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Gastric cancer is one of the most common causes of cancer death. Stromal fibroblasts in the tumor microenvironment have recently been implicated in tumor growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) have been suggested to be responsible for progression of undifferentiated scirrhous gastric cancers with vast fibrous stroma. However, the relationship between differentiated gastric cancer cells and CAFs in tumor progression remains unclear. In this study, we investigated whether differentiated and/or undifferentiated human gastric cancer cells induce CAFs. We used two differentiated human gastric cancer cells (MKN-7, MKN-74) and two undifferentiated human scirrhous gastric cancer cells (KATO-III, NUGC-4). Two types of human normal fibroblasts (WI-38, FEF-3) were used for induction of CAFs. The conditioned media (CM) from all cancer cells were obtained 48 hours after serum starvation. The morphological change and expression of CAF-specific markers (α-smooth muscle actin (α-SMA), fibroblast activation protein-α (FAP)) were analyzed by microscopy and Western blot analysis, respectively, in fibroblasts treated with CM for 120 hours. Moreover, the effect of CM from CAFs in the migration ability of cancer cells was evaluated using in vitro Migration assay. The CM from differentiated MKN-7 and MKN-74 cells induced the morphological change like CAFs in normal human fibroblasts, whereas CM from undifferentiated KATO-III and NUGU-4 cells did not induce. In fact, morphologically changed fibroblasts like CAFs showed increased α-SMA expression, whereas FAP expression was not increased. The CM from morphologically changed fibroblasts, but not normal fibroblasts, significantly enhanced the migration ability of MKN-7 cells. These results suggest that differentiated gastric cancer cells have a potential to induce CAFs in the invasion process of gastric cancers. CAF-targeting therapy may be a promising antitumor strategy in differentiated gastric cancers as well as undifferentiated scirrhous gastric cancers with vast fibrous stroma. Citation Format: Yuncheng Li, Hiroshi Tazawa, Nishizaki Masahiko, Yuuri Hashimoto, Naoto Hori, Ryoichi Katsube, Shinji Kuroda, Kazuhiro Noma, Shunsuke Kagawa, Toshiyoshi Fujiwara. Differentiated gastric cancer cells have a potential to induce cancer-associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2368. doi:10.1158/1538-7445.AM2015-2368

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.

Malignant mesothelioma is an extraordinarily challenging disease to treat; however, locoregional virotherapy may be applicable for this aggressive disease because of the accessibility by intrapleural and/or intratumoral virus delivery. We previously reported that an attenuated adenovirus OBP-301 (Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, could replicate in and causes selective lysis of human cancer cells. In the present study, we examined the antitumor effect of Telomelysin on human malignant mesothelioma cell lines in vitro and in vivo. The XTT assay demonstrated that Telomelysin could efficiently kill four human mesothelioma cell lines H2052, H2452, H28, and 211H, all of which expressed the coxsackievirus and adenovirus receptor (CAR), in a dose-dependent manner. In vivo antitumor effect was also evaluated in an orthotopic pleural dissemination model. Intrathoracic administration of 2 |[times]| 108 plaque forming units (PFU) of Telomelysin markedly reduced the size (1 |[plusmn]| 1.8 mg vs. 190 |[plusmn]| 75 mg, p < 0.01) of H2452 tumors intrathoracically implanted into BALB/c- nu/nu mice compared to mock-treated mice. These results suggest that locoregional administration of Telomelysin into the thoracic cavity may be efficacious in the prevention and treatment of pleural dissemination of human malignant mesothelioma.

