Li, S., Tokuyama, T., Yamamoto, J., Koide, M., Yokota, N., Namba, H., 2005a. Bystander effect-mediated gene therapy of gliomas
276 using genetically engineered neural stem cells. Cancer Gene Ther. 12, 600–607. Li, S., Tokuyama, T., Yamamoto, J., Koide, M., Yokota, N., Namba, H., 2005b. Potent bystander effect in suicide gene therapy using neural stem cells transduced with herpes simplex virus thymidine kinase gene. Oncology 69, 503–508. Li, S., Gao, Y., Tokuyama, T., Yamamoto, J., Yokota, N., Yamamoto, S., Terakawa, S., Kitagawa, M., Namba, H., 2007. Genetically engineered neural stem cells migrate and suppress glioma cell growth at distant intracranial sites. Cancer Lett. 251, 220–227. Mesnil, M., Piccoli, C., Tiraby, G., Willecke, K., Yamasaki, H., 1996. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc. Natl. Acad. Sci. U. S. A. 93, 1831–1835. Mohanam, S., Sawaya, R., McCutcheon, I., Ali-Osman, F., Boyd, D., Rao, J.S., 1993. Modulation of in vitro invasion of human glioblastoma cells by urokinase-type plasminogen activator receptor antibody. Cancer Res. 53, 4143–4147. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Nakamizo, A., Marini, F., Amano, T., Khan, A., Studeny, M., Gumin, J., Chen, J., Hentschel, S., Vecil, G., Dembinski, J., Andreeff, M., Lang, F.F., 2005. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res. 65, 3307–3318. Namba, H., Tagawa, M., Miyagawa, T., Iwadate, Y., Sakiyama, S., 2000. Treatment of rat experimental brain tumors by herpes simplex virus thymidine kinase gene-transduced allogeneic tumor cells and ganciclovir. Cancer Gene Ther. 7, 947–953. Namba, H., Iwadate, Y., Kawamura, K., Sakiyama, S., Tagawa, M., 2001. Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells. Cancer Gene Ther. 8, 414–420. Rainov, N.G., 2000. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an
S. Li et al. adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum. Gene Ther. 11, 2389–2401. Ram, Z., Culver, K.W., Oshiro, E.M., Viola, J.J., DeVroom, H.L., Otto, E., Long, Z., Chiang, Y., McGarrity, G.J., Muul, L.M., Katz, D., Blaese, R.M., Oldfield, E.H., 1997. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat. Med. 3, 1354–1361. Stewart, L.A., 2002. Chemotherapy in adult high-grade glioma: a systematic review and meta-analysis of individual patient data from 12 randomised trials. Lancet 359, 1011–1018. Stupp, R., Mason, W.P., Bent, M.J.v.d, Weller, M., Fisher, B., Taphoorn, M.J., Belanger, K., Brandes, A.A., Marosi, C., Bogdahn, U., Curschmann, J., Janzer, R.C., Ludwin, S.K., Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J.G., Eisenhauer, E., Mirimanoff, R.O., European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group, 2005. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987–996. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872. Vrionis, F.D., Wu, J.K., Qi, P., Waltzman, M., Cherington, V., Spray, D.C., 1997. The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions. Gene Ther. 4, 577–585. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., Thomson, J.A., 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920. Ziu, M., Schmidt, N.O., Cargioli, T.G., Aboody, K.S., Black, P.M., Carroll, R.S., 2006. Glioma-produced extracellular matrix influences brain tumor tropism of human neural stem cells. J. Neurooncol. 79, 125–133.
