Tomoki Todo

Autocrine TGF-β Signaling Maintains Tumorigenicity of Glioma-Initiating Cells through Sry-Related HMG-Box Factors

Despite aggressive surgery, radiotherapy, and chemotherapy, treatment of malignant glioma remains formidable. Although the concept of cancer stem cells reveals a new framework of cancer therapeutic strategies against malignant glioma, it remains unclear how glioma stem cells could be eradicated. Here, we demonstrate that autocrine TGF-beta signaling plays an essential role in retention of stemness of glioma-initiating cells (GICs) and describe the underlying mechanism for it. TGF-beta induced [corrected] expression of Sox2, a stemness gene, and this induction was mediated by Sox4, a direct TGF-beta target gene. Inhibitors of TGF-beta signaling drastically deprived tumorigenicity of GICs by promoting their differentiation, and these effects were attenuated in GICs transduced with Sox2 or Sox4. Furthermore, GICs pretreated with TGF-beta signaling inhibitor exhibited less lethal potency in intracranial transplantation assay. These results identify an essential pathway for GICs, the TGF-beta-Sox4-Sox2 pathway, whose disruption would be a therapeutic strategy against gliomas.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Systemic Antitumor Immunity in Experimental Brain Tumor Therapy Using a Multimutated, Replication-Competent Herpes Simplex Virus

Replication-competent, attenuated herpes simplex virus (HSV) vectors have been developed for viral oncolytic therapy of primary and metastatic malignant brain tumors. However, the role of the host immune responses in the brain has not been elucidated. N18 neuroblastoma cells were used as a tumor model in syngeneic A/J mice to test the therapeutic efficacy of G207, a conditionally replicating HSV vector, in an immunocompetent condition. G207 inoculated intraneoplastically exhibited a prominent oncolytic antitumor effect in mice harboring N18 tumors in the brain or subcutaneously, and, in addition, elicited a systemic antitumor immune response. Subcutaneous tumor therapy with G207 caused regression of a remote, established tumor in the brain or in the periphery, which was potentially mediated by the systemic antitumor immune response, and provided persistent tumor-specific protection against N18 tumor rechallenge in the brain as well as in the periphery. Antitumor immunity was associated with an elevation of specific CTL activity against N18 tumor cells that persisted for at least 13 months. The results suggest that the oncolytic antitumor action of replication-competent HSV may be augmented by induction of specific and systemic antitumor immunity effective both in the periphery and in the brain.

Glioma-initiating Cells Retain Their Tumorigenicity through Integration of the Sox Axis and Oct4 Protein

Background: Glioma-initiating cells are underlying causes of development and progression of glioblastoma. Results: Depletion of Oct4 expression suppresses tumorigenic activity of glioma-initiating cells through down-regulation of Sox2. Conclusion: Oct4 maintains tumorigenicity of glioma-initiating cells in cooperation with the Sox axis. Significance: This study uncovers the transcriptional network of stemness genes in cancer-initiating cells. Although the concept of cancer stem cells or cancer-initiating cells had created a new paradigm for the treatment of malignant tumors, it remains unclear how cancer-initiating cells can be eradicated. We have previously reported that the transforming growth factor-β (TGF-β)-Sox4-Sox2 pathway is essential for glioma-initiating cells to retain their stemness, and inhibition of TGF-β signaling may lead to differentiation of glioma-initiating cells (Ikushima, H., Todo, T., Ino, Y., Takahashi, M., Miyazawa, K., and Miyazono, K. (2009) Cell Stem Cell 5, 504–514). Here we demonstrate that Oct4 plays essential roles in retention of the stemness properties of glioma-initiating cells through positive regulation of Sox2 expression. We also show that, in glioma-initiating cells, Oct4 is associated with Sox4 and that Oct4-Sox4 complexes cooperatively activate the enhancer activity of the SOX2 gene. In contrast, in fetal neural progenitor cells, Sox2 expression is enhanced by transcriptional complex containing Sox2 protein itself, and this self-reinforcing loop of Sox2 appears to be disrupted in glioma-initiating cells, suggesting that Sox2 expression in glioma-initiating cells is differently regulated from that in neural progenitor cells. Our findings reveal differences between glioma-initiating cells and fetal neural progenitor cells and may open the way to depriving glioma-initiating cells of tumorigenic activity without affecting normal tissues.

