Disseminating disease of high grade gliomas is difficult to treat. We examined the therapeutic effect of intrathecal administration of mesenchymal stem cells transduced with herpes simplex virus-thymidine kinase gene (MSCtk) followed by systemic ganciclovir (GCV) administration in rat experimental leptomeningeal glioma model. First, to examine in vivo bystander effect, rats were intrathecally co-injected with a mixture of MSCtk and C6 cells and then, intraperitoneally administered with GCV or saline for 10days (co-injection model). Next, to examine the therapeutic effect of MSCtk/GCV therapy, MSCtk cells were intrathecally administered 1day after C6 injection and then, GCV or saline was administered (treatment model). GCV administration significantly reduced tumor size on day 14 both in the co-injection model (0.41+/-0.22 vs. 3.10+/-0.97mm(2), p<0.01) and in the treatment model (0.73+/-.29 vs. 2.84+/-0.82mm(2), p<0.01). Survival was also significantly prolonged in GCV group both in the co-injection model (29.2+/-3.3 vs. 18.8+/-0.8days, p<0.001) and in the treatment model (21.5+/-1.5 vs. 17.2+/-0.5days, p<0.001). This study provided a novel treatment strategy for leptomeningeal glioma dissemination using intrathecal MSCtk injection followed by systemic GCV administration.

In our previous rat study, an established intracranial C6 glioma was successfully treated using intratumoral injection of mesenchymal stem cells transduced with the herpes simplex virus-thymidine kinase gene (MSCtk) and systemic administration of ganciclovir (GCV). In the present study, effect of the "bystander effect" associated with the MSCtk/GCV strategy on the background normal brain tissues was examined in both in vitro and in vivo conditions. Rat MSCtk and C6 glioma cells were mixed and seeded on the rat primary neuron and glia co-culture in the medium containing GCV to generate the bystander effect and the numbers of background cells were counted on day 0, 2 and 7. Though the number of MSCtk and C6 cells decreased rapidly due to the bystander effect, most of the neurons and glias survived on day 7. Next, rats were intracranially injected with the MSCtk and C6 cells and then intraperitoneally administered with GCV for 7days. No remarkable histological abnormality including apoptosis was observed in the background brain tissues near the injection site. The present study has demonstrated that the tumoricidal bystander effect does not injure the background normal brain tissue significantly and that the suicide gene therapies are sufficiently safe.

Lung cancer is one of the most common cancers, and the number of patients with intracranial metastases is increasing. Previously, we developed an enzyme prodrug suicide gene therapy based on the herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system using various mesenchymal stem cells to induce apoptosis in malignant gliomas through bystander killing effects. Here, we describe stem cells from human exfoliated deciduous teeth (SHED) as gene vehicles of the TK/GCV system against a brain metastasis model of non-small cell lung cancer (NSCLC). We introduced the A168H mutant TK (TKA168H) into SHED to establish the therapeutic cells because of the latent toxicity of wild type. SHED expressing TKA168H (SHED-TK) exhibited chemotaxis to the conditioned medium of NSCLC and migrated toward implanted NSCLC in vivo. SHED-TK demonstrated a strong bystander effect in vitro and in vivo and completely eradicated H1299 NSCLC in the brain. SHED-TK cells implanted intratumorally followed by GCV administration significantly suppressed the growth of H1299 and improved survival time. These results indicate that the TKA168H variant is suitable for establishing therapeutic cells and that intratumoral injection of SHED-TK followed by GCV administration may be a useful strategy for therapeutic approaches.

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 co-culture 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.

Malignant glioma, the most common malignant brain tumor in adults, is difficult to treat due to its aggressive invasive nature. Enzyme/prodrug suicide gene therapy based on the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system is an efficient strategy for treating malignant gliomas. In the present study, we evaluated treatment with multilineage-differentiating stress-enduring (Muse) cells, which are endogenous non-tumorigenic pluripotent-like stem cells that are easily collectable from the bone marrow as SSEA-3+ cells, as carriers of the HSVtk gene. Human Muse cells showed potent migratory activity toward glioma cells both in vitro and in vivo. HSVtk gene-transduced Muse cells (Muse-tk cells) at a cell number of only 1/32 that of U87 human glioma cells completely eradicated U87 gliomas in nude mouse brains, showing a robust in vivo bystander effect. Pre-existing intracranial U87 gliomas in nude mouse brains injected intratumorally with Muse-tk cells followed by intraperitoneal GCV administration were significantly reduced in size within 2 weeks, and 4 of 10 treated mice survived over 200 days. These findings suggest that intratumoral Muse-tk cell injection followed by systemic GCV administration is safe and effective and that allogeneic Muse-tk cell-medicated suicide gene therapy for malignant glioma is clinically feasible.