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SHORT REPORT
Bystander effect in glioma suicide gene therapy using bone marrow stromal cells Shaoyi Li a, b , Chunyu Gu a, b , Yun Gao a , Shinji Amano a , Shinichiro Koizumi a , Tsutomu Tokuyama a , Hiroki Namba a,⁎ a
Department of Neurosurgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan b Department of Neurosurgery, Second Affiliated Hospital of China Medical University, Shenyang 110004, China
Received 29 February 2012; received in revised form 27 July 2012; accepted 8 August 2012 Available online 22 August 2012
Abstract An established rat intracranial glioma was successfully treated through the tumoricidal bystander effect generated by intratumoral injection of rat bone marrow stromal cells (BMSCs) transduced with the herpes simplex virus-thymidine kinase gene (BMSCtk cells) followed by systemic ganciclovir administration. In the present study, we tested the bystander effect of this treatment strategy when using human BMSCs as the vector cells. Human BMSCtk cells were mixed with various kinds of brain tumor cell lines (human and rat glioma cells) and examined in vitro and in vivo tumoricidal bystander effects, by coculture study and co-implantation study in the nude mouse, respectively. A significant in vitro bystander effect was observed between human BMSCtk cells and any of the tumor cells examined in the ganciclovir-containing medium. A potent in vivo bystander effect against human and rat glioma cells was also demonstrated when ganciclovir was administered. Migratory activity of the human BMSCs toward the tumor cells was enhanced by the conditioned media obtained from both human and rat glioma cells compared to the fresh media. The results of this study have demonstrated that the bystander effect generated by BMSCtk cells and ganciclovir is not cell type-specific, suggesting that the strategy would be quite feasible for clinical use. © 2012 Elsevier B.V. All rights reserved.
Introduction Glioblastomas, the most common and malignant primary brain tumor, have a dismal prognosis, despite modern refinement of diagnostic techniques and treatment strategy including surgery and radio/chemotherapy. Median survival of the patients with glioblastoma is generally about a year from the time of
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diagnosis and this has not significantly improved for more than three decades (DeAngelis, 2001; Stewart, 2002; Stupp et al., 2005). As a novel treatment strategy, gene therapies using the herpes simplex virus-thymidine kinase (HSVtk) gene and ganciclovir (GCV) were clinically tested (Rainov, 2000; Ram et al., 1997). However, the results were unsatisfactory partly due to limited migratory activity of the vector-producing cells derived from fibroblasts, which could not cover all of the glioma cells that widely infiltrated into the surrounding brain tissues. To improve the migratory activity of the effector cells, use of the neural stem cells and bone marrow stromal cells (BMSCs), that display extensive tropism for brain lesions including gliomas, has been introduced (Aboody et al., 2000;
Bystander effect using bone marrow stromal cells Benedetti et al., 2000; Li et al., 2007). In the previous study, we demonstrated that established intracranial glioma in the rat brain was successfully treated by intratumoral injection of BMSCs transduced with HSVtk gene (BMSCtk cells) followed by intraperitoneal GCV administration (Amano et al., 2009). This is mainly due to a very potent “bystander effect”, where tumor cells that are not transduced with HSVtk gene are eliminated when mixed with HSVtk-expressing cells by GCV administration (Culver et al., 1992), as well as an active tumor tracking ability of BMSCs (Lee et al., 2003; Nakamizo et al., 2005). In fact, we have demonstrated a surprisingly potent bystander effect between BMSCtk and C6 rat glioma cells (Amano et al., 2009). These observations suggest that “BMSCtk therapy” is promising as a novel clinical treatment strategy for glioblastomas. Bystander effect between HSVtk-positive cells and HSVtknegative cells has been extensively studied especially from the aspect of cell-to-cell communication. Connexin43, a major molecule in the connexin family gene products, has been reported to be related to the bystander effect in suicide gene therapies (Dilber et al., 1997; Elshami et al., 1886; Mesnil et al., 1996; Vrionis et al., 1997; Ziu et al., 2006). We previously tested bystander effect between HSVtk-positive and HSVtknegative tumor cells and found that the bystander effect was very potent in 9L cells with high connexin43 expression compared with in C6 cells with low connexin43 expression (Namba et al., 2001). However, we consequently found that the bystander effects between BMSCtk and C6 cells were very potent. In the present study, we examined bystander effect generated by human and rat BMSCtk cells on various kinds of human and rat brain tumor cells in both in vitro and in vivo conditions. The results have demonstrated that tumoricidal bystander effect in the suicide gene therapy using BMSCtk cells and GCV is not cell type- and species-specific, suggesting a possibility of “BMSCtk therapy” using pre-established BMSCtk cell.
