Potent bystander effect and tumor tropism in suicide gene therapy using stem cells from human exfoliated deciduous teeth.

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.

Interferon-ß and temozolomide combination therapy for temozolomide monotherapy-refractory malignant gliomas.

Interferon-ß (IFN-ß) has been found to downregulate O6-methyl-guanine-DNA methyltransferase and sensitize glioma cells to chemoradiation therapy. The effectiveness of IFN-ß and temozolomide (TMZ) combination therapy for newly diagnosed glioblastomas was previously reported. However, there is no clinical report of recurrent of malignant gliomas treated with the combination of IFN-ß and TMZ. In the present study, we reported 7 cases of gliomas classified as uncontrollable with adjuvant TMZ monotherapy, who were then treated with IFN-ß and TMZ combination therapy. The magnetic resonance imaging findings and clinical symptoms improved in the majority of the cases, with tolerable adverse events and minimal residual disability. The overall survival (OS) time from the date of the initial surgery exceeded 13 months, suggesting that this combination therapy was successful in improving the prognosis of malignant gliomas refractory to adjuvant TMZ monotherapy.

Potent tumor tropism of induced pluripotent stem cells and induced pluripotent stem cell-derived neural stem cells in the mouse intracerebral glioma model.

Although neural and mesenchymal stem cells have been well-known to have a strong glioma tropism, this activity in induced pluripotent stem cells (iPSCs) has not yet been fully studied. In the present study, we tested tumor tropic activity of mouse iPSCs and neural stem cells derived from the iPSC (iPS-NSCs) using in vitro Matrigel invasion chamber assay and in vivo mouse intracranial tumor model. Both iPSC and iPS-NSC had a similar potent in vitro tropism for glioma conditioned media. The migrated iPSCs to the gliomas kept expressing Nanog-GFP gene, suggesting no neuronal or glial differentiation. iPSCs or iPS-NSCs labeled with 5-bromo-2-deoxyuridine were intracranially implanted in the contralateral hemisphere to the GL261 glioma cell implantation in the allogeneic C57BL/6 mouse. Active migration of both stem cells was observed 7 days after implantation. Again, the iPSCs located in the tumor area expressed Nanog-GFP gene, suggesting that the migrated cells were still iPSCs. These findings demonstrated that both iPSCs and iPS-NSCs had potent glioma tropism and could be candidates as vehicles in stem cell-based glioma therapy.

Extraventricular neurocytoma of the sellar region with spinal dissemination.

Extraventricular neurocytoma (EVN) is a rare tumor that mainly occurs in the cerebral hemispheres and spinal cord. Sellar neurocytoma is extremely rare, with only two previously reported cases. We report a sellar EVN in a 48-year-old man presenting with visual impairment. This tumor was partially resected. The residual tumor disappeared on MRI with adjuvant radiotherapy. However, 2 years later the tumor recurred with craniospinal dissemination, which is also very rare, with only four previously reported cases. The recurrent tumor showed atypical features with an MIB-1 LI score of 3 %. It is suggested that postoperative adjuvant radiation therapy with long-term follow-up is required for incompletely resected EVN.

Dissecting Aneurysm at the Proximal Segment of the Anterior Cerebral Artery Associated with Infraoptic Course Anterior Cerebral Artery.

A 48-year-old man presented a subarachnoid hemorrhage caused by a rupture of a dissecting aneurysm at the proximal segment (A1 segment) of the right anterior cerebral artery (ACA). He also had an anomalous artery named infraoptic course ACA and an agenesis of the contralateral ACA A1 segment. Balloon occlusion test at the bifurcation of the right internal carotid artery demonstrated that the distal segments of the bilateral ACAs were perfused through the infraoptic course ACA. Therefore, we surgically trapped the A1 segment including the aneurysm. The patient got discharged without any neurological deficit. Natural course of ACA dissecting aneurysms is unclear because of rarity of the disease and treatment strategy is still controversial. Most of the dissecting aneurysms in the A1 segment are surgically treated, because they often present with massive hemorrhage and poor prognosis. In the present case, the contralateral A1 segment was absent but trapping of the dissecting aneurysm could be achieved without vascular reconstruction (e.g., bypass surgery) because of the presence of the infraoptic course ACA.

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.

Molecular targeting therapy against promyelocytic leukemia protein using arsenic acids in experimental intracranial medulloblastoma.

Our previous study using human Daoy medulloblastoma cells showed that the promyelocytic leukemia (PML) gene was significantly upregulated (2.5-fold) in cells positive to prominin-1 antigen (CD133), a possible marker for cancer initiating cells. Arsenic trioxide (As(2)O(3)) is known to degrade PML protein and has been used for the treatment of patients with acute PML. In the present study, the effect of PML targeting therapy with As(2)O(3) and cytarabine (Ara-C) on Daoy medulloblastoma cell proliferation was investigated. Daoy cells were pretreated with As(2)O(3) for 6 weeks. The As(2)O(3)-pretreated Daoy cells were cultured in medium containing Ara-C and cell viability was examined. Next, the As(2)O(3)-pretreated Daoy cells were inoculated into the nude mouse brain and the effect of Ara-C on the tumor size was evaluated. A significant increase in chemosensitivity to Ara-C was observed in the As(2)O(3)-pretreated Daoy cells in both in vitro and in vivo conditions. PML and CCND1 (cyclin D1) protein expression of Daoy medulloblastoma cells was downregulated by As(2)O(3) treatment. PML has been proposed as a novel therapeutic target to eradicate quiescent leukemia-initiating cells, and PML-expressing CD133-positive cells are similarly a potential therapeutic target of treatment for medulloblastoma.

