Yuuri Hashimoto

In vivo imaging of lymph node metastasis with telomerase-specific replication-selective adenovirus

Currently available methods for detection of tumors in vivo such as computed tomography and magnetic resonance imaging are not specific for tumors. Here we describe a new approach for visualizing tumors whose fluorescence can be detected using telomerase-specific replication-competent adenovirus expressing green fluorescent protein (GFP) (OBP-401). OBP-401 contains the replication cassette, in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of E1 genes, and the GFP gene for monitoring viral replication. When OBP-401 was intratumorally injected into HT29 tumors orthotopically implanted into the rectum in BALB/c nu/nu mice, para-aortic lymph node metastasis could be visualized at laparotomy under a three-chip color cooled charged-coupled device camera. Our results indicate that OBP-401 causes viral spread into the regional lymphatic area and selectively replicates in neoplastic lesions, resulting in GFP expression in metastatic lymph nodes. This technology is adaptable to detect lymph node metastasis in vivo as a preclinical model of surgical navigation.

A simple biological imaging system for detecting viable human circulating tumor cells

The presence of circulating tumor cells (CTCs) in the peripheral blood is associated with short survival, making the detection of CTCs clinically useful as a prognostic factor of disease outcome and/or a surrogate marker of treatment response. Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made it possible to detect a few CTCs in the blood; however, there is no sensitive assay to specifically detect viable CTCs. Here, we report what we believe to be a new approach to visually detect live human CTCs among millions of peripheral blood leukocytes, using a telomerase-specific replication-selective adenovirus expressing GFP. First, we constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401; TelomeScan). We then used OBP-401 to establish a simple ex vivo method that was able to detect viable human CTCs in the peripheral blood. The detection method involved a 3-step procedure, including the lysis of rbc, the subsequent addition of OBP-401 to the cell pellets, and an automated scan using fluorescence microscopy. OBP-401 infection increased the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal was amplified only in viable, infected human tumor cells, by viral replication. This GFP-expressing virus-based method is remarkably simple and allows precise enumeration of CTCs.

Autophagy-inducing agents augment the antitumor effect of telerase-selve oncolytic adenovirus OBP-405 on glioblastoma cells

Oncolytic adenoviruses are a promising tool in cancer therapy. In this study, we characterized the role of autophagy in oncolytic adenovirus-induced therapeutic effects. OBP-405, an oncolytic adenovirus regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad, OBP-301) with a tropism modification (RGD) exhibited a strong antitumor effect on glioblastoma cells. When autophagy was inhibited pharmacologically, the cytotoxicity of OBP-405 was attenuated. In addition, autophagy-deficient Atg5−/− mouse embryonic fibroblasts (MEFs) were less sensitive than wild-type MEFs to OBP-405. These findings indicate that OBP-405-induced autophagy is a cell killing effect. Moreover, autophagy-inducing therapies (temozolomide and rapamycin) synergistically sensitized tumor cells to OBP-405 by stimulating the autophagic pathway without altering OBP-405 replication. Mice harboring intracranial tumors treated with OBP-405 and temozolomide survived significantly longer than those treated with temozolomide alone, and mice treated with OBP-405 and the rapamycin analog RAD001 survived significantly longer than those treated with RAD001 alone. The observation that autophagy inducers increase OBP-405 antitumor activity suggests a novel strategy for treating patients with glioblastoma.

Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

Establishment of biological and pharmacokinetic assays of telomerase-specific replication-selective adenovirus

The use of replication‐selective tumor‐specific viruses represents a novel approach for the treatment of neoplastic disease. We constructed an attenuated adenovirus, telomerase‐specific replication‐selective adenovirus (TRAD), in which the human telomerase reverse transcriptase promoter element drives the expression of the E1A and E1B genes linked with an internal ribosome entry site (IRES). Forty‐eight hours after TRAD infection at a multiplicity of infection of 1.0, the cell viability of H1299 human lung cancer cells was consistently less than 50% and therefore this procedure could be used as a potency assay to assess the biological activity of TRAD. We also established a quantitative real‐time polymerase chain reaction (PCR) analysis with consensus primers for either the adenovirus E1A or IRES sequence. The linear ranges of quantitation with E1A and IRES primers were 103–108 and 102–108 plaque‐forming units/mL in the plasma, respectively. The PCR analysis demonstrated that the levels of E1A in normal tissues were more than 103 lower than in the tumors of A549 human lung tumor xenografts in nu/nµ mice at 28 days after intratumoral injection. Our results suggest that the cell‐killing assay against H1299 cells and real‐time PCR can be used to assess the biological activity and biodistribution of TRAD in clinical trials. (Cancer Sci 2008; 99: 385–390)

Telomerase-Dependent Oncolytic Adenovirus Sensitizes Human Cancer Cells to Ionizing Radiation via Inhibition of DNA Repair Machinery

The inability to repair DNA double-strand breaks (DSB) leads to radiosensitization, such that ionizing radiation combined with molecular inhibition of cellular DSB processing may greatly affect treatment of human cancer. As a variety of viral products interact with the DNA repair machinery, oncolytic virotherapy may improve the therapeutic window of conventional radiotherapy. Here, we describe the mechanistic basis for synergy of irradiation and OBP-301 (Telomelysin), an attenuated type-5 adenovirus with oncolytic potency that contains the human telomerase reverse transcriptase promoter to regulate viral replication. OBP-301 infection led to E1B55kDa viral protein expression that degraded the complex formed by Mre11, Rad50, and NBS1, which senses DSBs. Subsequently, the phosphorylation of cellular ataxia-telangiectasia mutated protein was inhibited, disrupting the signaling pathway controlling DNA repair. Thus, tumor cells infected with OBP-301 could be rendered sensitive to ionizing radiation. Moreover, by using noninvasive whole-body imaging, we showed that intratumoral injection of OBP-301 followed by regional irradiation induces a substantial antitumor effect, resulting from tumor cell-specific radiosensitization, in an orthotopic human esophageal cancer xenograft model. These results illustrate the potential of combining oncolytic virotherapy and ionizing radiation as a promising strategy in the management of human cancer.