Materials and methods Establishment of human BMSCtk cells The human BMSCs (from female of 19 years of age) and human BMSC growth medium were purchased from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). Thawing of cells and initiation of culture process were performed based on the manufacturer's instruction. Human BMSCs were plated in 10 cm tissue culture dishes in the concentration of 5 × 10 3 cells/cm 2 and cultured with human BMSC growth medium. Medium was changed every 2–3 days. The HSVtk retrovirus-producing cells (PA317; mouse fibroblast cell line with HSVtk gene) were kindly provided by Genetic Therapy Inc. (Gaithersburg, MD). The PA317 cells were cultured in the medium for 48 h and the supernatant was collected, filtered through 0.22 μm filter (Toyo Roshi Kaisha, Ltd) and the retrovirus containing supernatant was stored in −80 °C until following use. The method of infecting the BMSCs was described previously (Namba et al., 2000). Briefly, the BMSCs were plated at 5×10 3 cells/cm 2 in the human BMSC growth medium in a 25-cm 2 tissue culture flask and cultured for 24 h. Thereafter, the medium was replaced with 3 ml fresh medium and 1 ml of the supernatant of HSVtk retrovirus in the presence of 8 μg/ml Polybrene (Aldrich Chemical Company Inc., Milwaukee, WI) for 5 h. After washing,
271 the cells were maintained with fresh medium. The drug selection with 150 μg/ml G418 (Sigma-Aldrich Japan K.K., Tokyo, Japan) was performed for 1 week. The drug-resistant cells were collected and used for further experiments (BMSCtk cells).
Establishment of rat BMSCtk cells All the following experiments were performed according to the Rules of Animal Experimentation and the Guide for the Care and Use of Laboratory animals of the Hamamatsu University School of Medicine. BMSCs were obtained from 6-week Sprague–Dawley rats as previously described (Gu et al., 2010). Briefly, bone marrow from the femora and tibia bone was suspended in murine BMSC culture medium (Stem Cell Technologies Inc. Mesencult®) after aspiration by 5 ml syringe connected with 23 G needle. Depletion of macrophages was not performed. The total bone marrow cells were filtered through a 70 μm nylon cell strainer and plated in 10 cm tissue culture dished in the concentration of 5× 10 3 cells/cm 2. New medium was changed 24 h later and then every 2–3 days. BMSCs attach on the bottom of the dishes and form colonies. The HSVtk gene transfer methods and drug selection were the same as described above except using murine BMSC medium instead of human BMSC growth medium.
In vitro sensitivity of human BMSCtk cells to GCV To test the sensitivity to GCV, the obtained human BMSCtk cells and wild-type BMSCs were cultured in the medium containing various concentrations (0.01 to 1000 μg/ml) of GCV (Syntex Chemical Inc., Palo Alto, CA) in a 96-well plate (1 × 10 4 cells/well) and were incubated for 7 days. Viability of the cells was estimated using the tetrozolium-based colorimetric assay (MTT assay) (Mosmann, 1983).
In vitro bystander effect between BMSCtk and various glioma cell lines Human glioblastoma (A-172, YKG-1, T98G), medulloblastoma (Daoy, TE671) cell lines and rat glioma cell lines (C6, 9L) were obtained from the American Type Culture Collection (Manassas, VA) and the Human Science Research Bank (Osaka, Japan). Tumor cells (5 ×10 3) were co-cultured with the same number of human or rat BMSCtk in complete DMEM medium with/without containing 1 μg/ml GCV in a 96-well tissue culture plate. As another controls, 5×10 3 tumor cells were cultured alone in the medium with/without GCV. The conditional medium was changed every 2 days and the number of living cells was determined by MTT assay on day 7. Viability of the cells was expressed as the percent absorbance of the respective tumor cells alone cultured without GCV.
