Tsutomu Tokuyama

Bystander effect-mediated gene therapy of gliomas using genetically engineered neural stem cells

Since neural stem cells (NSCs) have the ability to migrate toward a tumor mass, genetically engineered NSCs were used for the treatment of gliomas. We first evaluated the “bystander effect” between NSCs transduced with the herpes simplex virus-thymidine kinase (HSVtk) gene (NSCtk) and C6 rat glioma cells under both in vitro and in vivo conditions. A potent bystander effect was observed in co-culture experiments of NSCtk and C6 cells. In the intracranial co-implantation experiments in athymic nude mice and Sprague–Dawley rats, the animals co-implanted with NSCtk and C6 cells and treated with ganciclovir (GCV) showed no intracranial tumors and survived more than 100 days, while those treated with physiological saline (PS) died of tumor progression. We next injected NSCtk cells into the pre-existing C6 tumor in rats and treated them with GCV or PS. The tumor volume was serially measured by magnetic resonance imaging. The tumor disappeared in six out of nine rats in the NSCtk/GCV group, while all the rats treated with PS died of tumor progression by day 21. The results indicate the feasibility of a novel gene therapy strategy for gliomas through a bystander effect generated by intratumoral injection of NSCtk cells and systemic GCV administration.

Monitoring of singlet oxygen is useful for predicting the photodynamic effects in the treatment for experimental glioma.

Purpose: Singlet oxygen (1O2) generated in photodynamic therapy (PDT) plays a very important role in killing tumor cells. Using a new near-IR photomultiplier tube system, we monitored the real-time production of 1O2 during PDT and thus investigated the relationship between the 1O2 production and photodynamic effects. Experimental Design: We did PDT in 9L gliosarcoma cells in vitro and in an experimental tumor model in vivo using 5-aminolevulinic acid and nanosecond-pulsed dye laser. During this time, we monitored 1O2 using this system. Moreover, based on the 1O2 monitoring, we set the different conditions of laser exposure and investigated whether they could affect the tumor cell death. Results: We could observe the temporal changes of 1O2 production during PDT in detail. At a low fluence rate the 1O2 signal gradually decreased with a low peak, whereas at a high fluence rate it decreased immediately with a high peak. Consequently, the cumulative 1O2 at a low fluence rate was higher, which thus induced a strong photodynamic effect. The proportion of apoptosis to necrosis might therefore be dependent on the peak and duration of the 1O2 signal. A low fluence rate tended to induce apoptotic change, whereas a high fluence rate tended to induce necrotic change. Conclusions: The results of this study suggested that the monitoring of 1O2 enables us to predict the photodynamic effect, allowing us to select the optimal laser conditions for each patient.

Incidentally identified syringomyelia associated with Chiari I malformations: is early interventional surgery necessary?

OBJECTIVEThe purpose of this study was to analyze clinical data and magnetic resonance imaging (MRI) findings for patients with asymptomatic, incidentally identified syringomyelia associated with Chiari I malformations who were monitored for more than 10 years, and to clarify the natural history of these lesions. METHODSThe clinical records of nine patients who had not been surgically treated and were regularly subjected to neurological and MRI examinations were analyzed. In MRI studies, the axial diameter of the syrinx at the widest level, the longitudinal extent of the syrinx, and the extent of tonsillar herniation into the spinal canal were analyzed. As a control, MRI findings for 11 patients with symptomatic syringomyelia associated with Chiari I malformations who had been surgically treated were also analyzed, and these MRI parameters were statistically compared between the asymptomatic and symptomatic groups. RESULTSOne patient underwent surgery, because of neurological changes, 7 years after the first visit. None of the remaining patients demonstrated any neurological change during the follow-up period (11.2 ± 0.7 yr), and all of them have been faring well without surgery. No statistically significant differences in MRI findings between the asymptomatic and symptomatic groups were observed. CONCLUSIONThe long-term clinical courses of patients with asymptomatic, incidentally identified syringomyelia associated with Chiari I malformations were observed to be benign. MRI parameters did not provide predictable values to recommend interventional surgery. Unless changes in neurological or MRI findings are detected, early interventional surgery is not necessary.