The motor-evoked potential threshold evaluated by tractography and electrical stimulation

OBJECT To validate the corticospinal tract (CST) illustrated by diffusion tensor imaging, the authors used tractography-integrated neuronavigation and direct fiber stimulation with monopolar electric currents. METHODS Forty patients with brain lesions adjacent to the CST were studied. During the operation, the motor responses (motor evoked potential [MEP]) elicited at the hand by the cortical stimulation to the hand motor area were continuously monitored, maintaining the consistent stimulus intensity (mean 15.1 +/- 2.21 mA). During lesion resection, direct fiber stimulation was applied to elicit MEP (referred to as fiber MEP) to identify the CST functionally. The threshold intensity for the fiber MEP was determined by searching for the best stimulus point and changing the stimulus intensity. The minimum distance between the resection border and illustrated CST was measured on postoperative isotropic images. RESULTS Direct fiber stimulation demonstrated that tractography accurately reflected anatomical CST functioning. There were strong correlations between stimulus intensity for the fiber MEP and the distance between the CST and the stimulus points. The results indicate that the minimum stimulus intensity of 20, 15, 10, and 5 mA had stimulus points approximately 16, 13.2, 9.6, and 4.8 mm from the CST, respectively. The convergent calculation formulated 1.8 mA as the electrical threshold of the CST for the fiber MEP, which was much smaller than that of the hand motor area. CONCLUSIONS The investigators found that diffusion tensor imaging-based tractography is a reliable way to map the white matter connections in the entire brain in clinical and basic neuroscience applications. By combining these techniques, investigating the cortical-subcortical connections in the human CNS could contribute to elucidating the neural networks of the human brain and shed light on higher brain functions.

Oncolytic virus therapy: A new era of cancer treatment at dawn

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. Oncolytic viruses are defined as genetically engineered or naturally occurring viruses that selectively replicate in and kill cancer cells without harming the normal tissues. T‐Vec (talimogene laherparepvec), a second‐generation oncolytic herpes simplex virus type 1 (HSV‐1) armed with GM‐CSF, was recently approved as the first oncolytic virus drug in the USA and Europe. The phase III trial proved that local intralesional injections with T‐Vec in advanced malignant melanoma patients can not only suppress the growth of injected tumors but also act systemically and prolong overall survival. Other oncolytic viruses that are closing in on drug approval in North America and Europe include vaccinia virus JX‐594 (pexastimogene devacirepvec) for hepatocellular carcinoma, GM‐CSF‐expressing adenovirus CG0070 for bladder cancer, and Reolysin (pelareorep), a wild‐type variant of reovirus, for head and neck cancer. In Japan, a phase II clinical trial of G47∆, a third‐generation oncolytic HSV‐1, is ongoing in glioblastoma patients. G47∆ was recently designated as a “Sakigake” breakthrough therapy drug in Japan. This new system by the Japanese government should provide G47∆ with priority reviews and a fast‐track drug approval by the regulatory authorities. Whereas numerous oncolytic viruses have been subjected to clinical trials, the common feature that is expected to play a major role in prolonging the survival of cancer patients is an induction of specific antitumor immunity in the course of tumor‐specific viral replication. It appears that it will not be long before oncolytic virus therapy becomes a standard therapeutic option for all cancer patients.

Functional Monitoring for Visual Pathway Using Real-time Visual Evoked Potentials and Optic-radiation Tractography

OBJECTIVE: It has been difficult to obtain anatomic and functional information about the visual pathway during neurosurgical operations. The aim of this study was to combine the information of the visual evoked potentials (VEPs) and the anatomic navigation of the optic radiation by diffusion tensor imaging-based tractography for functional monitoring of the visual pathway. METHODS: The subjects were two patients with brain lesions adjacent to the visual pathway. Diffusion tensor imaging-based tractography of the optic radiation was performed by selecting appropriate regions of interest and by fractional anisotropy. During surgery, cortical VEPs were recorded continuously under general anesthesia with sevoflurane. In Patient 2, the results of optic radiation tractography were imported to a neuronavigation system to better understand the spatial relationships between the lesions and the visual pathway (functional neuronavigation). RESULTS: In Patient 1, the lesion did not seem to be attached to the optic radiation, and VEP profiles remained stable during resection. In Patient 2, who had a lesion adjacent to the posterior horn of the lateral ventricle, VEPs suddenly diminished when resection reached the optic radiation as illustrated on the neuronavigation system. As a result, complete left hemianopia developed after surgery in Patient 2. CONCLUSION: We confirmed functional correlations of the results of diffusion tensor imaging-based tractography by monitoring intraoperative VEPs. The combination of continuous VEP and optic-radiation tractography is reliable to monitor the visual function and is helpful in performing neurosurgical planning near the visual pathway.