In vivo bystander effect between human BMSCtk and glioma cells in nude mice We anesthetized 60 female BALB/c nude mice (19–21 g, 9 weeks old, Nippon SLC, Hamamatsu, Japan) with 0.4 ml/100 g Equithesin and placed in a stereotaxic apparatus
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Figure 1 In vitro bystander effect between human BMSCtk and various tumor cells (human and rat glioma, human medulloblastoma cell lines) tested with MTT assay (A). The proliferation of glioma cells co-cultured with BMSCtk cells was significantly inhibited when they were cultured in the presence of GCV (1 μg/ml, gray column, triplicate, mean ± standard error) compared to when cultured without GCV (open column, p b 0.01). In vitro bystander effect with rat BMSCtk cells showed similar results as human BMSCtk cells (B).
Figure 2 The tumor volume for each group (n = 5, mean± standard error) on day 21 (A). When treated with GCV for 10 days, no tumor was observed in the mice co-implanted with human BMSCtk and human glioma cells (A-172 and T98G) and only small tumors were observed in the mice co-implanted with human BMSCtk and rat C6 glioma cells (arrow). When treated with PBS, a large tumor (5–6 mm3) developed in the mice co-implanted with BMSCtk and glioma cells, which was not significantly different from the sizes of tumors in the mice implanted with tumor cells only regardless of GCV administration. Representative photomicrographs of coronal brain sections are shown (B, bar: 1 mm).
Bystander effect using bone marrow stromal cells
273 was analyzed by a log-rank test based on the Kaplan–Meier test using Statview 5.0 software. Another 10 mice were implanted with 2× 10 4 human BMSCtk alone to test tumorigenicity of BMSCtk cells. When the animals survived more than 100 days, they were euthanized and the brains were taken for histological analyses.
In vitro migration assay
Figure 3 Kaplan–Meier survival curves of the mice for each tumor cell line. The mice co-implanted with a mixture of human BMSCtk and human glioma cells (A-172 and T98G) and treated with GCV for 10 days survived more than 100 days (red lines), while those co-implanted with a mixture but treated with PBS (black lines) and those implanted with tumor cells only and treated with GCV (pink lines) or PBS (green lines) died in 3 weeks (there were no difference among those three groups). The mice co-implanted with a mixture of human BMSCtk and rat C6 glioma cells and treated with GCV for 10 days survived significantly longer than the other three groups and 2 out of 5 mice survived more than 100 days (red line).
(Narishige Scientific Instrument Lab., Tokyo, Japan). The method of cell implantation was the same as previously described (Li et al., 2007). Briefly, 2 ×10 4 tumor cells (A-172, T98G, or C6 cells) with/without 2 ×10 4 human BMSCtk were infused with a 10 μl microsyringe (Hamilton Company, Reno, NV) to the point of 3 mm ventral from the dura through the burr hole (co-ordinates with respect to bregma: 0.2 mm posterior, 2 mm right) for 5 min. Half of the animals (n = 5) for each group underwent intraperitoneal administration of 50 mg/kg of GCV twice daily (100 mg/kg/day) from day 0 for 10 days, and the other half (n = 5), phosphate-buffered saline (PBS). All the animals were killed on day 21 for histological analyses. Serial coronal sections (5 μm) were obtained and stained with hematoxylin and eosin. The tumor area of each section was measured using NIH Image software (rsbweb.nih.gov). The total volume of the tumor (mm 3) was calculated by summing up the cross-sectional areas. Another 60 mice were treated in the same way as above for the survival study. When the mice developed symptoms such as severe paresis and/or ataxia, or when their body weight decreased to less than 80%, they were euthanized. Survival