Preclinical evaluation of synergistic effect of telomerase-specific oncolytic virotherapy and gemcitabine for human lung cancer

A phase I dose-escalation study of telomerase-specific oncolytic adenovirus, OBP-301 (Telomelysin), is now under way in the United States to assess feasibility and to characterize its pharmacokinetics in patients with advanced solid tumors. The present preclinical study investigates whether OBP-301 and a chemotherapeutic agent that is commonly used for lung cancer treatment, gemcitabine, are able to enhance antitumor effects in vitro and in vivo. The antitumor effects of OBP-301 infection and gemcitabine were evaluated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. In vivo antitumor effects of intratumoral injection of OBP-301 in combination with systemic administration of gemcitabine were assessed on nu/nu mice s.c. xenografted with human lung tumors. OBP-301 infection combined with gemcitabine resulted in very potent synergistic cytotoxicity in human lung cancer cells. The three human lung cancer cell lines treated with OBP-301 for 24 hours tended to accumulate in S phase compared with controls. The proportion of cells in S phase increased from 43.85% to 56.41% in H460 cells, from 46.72% to 67.09% in H322 cells, and from 38.22% to 57.67% in H358 cells. Intratumoral injection of OBP-301 combined with systemic administration of gemcitabine showed therapeutic synergism in human lung tumor xenografts. Our data suggest that the combination of OBP-301 and gemcitabine enhances the antitumor effects against human lung cancer. We also found that the synergistic mechanism may be due to OBP-301–mediated cell cycle accumulation in S phase. These results have important implications for the treatment of human lung cancer.[Mol Cancer Ther 2009;8(4):980–7]

A genetically engineered oncolytic adenovirus decoys and lethally traps quiescent cancer stem-like cells in S/G2/M phases.

Purpose: Because chemoradiotherapy selectively targets proliferating cancer cells, quiescent cancer stem–like cells are resistant. Mobilization of the cell cycle in quiescent leukemia stem cells sensitizes them to cell death signals. However, it is unclear that mobilization of the cell cycle can eliminate quiescent cancer stem–like cells in solid cancers. Thus, we explored the use of a genetically-engineered telomerase-specific oncolytic adenovirus, OBP-301, to mobilize the cell cycle and kill quiescent cancer stem–like cells. Experimental Design: We established CD133+ cancer stem–like cells from human gastric cancer MKN45 and MKN7 cells. We investigated the efficacy of OBP-301 against quiescent cancer stem–like cells. We visualized the treatment dynamics of OBP-301 killing of quiescent cancer stem–like cells in dormant tumor spheres and xenografts using a fluorescent ubiquitination cell-cycle indicator (FUCCI). Results: CD133+ gastric cancer cells had stemness properties. OBP-301 efficiently killed CD133+ cancer stem–like cells resistant to chemoradiotherapy. OBP-301 induced cell-cycle mobilization from G0–G1 to S/G2/M phases and subsequent cell death in quiescent CD133+ cancer stem–like cells by mobilizing cell-cycle–related proteins. FUCCI enabled visualization of quiescent CD133+ cancer stem–like cells and proliferating CD133− non–cancer stem–like cells. Three-dimensional visualization of the cell-cycle behavior in tumor spheres showed that CD133+ cancer stem–like cells maintained stemness by remaining in G0–G1 phase. We showed that OBP-301 mobilized quiescent cancer stem–like cells in tumor spheres and xenografts into S/G2/M phases where they lost viability and cancer stem–like cell properties and became chemosensitive. Conclusion: Oncolytic adenoviral infection is an effective mechanism of cancer cell killing in solid cancer and can be a new therapeutic paradigm to eliminate quiescent cancer stem–like cells. Clin Cancer Res; 19(23); 6495–505. ©2013 AACR.

Dual Programmed Cell Death Pathways Induced by p53 Transactivation Overcome Resistance to Oncolytic Adenovirus in Human Osteosarcoma Cells

Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301–resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301–resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301–sensitive (U2OS and HOS) and OBP-301–resistant (SaOS-2 and MNNG/HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702–mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301–resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301–resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301–sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Ad-p53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702–infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301–resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702–mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells. Mol Cancer Ther; 12(3); 314–25. ©2012 AACR.

Preclinical Evaluation of Telomerase-Specific Oncolytic Virotherapy for Human Bone and Soft Tissue Sarcomas

Purpose: Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy for induction of cell death in tumor cells but not of normal cells. We previously developed an oncolytic adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner. Recent evidence suggests that nonepithelial malignant cells, which have low telomerase activity, maintain telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear whether OBP-301 is cytopathic for nonepithelial malignant cells. Here, we evaluated the antitumor effect of OBP-301 on human bone and soft tissue sarcoma cells. Experimental Design: The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR) expression, and telomerase activity were examined in 10 bone (OST, U2OS, HOS, HuO9, MNNG/HOS, SaOS-2, NOS-2, NOS-10, NDCS-1, and OUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1, and NMFH-1) sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The fiber-modified OBP-301 (termed OBP-405) was used to confirm an antitumor effect on OBP-301–resistant sarcomas. Results: OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non–CAR-expressing OUMS-27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression and not on telomerase activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301 significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405 showed antitumor effects on OBP-301–resistant OUMS-27 and NMFH-1 cells. Conclusions: A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the treatment of bone and soft tissue sarcomas. Clin Cancer Res; 17(7); 1828–38. ©2011 AACR.