Potent Bystander Effect in Suicide Gene Therapy Using Neural Stem Cells Transduced with Herpes Simplex Virus Thymidine Kinase Gene

Objective: The herpes simplex virus thymidine kinase (HSVtk)/ganciclovir suicide gene therapy system has been considered as one of the most promising therapeutic strategies for malignant gliomas. We have been using HSVtk gene-transduced neural stem cells (NSCtk) that possess an ability to migrate toward a tumor mass for the treatment of experimental brain tumors. In the present study, we evaluated the potency of anti-tumor effect mediated by the bystander effect between NSCtk and C6 glioma cells in the HSVtk/ganciclovir suicide gene therapy system. Methods: NSCtk and C6 glioma cells were mixed at various ratios (NSCtk:C6 cell ratios of 1:1 to 1:64) and the bystander effect was evaluated both under in vitro and in vivo conditions. Results: In vitro co-culture experiment showed a complete tumor growth inhibition at the NSCtk:C6 ratios as low as 1:16. In vivo co-implantation study in the rat brain showed no visible tumors at the NSCtk:C6 ratios as low as 1:16 and all those rats survived more than 100 days. Conclusion: The results clearly demonstrated an extremely potent bystander effect between NSCtk and C6 cells, and the minimum number of NSCtk cells needed for the treatment of tumors was roughly estimated.

Therapeutic effect of genetically engineered mesenchymal stem cells in rat experimental leptomeningeal glioma model

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.

Bystander effect in glioma suicide gene therapy using bone marrow stromal cells.

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.

The two types of mRNAs for neurofibromin isoforms produced by Von Recklinghausen neurofibromatosis (NF1) gene : analysis in human astrocytic tumors

Two types of mRNAs for neurofibromin isoforms, which are produced by Von Recklinghausen neurofibromatosis (NF1) gene, have so far been identified. In the present study, we analyzed the differential expression of the two NF1 mRNAs in 16 cases of human astrocytic tumors and in surrounding normal tissues by the RNA-polymerase chain reaction. Astrocytic tumors predominantly expressed type II NF1 mRNA, whereas it was type I isoform that was predominantly expressed in the normal tissues. These results suggested that the increased type II NF1 protein might play an important role in the growth of astrocytic tumors.

Primary Neurolymphomatosis of the Lower Cranial Nerves Presenting as Dysphagia and Hoarseness: A Case Report

Primary neurolymphomatosis is an extremely rare tumor. We report the case of a 74-year-old patient presenting with dysphagia and hoarseness. Initial contrast-enhanced computed tomography of the head, neck, and chest did not reveal any lesions. His symptoms improved with short-term administration of prednisone but recurred and deteriorated. Magnetic resonance (MR) imaging revealed a tumor along the ninth and tenth cranial nerves across the jugular foramen. Fluorine-18 fluorodeoxyglucose positron emission tomography indicated this was a primary tumor. Repeated MR imaging after 2 months revealed considerable tumor enlargement. A left suboccipital craniotomy was performed to remove the tumor that infiltrated the ninth and tenth cranial nerves. The histopathologic diagnosis was diffuse large B-cell lymphoma. Although focal radiation therapy was administered to ensure complete eradication of the tumor, the patient died of aspiration pneumonia with systemic metastasis. To our knowledge, this is the first reported case of primary neurolymphomatosis in the lower cranial nerves.

Image-Guided Surgery for Gliomas

The significance of the extent of surgical resection on the prognosis of glioma still remains controversial even at the present time with modern computerized surgical facilities. Low-grade tumors are in general relatively slow-growing and most tumors remain stable for a quite long period even after partial resection with/without radiotherapy. Glioblastoma multiforme, the most malignant histological type of gliomas, on the other hand, is highly invasive and virtually incurable even after removal of the whole tumor mass with contrast-enhancement on MRI. The median survival of the patients with glioblastoma is generally less than one year from the time of diagnosis and this has not significantly improved for more than three decades despite continuous refinement of treatment strategies. Since the tumor recurrence usually occurs from the resection border where some residual tumor cells have been left behind and distant metastasis is seldom observed in gliomas, local tumor control would prolong the survival of those patients. Continuous efforts have been paid to develop novel strategies and some promising experimental data have been demonstrated. Therefore, when the effective adjuvant therapies after surgical removal become available, the extent of surgical removal would become one of the most important prognostic factors, which is already the case in medulloblastoma. In the present paper, current standard therapies for gliomas are reviewed and newly developed methods to improve extent of tumor removal are introduced.