Timing of ganciclovir administration in glioma gene therapy using HSVtk gene-transduced mesenchymal stem cells.

BACKGROUND: An established C6 glioma was successfully treated with intratumoral injection of mesenchymal stem cells transduced with HSVtk gene (MSCtk) and systemic administration of ganciclovir (GCV). The best timing of GCV administration after the MSCtk implantation was studied. MATERIALS AND METHODS: GCV administration was started from 2 days before and 1, 3 and 7 days after the MSCtk administration under both in vitro and in vivo conditions. RESULTS: The C6 cells were completely eradicated in vitro when GCV administration was started from day -2, 1, and 3. Animals with intracranial tumor survived longer when GCV was administered earlier after MSCtk administration. This may, mainly, reflect the difference in the MSCtk/C6 ratio at the time of GCV administration because this ratio drastically decreases during the delay of GCV administration. CONCLUSION: When using a slowly growing vector cell as MSCtk, GCV should be administered soon after MSCtk implantation.

Tumoricidal bystander effect in the suicide gene therapy using mesenchymal stem cells does not injure normal brain tissues.

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.

Migration of mouse-induced pluripotent stem cells to glioma-conditioned medium is mediated by tumor-associated specific growth factors.

Neural and mesenchymal stem cells have extensive tropism for malignant glioma. The tumor tropism of induced pluripotent stem (iPS) cells was tested using the Matrigel invasion assay. Mouse iPS cells showed a significant tropism to the conditioned media prepared from six rodent and human glioma cell lines and this tropism to the glioma conditioned media was partially blocked by the neutralizing antibodies for four major tumor-associated growth factors [stem cell factor (SCF), platelet-derived growth factor BB (PDGF-BB), stromal-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF)], which are secreted from the malignant gliomas. The tropism of the iPS cells was enhanced by the growth factors in a concentration-dependent manner from 0.1 to 100 ng/ml. The receptors for those growth factors (c-Kit, ICAM-1, CXCR4 and VEGFR2), measured by reverse transcriptase-polymerase chain reaction, were highly up-regulated in the mouse iPS cells compared to the mouse fibroblasts. The results showed that the specific growth factors secreted from the gliomas strongly attracted the iPS cells. Therefore, gene therapies using iPS cells as vectors to deliver anti-tumor agents are novel strategies for the treatment of malignant gliomas that deeply infiltrate the brain.

Use of genetically engineered bone marrow-derived mesenchymal stem cells for glioma gene therapy.

In our previous study, we successfully treated an established C6 brain tumor using neural stem cells transduced with the herpes simplex virus-thymidine kinase gene (HSVtk) and ganciclovir in the rat. In the present study, we investigated the use of mesenchymal stem cells (MSCs), obtained from adult rats and transduced with HSVtk (MSCtk cells), instead of neural stem cells because MSCs are much easier to obtain from the adult subjects. Those cells were used for in vitro co-culture study and in vivo co-implantation study with C6 rat glioma cells to examine bystander tumoricidal effect, which revealed a sufficient bystander effect and only 1/32 MSCtk cells were needed for complete tumor eradication. In vitro bystander effect was also observed in a real-time fashion using a culture microscope and it was shown that only tumor cells that had contact with MSCtk cells died. In vivo treatment study of an established C6 brain tumor with an intratumoral injection of MSCtk cells followed by systemic ganciclovir administration demonstrated a significant reduction of the tumor size and a significant survival prolongation. The treatment strategy using MSCtk and ganciclovir (MSCtk therapy) is more feasible and practical for clinical application than the method using neural stem cells.

Mixed testicular germ cell tumor in an adult with cryptorchidism and Down's syndrome.

We here present a case of mixed testicular germ cell tumor in an adult with cryptorchidism and Down's syndrome. A 20-year-old Japanese man with a mass in the left inguinal region underwent orchidectomy as a left testicular tumor was suspected. Histology showed a mixed germ-cell tumor with embryonal carcinoma and yolk sac tumor with syncytiotrophoblastic giant cells occurring in a cryptorchid testis. Chromosomal analysis of peripheral lymphocytes disclosed a karyotype of 47,XY,+21[20]. Our case provides further evidence that these three conditions-Down's syndrome, cryptorchidism and testicular germ cell tumor-may be closely associated. To our knowledge this is the first case of mixed germ cell tumor arising in a patient with Down's syndrome and cryptorchidism.