Endoscopic resection is now one of the options as minimally invasive therapy for early colorectal cancer, However, surgical resection has been still standard for submucosal invasive colorectal cancer, because about 10% of that have LN metastasis. To secure endoscopic resection for submucosal invasive colorectal cancer, an alternative, innovational strategy to purge potential LN metastasis is required. We previously reported that a telomerase-dependent oncolytic adenovirus (OBP-301) spreads to the regional LNs, selectively replicates, and eradicates metastatic cancer cells in LNs, when administered into the orthotopically xenotransplanted rectal tumor in mouse model. In this study we evaluated whether OBP-301 could inhibit LN metastasis after the local resection mimicking endoscopic resection in a submucosal invasive rectal tumor mouse model. Human colon cancer HCT-116 cells expressing green fluorescent protein (GFP) were orthotopically implanted into the submucosal layer of the rectum in nude mouse, to develop resultant rectal tumor mimicking submucosal invasive one. LN metastasis was observed in 78.5% of mice as early as 7 days after cancer cell inoculation. The rectal tumor was injected with OBP-301 peritumorally, and then got locally excised mimicking Endoscopic submucosal dissection (ESD) technique. Seven days after tumor resection, the treatment with OBP-301 showed a significant inhibition in LN metastasis, whereas the other control treatment did not. Moreover, LN metastatic recurrence was not detected in OBP-301-treated group even 4 weeks after tumor resection. Similar results were also observed in another model with human colon cancer Colo-205-GFP cells. These results suggest that a novel combination of ESD and OBP-301 might have potential to take place of the conventional surgical approach in treating submucosal invasive colorectal cancer. Citation Format: Naoto Hori, Satoru Kikuchi, Hiroyuki Kishimoto, Hiroshi Tazawa, Yuuri Hashimoto, Shinji Kuroda, Shunsuke Kagawa, Yasuo Urata, Robert M. Hoffman, Toshiyoshi Fujiwara. Combination strategy of endoscopic resection and telomerase-targeting oncolytic virus for eradicating lymph node metastasis of submucosal invasive colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2014-4025

Melanoma cells in vivo are well known to express varying inflammatory features, which is not limited by somatic mutational subclass. Among the most prevalent of inflammatory markers are iNOS (inducible Nitric Oxide Synthase) generated NO production and COX-2/PGE2, and for which their active pathways have been shown to enhance carcinogenesis and tumor progression, stimulate angiogenesis, support tumor growth, and promote metastasis. COX-2 inhibitors are well studied agents that inhibit tumor growth and prevent tumor development including melanoma, but the focus of research has shifted to develop inhibitors for enzymes, which are downstream of COX-2, especially microsomal PGE2 synthase-1 (mPGES-1), due to the cardiovascular risk associated with the initial COX-2 inhibitory drugs. Here, we demonstrate crosstalk between iNOS/NO and mPGES-1/PGE2 signaling pathways and suggest that mPGES-1 is a novel potential therapeutic target in human melanoma employing a spectrum of well-defined human cell lines, as well as human tissues. Our melanoma stage III survival Tissue MicroArray analysis shows that the expression of mPGES-1 is elevated in 65% of human stage III melanomas and intense expression of mPGES-1 is significantly associated with the short-term survival of patients. More interestingly, about 90 % of patient samples which express mPGES-1 were also positive for iNOS. In addition, treatment of human melanoma cells with NO donors or constitutively-driven expression of iNOS resulted in increased PGE2 production, which was suppressed by mPGES-1 inhibition, indicating that the pathway is active and targetable. Moreover, iNOS was found to interact directly with mPGES-1, and iNOS or NO donors enhance mPGES-1 activity in melanoma cells and in vitro, suggesting that iNOS-mediated NO promotes PGE2 production through mPGES-1 activation. mPGES-1 inhibition by its shRNA or specific inhibitors suppressed human melanoma cell viability and migration. Collectively, these findings suggest that the interaction between iNOS and mPGES-1 is associated with human melanoma progression and could be considered as a novel target for translational application in melanoma growth regulation. Citation Format: Sun-Hee Kim, Yuuri Hashimoto, Suhendan Ekmekcioglu, Elizabeth A. Grimm. Interaction between mPGES-1 and iNOS promotes human melanoma progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4200. doi:10.1158/1538-7445.AM2014-4200