The in vitro migratory capacity of human BMSCtk cells, another important factor for the success of “BMSCtk therapy”, was then assessed using the 24-well Matrigel Invasion Chamber (BD Biosciences Discovery Labware, Bedford, MA), which contains an 8-μm pore size PET membrane that has been treated with Matrigel Basement Membrane Matrix in the insert (Li et al., 2007; Mohanam et al., 1993). First, 0.5 ml Dulbecco's Modified Eagle's Medium (DMEM) (without serum) was added to the interior of the inserts and bottom of wells and allowed to rehydrate for 2 h at 37 °C under 5% CO2. Then, the medium was carefully removed without disturbing the layer of Matrigel Matrix on the membrane. The BMSCtk cells were washed twice in BMSC growth medium and resuspended to 1× 10 5 cells/ml. 0.5 ml of the cell suspension (5 × 10 4 cells) was added to the upper insert. The lower chamber was filled with 0.75 ml conditioned medium obtained by incubating C6 cells for 24 h in DMEM (with or without 10% fetal bovine serum (FBS)) or unconditioned fresh DMEM (with or without 10% FBS). After incubating the Matrigel Invasion Chambers for 24 h at 37 °C under 5% CO2, the non-invading cells and/or Matrigel Matrix were removed from the upper surface of the membrane in the inserts with a cotton swab. The cells migrating to the lower surface of the membrane were stained with the DiffQuik kit (International Reagents), which was accomplished by sequentially transferring the inserts through three solutions and allowing the inserts to air dry. Ten random fields per membrane were counted using × 200 magnification. Each assay was performed in triplicate, and results were expressed as the mean number of cells migrating per field ± standard error. At the same time, 0.5 ml of the cell suspension (5 × 10 4 cells) of PA317 or C6 cells in DMEM (containing 0.1% BSA) was also tested for their migratory capacity using the same conditioned medium as described above.
Results In vitro sensitivity of human BMSCtk cells to GCV
Human BMSCtk cells were cultured in the medium containing various concentrations (0.01 to 1000 μg/ml) of GCV and the viability of the cells was tested with MTT assay (Mosmann, 1983). At a GCV concentration of 1 μg/ml, almost no BMSCtk cells survived, while no significant toxicity was observed on wild-type BMSCs. The 100% lethal dose of GCV for wild-type BMSCs was 300 μg/ml, 100 times higher than that of BMSCtk cells (Supplementary Fig. 1). We used a 1 μg/ml GCV in the following in vitro studies, because this concentration of GCV killed all BMSCtk cells but not wild-type BMSCs and tumor cells.
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Figure 4 In vitro migratory capacity of human BMSCtk cells when they were exposed to different media. 0.5 ml of the BMSCtk cell suspension was added to the upper insert of Matrigel Invasion Chamber and the lower chamber was filled with 0.75 ml of the conditioned DMEM obtained by incubating human glioma cells (A-172, T98G) or rat C6 cells for 24 h or fresh DMEM (with or without 10% FBS). BMSCtk cells showed a potent migratory capacity when the conditioned medium was used as attractant in the lower chamber, while only a few migrating BMSCtk cells could be observed when they were exposed to the fresh DMEM. The migration was further enhanced with 10% FBS. A: Mean number of migrating cells ± standard error (triplicate). B: Representative photomicrographs of the micropore membrane after cell migration.
In vitro bystander effect between BMSCtk and various tumor cell lines The proliferation of tumor cells, irrespective of human or rat cells, co-cultured with human BMSCtk cells in the presence of GCV (1 μg/ml) (Fig. 1A, closed column) was significantly inhibited as compared to that without GCV (Fig. 1A, open column), which was not different from the proliferation of tumor cells cultured alone with or without GCV (data not shown). This is due to a significant bystander effect between human BMSCtk and tumor cells. Relative viability of “tumor + BMSCtk” groups was around 100% (Fig. 1A, open column), suggesting that BMSCtk did not have significant stimulatory or inhibitory effects on tumor cell proliferation. When BMSCs (and not BMSCtk cells) were co-cultured with tumor cells in the GCV containing medium, no bystander effect was exerted (data not shown). The similar bystander effect was also observed between rat BMSCtk and various tumor cells (Fig. 1B). These observations suggested that the in vitro bystander effect generated by BMSCtk cells and GCV was not specific to tumor cell types and species.