Expressive and receptive language areas determined by a non-invasive reliable method using functional magnetic resonance imaging and magnetoencephalography

OBJECTIVEIt is known that functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG) are sensitive to the frontal and temporal language function, respectively. Therefore, we established combined use of fMRI and MEG to make reliable identification of the global language dominance in pathological brain conditions. METHODSWe investigated 117 patients with brain lesions whose language dominance was successfully confirmed by the Wada test. All patients were asked to generate verbs related to acoustically presented nouns (verb generation) for fMRI and to read three-letter words for fMRI and MEG. RESULTSfMRI typically showed prominent activations in the inferior and middle frontal gyri, whereas calculated dipoles on MEG typically clustered in the superior temporal region and the fusiform gyrus of the dominant hemisphere. A total of 87 patients were further analyzed using useful data from both the combined method and the Wada test. Remarkably, we observed a 100% match of the combined method results with the results of the Wada test, including two patients who showed expressive and receptive language areas dissociated into bilateral hemispheres. CONCLUSIONThe results demonstrate that this non-invasive and repeatable method is not only highly reliable in determining language dominance, but can also locate the expressive and receptive language areas separately. The method may be a potent alternative to invasive procedures of the Wada test and useful in treating patients with brain lesions.

Vasospasm prevention with postoperative intrathecal thrombolytic therapy : A retrospective comparison of urokinase, tissue plasminogen activator, and cisternal drainage alone

The authors report the results of a retrospective review, between January 1986 and December 1991, of the results of early surgery and intrathecal thrombolytic therapy in 111 patients with aneurysmal subarachnoid hemorrhage. Effects on clot lysis, angiographic and symptomatic vasospasm, cerebral infarction, and clinical outcome were compared in 60 patients treated with urokinase (UK) 60,000 IU/d for 7 days (UK group), 22 patients treated with 0.042 to 1 mg tissue plasminogen activator (tPA) every 6 to 8 hours for 5 days (tPA group), and 29 patients who did not receive treatment with either thrombolytic agent (no-treatment group). The no-treatment group consisted of all patients treated before July 1986 and of patients in whom thrombolytic therapy was attempted but failed to start or in whom the therapy was not used intentionally because of small subarachnoid clot. Treatment with UK was employed between July 1986 and March 1991, and tPA was employed during the remainder of the study for patients at a higher risk for vasospasm. The severity of angiographic vasospasm and the incidence of infarction in the UK and the tPA groups were less than those of the no-treatment group (P < 0.01), in spite of a larger amount of initial subarachnoid blood clot in both thrombolytic groups. This appears to be the result of the more rapid clearance of cisternal clot in the thrombolytic groups than the no-treatment group (P < 0.01). Only tPA therapy reduced the incidence of symptomatic vasospasm (P < 0.05). No serious complications were observed, although in the tPA group, asymptomatic intraventricular hemorrhage occurred in one patient, and transient confusion in another. Both received 4 mg tPA/d. Meningitis was suspected in 16 patients of the UK group. However, in this relatively small retrospective series, there were no differences among the three groups in overall outcome at 3 months. This study indicates that postoperative intrathecal thrombolytic therapies, especially with less than 4 mg/d of tPA, are effective in lysing subarachnoid clot and preventing vasospasm and infarction safely.

Preclinical safety evaluation of G207, a replication-competent herpes simplex virus type 1, inoculated intraprostatically in mice and nonhuman primates.

G207, a replication-competent herpes simplex virus type 1 (HSV-1) virus, has been previously shown to be effective against human prostate cancer xenografts in mice. This study assesses its safety in the prostate of two animal models known for their sensitivity to HSV-1. BALB/c mice were injected intraprostatically with either HSV-1 G207 or strain F and observed for 5 months. None of the G207-injected animals exhibited any clinical signs of disease or died. However, 50% of strain F-injected mice displayed sluggish, hunched behavior and died by day 13. Histopathologically, the G207-injected prostates were normal whereas strain F-injected prostates showed epithelial flattening, sloughing, and stromal edema. Four Aotus nancymae monkeys were also injected with G207 intraprostatically and observed short term (up to 21 days) and long term (56 days). Safety was assessed on the basis of clinical observations, viral biodistribution, virus shedding, and histopathology. None of the injected monkeys displayed evidence of clinical disease, shedding of infectious virus, or spread of the virus into other organs. Except for minor histological changes unrelated to the study, no significant abnormalities were observed. These results demonstrate that G207 can be safely inoculated into the prostate and should be considered for human trials for the treatment of prostate cancer.