Photodynamic therapy (PDT) is an antitumor strategy to induce tumor-specific cytotoxicity through accumulation of photosensitizers (PSs) and following light irradiation. Specific wavelength light irradiation induces the PS-mediated generation of reactive oxygen species (ROS) and subsequent cytotoxicity in tumor cells. Currently, some kinds of PSs, such as photofrin and laserphyrin, are used for PDT on clinical settings. However, the lack of sufficient and continuous accumulation of PSs in tumor tissues makes difficult to eradicate tumor cells by PDT. Therefore, the development of novel strategy for accumulation of PS in tumor tissues more efficiently and persistently is required to improve the antitumor effect of PDT. A bioengineered fluorescent protein KillerRed has been previously reported to generate ROS upon green light irradiation, suggesting a potential of KillerRed fluorescent protein as a novel PS. We recently confirmed that transient or stable transfection with KillerRed-expressing plasmid vector suppressed the cell viability through induction of ROS-mediated apoptosis in various types of human cancer cells when combined with light irradiation. In this study, we generated non-replicative adenovirus expressing KillerRed (Ad-KillerRed) and telomerase-specific conditionally replicating adenovirus expressing KillerRed (CRAd-KillerRed) to induce efficient accumulation of KillerRed in tumor cells. The in vitro antitumor effect of these viruses was investigated in human cancer H1299, HCT116 and HT29 cells in combination with light irradiation. Ad-KillerRed at high dose suppressed cell viability of human cancer cells when combined with light irradiation. In contrast, CRAd-KillerRed suppressed cell viability of human cancer cells more efficiently compared to Ad-KillerRed in combination with light irradiation. These results suggest that virus-mediated delivery system of KillerRed is a promising strategy for novel PDT. Now, in vivo experiments are under way to investigate antitumor effect of these viruses. Citation Format: Kiyoto Takehara, Hiroshi Tazawa, Yuuri Hashimoto, Hiroyuki Kishimoto, Toshiyoshi Fujiwara, Nobuhiro Narii, Hiroyuki Mizuguchi. A novel photodynamic therapy with virus-mediated delivery of photosensitive cytotoxic fluorescent protein KillerRed for human cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 706. doi:10.1158/1538-7445.AM2014-706

Cancer gene therapy and oncolytic virotherapy have been studied extensively. However, the clinical application is hampered by its weak anticancer activity. We constructed a replicating adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of the adenoviral E1 genes, could replicate in and causes selective lysis of human cancer cells. We hypothesized that combination adenoviral therapy containing OBP-301 and a non-replicating E1-deleted adenovirus expressing the proapoptotic Bax gene (Ad/Bax) could overcome the weakness and augment anticancer efficacy of each modality.

Introduction: CAFs are thought to play an essential role in cancer invasion and metastasis. We have developed the novel therapy targeting CAFs. Originally, CAFs have been defined as fibroblasts which express α-SMA in cancer tissue. As α-SMA express in cytoplasm, it can't become the target by itself. However recently it was reported that fibroblast activation protein (FAP) is functional and specific surface protein for CAFs. The aim of this study is to analyze CAFs and FAP expression of esophageal cancer and to develop the new therapy that targeted CAFs by PIT for esophageal cancer. Here we suggest a novel method of an antitumor effect by killing CAF itself. Matherials and Methods: TE-4 and TE-8 (esophageal squamous cell carcinoma), FEF3 (fetal esophageal fibroblast 3),GEP-FEF3 cell lines were used in this study. 1) The specimens of esophageal cancer were double immunostained with α-SMA and FSP1 antibodies to count CAFs. 2) In vivo study, TE cells and TE cells+activated FEF3 in the two groups were compared with tumor progression. 3) The interaction of activated FEF3 and TE cells was examined about the expression of α-SMA and FAP by western blotting and flowcytometry. 4) We examined the surface expression of FAP in activated FEF3 by immunohistochemistory(IHC). 5) We used a target-specific photosensitizer based on a near-infrared(NIR) phthalocyanine dye, IR700, conjugated to monoclonal antibodies targeting FAP. In vitro and in vivo, we observed whether cell proliferation and tumorigenicity are controlled after irradiation with NIR light in FEF3 expressing FAP conjugated FAP-IR700. Results: 1) The expression of CAFs was recognized as a deeper invasion depth, moreover the CAFs has tended to express more in the periphery and the leading parts. 2) In vivo, the tumors co-injected TE cells and CAFs tended to grow more than cancer cell alone. 3) In western blotting, FAP expression of FEF3 activated by TGF-β and co-cultured with TE cells was detected more than that of normal status.In flowcytometry, FAP expression of FEF3 activated by TGF-β, co-cultured with TE cells and cultured in TE cells conditioned medium was detected more than that of normal status. 4) In IHC, FAP expression of FEF3 activated by TGF-β and co-cultured with TE cells was increased more than that of normal status.5.We could suppress the proliferation of activated FEF3 cells in vitro with FAP-IR700 mediated PIT. And in vivo, the tumor with FAP-IR700 was also suppressed. Conclusion: In conclusion, we demonstrate that CAF contributed to tumor invasion and progression in surgical specimens and it was represented in vivo, which indicated that CAF is essential for cancer development. Furthermore, here we suggest novel therapeutic concept of “Targeting CAF by PIT” for cancer progression. The PIT for CAF is a unique strategy and can bring us a new type theory as combination targeting cancer cells and their fundamental microenvironment. Citation Format: Shinichiro Watanabe, Kazuhiro Noma, Shinichi Urano, Toshiaki Ohara, Yuuri Hashimoto, Hiroshi Tazawa, Toshiyoshi Fujiwara. Novel photoimmunotherapy (PIT) targeting cancer-associated fibroblasts (CAFs) for esophageal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4946. doi:10.1158/1538-7445.AM2013-4946