In vivo bystander effect between human BMSCtk and glioma cell lines Since a potent in vitro bystander effect was observed between human BMSCtk and various tumor cells, we then tested in vivo bystander effect by an intracranial co-implantation study of human BMSCtk and glioma cells in the nude mouse. When
tumor cells alone were implanted, a large tumor (5-6 mm 3) developed regardless of GCV administration (Fig. 2A). When a mixture of BMSCtk and human glioma cells (A-172 and T98G) was co-implanted, no tumor was observed in the GCV group, while tumors of similar sizes as those of the tumoralone groups developed in the PBS group (Fig. 2A). Representative mouse brain sections of each group were shown in Fig. 2B (A-172, T98G). The survival times of the mice implanted with a mixtures of BMSCtk and human glioma cells and treated with GCV (Fig. 3, A-172 and T98G, red lines) were more than 100 days, while those treated with PBS were about 3 weeks (black lines), which were similar to those of the tumor-alone groups treated with GCV (pink lines) or PBS (green lines). All the mice implanted with BMSCtk alone survived more than 100 days and no tumor formation was observed (Fig. 2A). Surprisingly, a similar in vivo bystander effect was observed between BMSCtk and rat C6 glioma cells (Fig. 2, C6). When human BMSCtk cells were mixed with rat C6 cells and coimplanted into the mice brain, small tumors developed in some mice of the GCV group (Fig. 2B, arrow). The survival was also significantly prolonged in the GCV group and 2 out of 5 mice survived more than 100 days (Fig. 3, C6).
In vitro migration of human BMSCtk cells to the conditioned medium Migratory activity of human BMSCtk cells was evaluated in Matrigel invasion assay (Li et al., 2007; Mohanam et al.,
Bystander effect using bone marrow stromal cells 1993). BMSCtk cells actively migrated through the Matrigel Matrix layer and micropore when the lower chamber was filled with the conditioned medium from the tumor cells, while they hardly migrated when the lower chamber was filled with the fresh DMEM (p b 0.01, Fig. 4). The migratory capacity of BMSCtk cells to the tumor conditioned medium was further enhanced when 10% FBS was added in the lower chamber compared with respective controls (p b 0.01, Fig. 4).
Discussion We used commercially-available human BMSCs as the vector cells in the present study. Thus, a limitation of the present study is that the commercially-available BMSCs do not represent general BMSCs that are obtained from the patients. They may not behave as “stem cells” that have multipotent differentiation capacity, and therefore, more studies are obviously needed to establish ideal vector cells for clinical use. Induced pluripotent stem cells, recently established and vigorously examined in the field of reconstructive neurosciences, could be also suitable vehicles for gene therapy (Takahashi et al., 2007; Yu et al., 2007). We have recently confirmed that induced pluripotent stem cells have a potent migratory activity toward the conditioned medium of brain tumors and this activity is also not tumor cell type-specific, or dependent on the species as shown in the present migration assay (Koizumi et al., 2011). In the present study, we used the same number of BMSCtk cells as that of tumor cells (BMSCtk/tumor cell ratio of 1:1). The previous in vitro study testing the potency of the bystander effect, using rat BMSCtk or HSVtk-transduced neural stem cells and C6 cells at various ratios (BMSCtk/tumor cell ratio of 1:1 to 1:128) showed a very potent bystander effect up to BMSCtk/ tumor cell ratio of 1:16 (Amano et al., 2009; Li et al., 2005b). Therefore, a dose response study at different BMSCtk/tumor cell ratios should be also performed