One of the biggest challenges in endoscopic therapeutic approach for early colorectal cancer with submucosal invasion is how to treat regional lymph nodes (LNs) possibly metastasized. The rate of LN metastasis in submucosal invasive colorectal cancer are estimated approximately 10%. Since endoscopic treatment can't clean LNs outside the bowel physically, open or laparoscopic surgical treatment is still gold standard for submucosal invasive colorectal cancer. We previously reported that in an orthotopic rectal tumor mouse model, a telomerase-dependent tumor killing adenoviral agent (OBP-301) spreads to the regional LNs, selectively replicates in cancer cells in metastasized LNs, and inhibits LN metastasis, when administered into the primary advanced rectal tumor. In this study we evaluated the effectiveness of novel less invasive treatment against submucosal invasive colorectal cancer, using local tumor resection such as endoscopic submucosal dissection (ESD) and OBP-301. HCT-116-GFP human colon cancer cells were orthotopically implanted into the submucosal layer of nude mouse rectum with resultant submucosal invasive early rectal tumor. LN metastasis was observed in 78.5% of mice as early as 7 days after cancer cell inoculation, and the rectal tumor of mice with LN metastasis got locally excised with prior peritumoral injection of OBP-301 solution, mimicking ESD technique. Seven days after tumor resection, treatment of OBP-301 showed a significant inhibition in LN metastasis status compared to control group. Moreover, LN metastatic recurrence was not detected in OBP-301 treated group even 4 weeks after tumor resection. The combination of ESD and OBP-301 has promising potential to take place of the conventional surgical approach in treating submucosal invasive colorectal cancer. Citation Format: Satoru Kikuchi, Hiroyuki Kishimoto, Hiroshi Tazawa, Yuuri Hashimoto, Futoshi Uno, Masahiko Nishizaki, Shunsuke Kagawa, Yasuo Urata, Robert M. Hoffman, Toshiyoshi Fujiwara. Novel strategy for eradicating lymph node metastasis of early-stage colorectal cancers using telomerase-dependent replicating adenoviral agent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1570. doi:10.1158/1538-7445.AM2013-1570