between different BMSCtk/tumor cell combinations. In the present study, we only performed a co-implantation study, because the aim of the study was to test cell type- and species-specificity. We obviously have to test the effect on an established intracranial tumor as in our previous treatment studies (Amano et al., 2009; Li et al., 2005a). In vivo bystander effect between human BMSCtk and rat glioma cells was a little weaker than that between human BMSCtk and human glioma cells (Figs. 2 and 3), though the difference was not very obvious in in vitro conditions (Fig. 1). Precise mechanism is unknown but in vivo cell-to-cell communication, which is important for generation of bystander effect, may be different between human and rat cells. Bystander effect is still potent between cells of different species, but use of human BMSCs is recommended for treatment of human gliomas. In conclusion, we have demonstrated that the bystander effect by the human BMSCtk cells and GCV exerts against various kinds of human brain tumor cells, and also against rat glioma cells. Even after we changed the vector cells from neural stem cells to BMSCs for convenience for clinical use, it is still time-consuming to obtain sufficient number of BMSCtk cells from the patients when considering the duration for life-expectancy of glioblastoma patients. If pre-established
275 allogeneic or heterogeneic vector cells can be used, the strategy becomes much more feasible because those cells can be used at the time of the initial operation of glioblastoma. The results suggest that several established BMSCtk cell lines prepared in advance can be used for the BMSC-based HSVtk/ GCV gene therapy of glioma patients provided that safety issues due to immunological reactions are properly solved. Then “BMSCtk therapy” would become easier and more feasible as a treatment strategy for malignant gliomas that is inevitably fatal at the present time. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scr.2012.08.002.
Conflict of interest The authors have no conflict of interest.
References Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., Snyder, E.Y., 2000. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U. S. A. 97, 12846–12851. Amano, S., Li, S., Gu, C., Gao, Y., Koizumi, S., Yamamoto, S., Terakawa, S., Namba, H., 2009. Use of genetically engineered bone marrow-derived mesenchymal stem cells for glioma gene therapy. Int. J. Oncol. 35, 1265–1270. Benedetti, S., Pirola, B., Pollo, B., Magrassi, L., Bruzzone, M.G., Rigamonti, D., Galli, R., Selleri, S., DiMeco, F., DeFraja, C., Vescovi, A., Cattaneo, E., Finocchiaro, G., 2000. Gene therapy of experimental brain tumors using neural progenitor cells. Nat. Med. 6, 447–450. Culver, K.W., Ram, Z., Walbridge, S., Ishii, H., Oldfield, E.H., Blaese, R.M., 1992. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256, 1550–1552. DeAngelis, L.M., 2001. Brain tumors. N. Engl. J. Med. 344, 114–123. Dilber, M.S., Abedi, M.R., Christensson, B., Björkstrand, B., Kidder, G.M., Naus, C.C., Gahrton, G., Smith, C.I., 1997. Gap junctions promote the bystander effect of herpes simplex virus thymidine kinase in vivo. Cancer Res. 57, 1523–1528. Elshami, A.A., Saavedra, A., Zhang, H., Kucharczuk, J.C., Spray, D.C., Fishman, G.I., Amin, K.M., Kaiser, L.R., Albelda, S.M., 1886. Gap junctions play a role in the ‘bystander effect’ of the herpes simplex virus thymidine kinase/ganciclovir system in vitro. Gene Ther. 3, 85–92. Gu, C., Li, S., Tokuyama, T., Yokota, N., Namba, H., 2010. Therapeutic effect of genetically engineered mesenchymal stem cells in rat experimental leptomeningeal glioma model. Cancer Lett. 291, 256–262. Koizumi, S., Gu, C., Amano, S., Yamamoto, S., Ihara, H., Tokuyama, T., Namba, H., 2011. Migration of mouse-induced pluripotent stem cells to glioma-conditioned medium is mediated by tumorassociated specific growth factors. Oncol. Lett. 2, 283–288. Lee, J., Elkahloun, A.G., Messina, S.A., Ferrari, N., Xi, D., Smith, C.L., Cooper, R., Albert, P.S., Fine, H.A., 2003. Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: a potential antiglioma cellular vector. Cancer Res. 63, 8877–8889. Li, S., Tokuyama, T., Yamamoto, J., Koide, M., Yokota, N., Namba, H., 2005a. Bystander effect-mediated gene therapy of gliomas