Photodynamic therapy (PDT) is an anticancer therapy to induce tumor-specific cytotoxicity by accumulation of photosensitizers (PSs) and following non-toxic light irradiation to tumor tissues. Non-toxic light irradiation induces the PS-mediated generation of reactive oxygen species (ROS) and subsequent cytotoxicity in tumor cells. For clinical application, some kinds of PSs, such as photofrin and laserphyrin, are intravenously injected before light irradiation in anticancer PDT. However, the lack of sufficient and continuous accumulation of PSs in tumor tissues makes difficult to eradicate tumor cells by PDT. Therefore, the development of novel PSs that accumulate to tumor tissues more efficiently and persistently is required to improve the antitumor effect of PDT. A bioengineered fluorescent protein KillerRed has been previously reported to generate ROS upon green light irradiation, suggesting a potential of KillerRed fluorescent protein as a novel PS. We recently confirmed that transient or stable transfection with KillerRed-expressing plasmid vector efficiently suppressed the cell viability through induction of ROS-mediated apoptosis in various types of human cancer cells when combined with light irradiation. In this study, we constructed non-replicative adenovirus expressing cytotoxic KillerRed (Ad-KillerRed) or non-cytotoxic red fluorescent protein Katushka (Ad-Katushka). The cytotoxic effect of these viruses was investigated in human cancer H1299, HCT116 and HT29 cells in combination with light irradiation. Infection with Ad-KillerRed or Ad-Katushka did not induce any cytotoxic effect in human cancer cells without light irradiation. When combined with light irradiation, human cancer cells infected with Ad-KillerRed, but not Ad-Katushka, showed morphological change like round-shape. However, the cell viability was not significantly suppressed in Ad-KillerRed-infected cancer cells after light irradiation. These results suggest that replication-competent adenovirus is necessary to efficiently induce KillerRed-mediated cytotoxic effect in human cancer cells in combination with light irradiation. Now we are planning to generate the replication-competent oncolytic adenovirus expressing KillerRed and investigate its antitumor effect on the in vitro and in vivo settings. Citation Format: Kiyoto Takehara, Hiroshi Tazawa, Yuuri Hashimoto, Hiroyuki Kishimoto, Nobuhiro Narii, Hiroyuki Mizuguchi, Toshiyoshi Fujiwara. Cytotoxic effect of photosensitive fluorescent protein KillerRed-expressing adenovirus against human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3318. doi:10.1158/1538-7445.AM2013-3318

Bone and soft tissue sarcomasare the third most common cancer in children and account for 15.4% of all childhood malignancies. About one fourth of the patients show a poor response to conventional chemo-radiotherapy, resulting in subsequent recurrence and leading to a poor prognosis. Therefore, the development of a novel therapeutic strategy is required. We previously developed an oncolytic adenovirus, OBP-301 (Telomelysin), in which the hTERT gene promoter drives the expression of the E1A and E1B genes linked to an internal ribosome entry site (IRES). Recently, we revealed that OBP-301 shows the cytopathic activity in human bone and soft tissue sarcoma cells. However, some human osteosarcoma cells (MNNG/HOS, SaOS-2) were less sensitive to cytopathic activity of OBP-301. Therefore, we developed a telomerase-specific p53-armed oncolytic adenovirus (OBP-702). In this study, we examined the antitumor effect of OBP-702 in OBP-301-resistant osteosarcoma cells, and revealed the mechanism. We compared the antitumor effects of OBP-702 and OBP-301 using XTT assay. The 50% inhibiting dose (ID50) value of OBP-702 and OBP-301 for each cell was calculated using cell viability data obtained on day 5 after virus infection. We further evaluated the expression of p53, p21 and cleaved-PARP proteins using western blot analysis in OBP-301-resistant osteosarcoma cells after infection with OBP-702 or Ad-p53, which is a p53-expressing replication-defective adenovirus. OBP-702 showed more cytopathic activity compared to OBP-301. The ID50 value of OBP-702 was much lower than that of OBP-301 in all cell lines. Western blot analysis showed OBP-702 infection induced the cleavage of PARP in a dose-dependent manner and cell cycle analysis also showed that OBP-702 induced the highest percentages of sub-G1 population in MNNG/HOS cells compared to Ad-p53 or OBP-301. These results suggest that OBP-702 induces increased apoptosis compared to Ad-p53 or OBP-301. OBP-702 infection induced more profound p53 expression than Ad-p53, but p53-downstream target p21 proteins were not activated by OBP-702. The expression levels of miR-93 and miR-106b increased after OBP-702 infection. When SaOS-2 cells were infected with Ad-p53 at an MOI of 10 for 48 hours, pre-transfection with miR-93, miR-106b or both efficiently suppressed Ad-p53-induced p21 expression. These results suggest that OBP-702 suppresses p21 expression through E1A-dependent upregulation of miR-93 and miR-106b. In conclusion, we have clearly demonstrated that the p53-expressing oncolytic adenovirus OBP-702 has a much stronger antitumor effect than OBP-301 and Ad-p53. Oncolytic adenovirus-mediated p53 gene transduction would induce profound p53 expression without p21 activation regulated by miR-93 and 106b, resulting in the enhancement of antitumor effect. Citation Format: Joe Hasei, Tsuyoshi Sasaki, Hiroshi Tazawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic adenovirus-armed p53 induces apoptosis significantly through upregulating miR-93 and 106b in human osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2013-3316