276 using genetically engineered neural stem cells. Cancer Gene Ther. 12, 600–607. Li, S., Tokuyama, T., Yamamoto, J., Koide, M., Yokota, N., Namba, H., 2005b. Potent bystander effect in suicide gene therapy using neural stem cells transduced with herpes simplex virus thymidine kinase gene. Oncology 69, 503–508. Li, S., Gao, Y., Tokuyama, T., Yamamoto, J., Yokota, N., Yamamoto, S., Terakawa, S., Kitagawa, M., Namba, H., 2007. Genetically engineered neural stem cells migrate and suppress glioma cell growth at distant intracranial sites. Cancer Lett. 251, 220–227. Mesnil, M., Piccoli, C., Tiraby, G., Willecke, K., Yamasaki, H., 1996. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc. Natl. Acad. Sci. U. S. A. 93, 1831–1835. Mohanam, S., Sawaya, R., McCutcheon, I., Ali-Osman, F., Boyd, D., Rao, J.S., 1993. Modulation of in vitro invasion of human glioblastoma cells by urokinase-type plasminogen activator receptor antibody. Cancer Res. 53, 4143–4147. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Nakamizo, A., Marini, F., Amano, T., Khan, A., Studeny, M., Gumin, J., Chen, J., Hentschel, S., Vecil, G., Dembinski, J., Andreeff, M., Lang, F.F., 2005. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res. 65, 3307–3318. Namba, H., Tagawa, M., Miyagawa, T., Iwadate, Y., Sakiyama, S., 2000. Treatment of rat experimental brain tumors by herpes simplex virus thymidine kinase gene-transduced allogeneic tumor cells and ganciclovir. Cancer Gene Ther. 7, 947–953. Namba, H., Iwadate, Y., Kawamura, K., Sakiyama, S., Tagawa, M., 2001. Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells. Cancer Gene Ther. 8, 414–420. Rainov, N.G., 2000. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an
S. Li et al. adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum. Gene Ther. 11, 2389–2401. Ram, Z., Culver, K.W., Oshiro, E.M., Viola, J.J., DeVroom, H.L., Otto, E., Long, Z., Chiang, Y., McGarrity, G.J., Muul, L.M., Katz, D., Blaese, R.M., Oldfield, E.H., 1997. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat. Med. 3, 1354–1361. Stewart, L.A., 2002. Chemotherapy in adult high-grade glioma: a systematic review and meta-analysis of individual patient data from 12 randomised trials. Lancet 359, 1011–1018. Stupp, R., Mason, W.P., Bent, M.J.v.d, Weller, M., Fisher, B., Taphoorn, M.J., Belanger, K., Brandes, A.A., Marosi, C., Bogdahn, U., Curschmann, J., Janzer, R.C., Ludwin, S.K., Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J.G., Eisenhauer, E., Mirimanoff, R.O., European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group, 2005. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987–996. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872. Vrionis, F.D., Wu, J.K., Qi, P., Waltzman, M., Cherington, V., Spray, D.C., 1997. The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions. Gene Ther. 4, 577–585. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., Thomson, J.A., 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920. Ziu, M., Schmidt, N.O., Cargioli, T.G., Aboody, K.S., Black, P.M., Carroll, R.S., 2006. Glioma-produced extracellular matrix influences brain tumor tropism of human neural stem cells. J. Neurooncol. 79, 125–133.