The prognosis of HER2 positive breast cancer has been improved by Trastuzumab which features high specificity and limited side effects. Numerous randomized trials have confirmed the efficacy and safety of Trastuzumab in HER2-overexpressing cancers. However, many patients who initially respond to Trastuzumab ultimately develop disease progression. Strategies to overcome Trastuzumab-refractory cancer are sought, and drug-conjugation is one of candidates. Trastuzumab-IR700 which is Trastuzumab conjugated with a near-infrared (NIR) photosensitizer, phthalocyanine dye, IR700, has recently been developed as a new type of molecular-target photoimmunotherapy(PIT). This PIT can induce target-selective necrotic cell death only at the place which have Trastuzumab's target part and where exposure of NIR light. Disappointingly, HER2-targeting therapy is applicable to only Her2-expressig tumors which consist of approximately 20-25% of primary breast cancers. In previous study, we developed the adenoviral vector to express HER2-extracellular domain (HER2-ECD), Ad/HER-ECD, and confirmed that it efficiently induced HER2 expression for HER2 negative and HER2-downregulated human cancer cells. So, we hypothesized that Ad/HER-ECD expand the range of tumor entities suitable for Trastuzumab-based PIT. By using Ad/HER-ECD and Trastuzumab-IR700 with PIT we revealed that the direct cell membrane destruction are induced at HER2 negative cancer cells, similarly at HER2 positive cancer cells, in vitro. These results suggest that combination of virally-transduction of target antigen and an antibody-based PIT would expand and potentiate target therapy even for target-negative or attenuated cancer cells. Citation Format: Michihiro Ishida, Shunsuke Kagawa, Kyoko Shimoyama, Shinichiro Watamabe, Kazuhiro Noma, Hiroshi Tazawa, Yuuri Hashimoto, Junji Matsuoka, Hisataka Kobayashi, Toshiyoshi Fujiwara. Neotargeting HER2 negative cancer cells with Trastuzumab-based photoimmunotherapy by viral transduction of HER2-extracellular domain. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3298. doi:10.1158/1538-7445.AM2013-3298

Osteosarcomas are the most common malignant bone tumors. Although the multi-agent chemotherapy has improved the long-term survival rate of osteosarcoma patients, some patients show a poor response to chemotherapy, leading to recurrence and poor prognosis. Therefore, the enhancement of chemosensitivity is required to cure patients with osteosarcomas. We recently developed a telomerase-specific replication-competent oncolytic adenovirus, Telomelysin (OBP-301), and confirmed the antitumor effect of OBP-301 in human osteosarcoma cells. A phase I clinical trial in US has also shown the safety of OBP-301 in cancer patients. In this study, we investigated whether OBP-301 enhances the antitumor effect of chemotherapeutic agents, doxorubicin and cisplatin, that are used for the treatment of osteosarcomas, and the molecular mechanism of chemotherapy- and OBP-301-mediated cell death pathway, autophagy and apoptosis, in human osteosarcoma cell lines. We used four human osteosarcoma cell lines, HOS, MNNG/HOS, 143B and SaOS2. OBP-301 is an attenuated adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter element drives expression of E1A and E1B genes, and causes tumor-selective lysis in a variety of human malignant tumor cells with high telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents and the calculation of combination index revealed the synergistic effects in all human osteosarcoma cell lines. To analyze the molecular mechanism in the synergistic effect induced by combination therapy, western blot analysis for apoptosis (PARP) and autophagy (LC3, p62) was performed. Combination of OBP-301 increased both apoptosis (the cleavage of PARP) and autophagy (conversion of LC3-I to LC3-II and p62 downregulation) in the chemotherapeutic agents-treated human osteosarcoma cells. In contrast, the replication of OBP-301 was not suppressed by chemotherapeutic agents. Moreover, combination therapy of chemotherapeutic agents with OBP-301 significantly suppressed tumor growth in a subcutaneous xenograft tumor model compared to monotherapy with chemotherapeutic agents or OBP-301. These results suggest that combination therapy of chemotherapeutic agents with OBP-301 provides a novel therapeutic strategy for human osteosarcomas. Citation Format: Shuhei Osaki, Tsuyoshi Sasaki, Hiroshi Tazawa, Joe Hasei, Yasuaki Yamakawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Enhanced chemosensitivity of osteosarcoma cells by telomerase-specific oncolytic adenovirus in combination therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 584. doi:10.1158/1538-7445.AM2013-584

[Introduction] Epithelial-Mesenchymal-Transition(EMT) plays a great role in cancer metastasis and invasion. Mechanism of EMT still remains unclear, However E-cadherin and N-cadherin are known to have contribution to EMT. In particular, down-regulation of N-cadherin has been reported to reduce cancer metastasis and invasiveness. Iron metabolism and its relationship with cancer cell were studied for a long time. Iron overload is known to induce some kinds of cancer, and iron deficiency is also known to suppress the tumor growth in vivo study. But the correlation of iron metabolism and tumor progression isn't unclear. Recently, it was reported that iron metabolism was essential for EMT and iron deficiency down-regulated EMT differentiation. In this study, we hypothesized that iron deficiency down-regulated the expression of N-cadherin, and suppressed the ability of metastasis and invasiveness. Here we demonstrate our results. [Materials and Methods] In vitro study, TE-4 and TE-10 esophageal squamous cell carcinoma and OE19 esophageal adenocarcinoma cell line were used in this study. Cell viability was determined by XTT assay. Iron chelator deferasirox was used to simulate iron deficient condition. To determine that iron deficiency suppresses EMT and invasiveness of cancer cells, we investigated western blotting analysis, three-dimensional spheroid model analysis and migration assay. Moreover, to determine the expression N-cadherin in mRNA level, PCR assay was performed. In vivo study, iron depletion condition was made by iron deficient diet. Normal and iron deficient diet were fed for 3 weeks before implantation of TE-4 cells in athymic male nude mice(n=8/group). After implantation, each diet was fed continuously for a month. [Results] In vivo study, iron deficient diet suppressed the tumor growth(tumor volume: normal diet vs iron deficient diet = 30.7±1.1 mm3 vs 10.9±2.2 mm3). In vitro study, deferasirox reduces proliferation of all cell lines in a concentration manner. According to western blotting analysis, deferasirox induces reduction of N-cadherin protein in TE-4 and TE-10 cell lines. N-cadherin down-regulation declines invasiveness of TE-4 and TE-10 cell lines in the three-dimensional spheroid model analysis and migration assay. Real time PCR assay shows that down-regulation of N-cadherin is dependent on reduction of N-cadherin mRNA. [Conclusion] Iron controlled treatment can be a novel therapy for invasiveness of cancer under EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2421. doi:1538-7445.AM2012-2421

Photodynamic therapy (PDT) is an anticancer therapy to induce tumor-specific cytotoxicity by accumulation of photosensitizers (PSs) and following non-toxic light irradiation to tumor tissues. Non-toxic light irradiation induces the PS-mediated generation of reactive oxygen species (ROS) and subsequent cytotoxicity in tumor cells. For clinical application, some kinds of PSs, such as photofrin and laserphyrin, are intravenously injected before light irradiation in anticancer PDT. However, the lack of sufficient and continuous accumulation of PSs in tumor tissues makes difficult to eradicate tumor cells by PDT. Therefore, the development of novel PSs that accumulate to tumor tissues more efficiently and persistently is required to improve the antitumor effect of PDT. Recently, a bioengineered fluorescent protein KillerRed has been reported to generate ROS upon green light irradiation, suggesting a potential of KillerRed fluorescent protein as a novel PS. In this study, we investigated the antitumor effect of KillerRed fluorescent protein in human cancer cells in combination with PDT. Transient transfection with KillerRed expression vector induced the photosensitive cytotoxicity upon green light irradiation within a few hours in human cancer HeLa and OST cells. To further evaluate the cytotoxic effect of KillerRed fluorescent protein in human cancer cells, we obtained the stable transfectants expressing cytotoxic KillerRed or non-cytotoxic Katushka fluorescent protein from human lung cancer H1299 cells. Green light irradiation induced the cytotoxic effect within one hour in H1299-KillerRed cells, but not in H1299-Katushka or H1299 cells. H1299-KillerRed cells irradiated with green light showed significantly higher levels of ROS, which was confirmed by intracellular fluorescence level of 2′,7′-Dichlorodihydrofluorescein Diacetate, than H1299-Katushka or H1299 cells. Immunocytochemistry further revealed that green light-irradiated H1299-KillerRed cells showed the apoptotic cell death with the cleavage of caspase-3 24 hours after irradiation. These results suggest that KillerRed fluorescent protein is a potent novel PS inducing ROS generation and apoptotic cell death in tumor cells in anticancer PDT. The development of virus-mediated delivery system for tumor-specific induction of KillerRed fluorescent protein is under way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5656. doi:1538-7445.AM2012-5656

[Introduction] Iron is essential for cancer to survive. Cancer cell is suppressed to proliferate under the iron depletion condition using iron chelator. However, only iron depletion can't suppress tumor growth completely. The reason is that cancer cell has abilities to escape the severe environment. These abilities are known as angiogenesis, migration, etc. Thus, to inhibit not only cancer cell proliferation but also these abilities are important for anti-cancer therapy. Bevacizumab, an antibody against vascular endothelial growth factor, is the first clinical anti-angiogenic drug. It's used for many kinds of cancer patients and revealed clinical benefit. According to spread widely in use, the resistance of Bevacizumab has been recognized. Recently, patients in resistance of Bevacizumab were reported to be high concentration of PIGF, VEGF-C and VEGF-D. On the other hand, we have demonstrated a synergistic therapeutic effect of iron depletion on anti-angiogenic therapy for human cancer in this meeting. In vitro study, iron chelator was used to simulate the iron depletion condition. Our result showed that iron depletion has anti-proliferative effect and induction of VEGF-A. Therefore, Bevacizumab revealed a synergistic therapeutic effect in iron depletion condition. In this study, we hypothesized that iron chelation therapy has anti-proliferative effect and induction of only VEGF-A(not PIGF, VEGF-C and VEGF-D). [Matherials and Methods] A549, H1299 NSCLC cell lines were used. Iron chelator deferasirox and deferoxamine were prepared to simulate iron depletion condition. Cell viability was determined by WST-1 assay. To determine induction of iron chelator, PIGF, VEGF-A, C, D ELISA assays were performed. In vivo Bevacizumab administration study, we used A549 subcutaneous tumor xenograft mice(n=5/group). Deferasirox(50mg/kg) was administrated orally 5days in a week. Bevaicuzmab(5 mg/kg) was administrated twice a week intravenously. [Results] Iron chelator suppressed cancer cell proliferation and induced only VEGF-A(not PIGF, VEGF-C and VEGF-D). VEGF-A was induced by iron chelator in dose dependent manner. Deferasirox revealed synergistic anti-tumor effect with Bevacizumab in vivo study. [Conclusion] Iron chelator contributes to anti-angiogenic therapy via selectively inducing VEGF-A. Iron chelator can be a new therapeutic strategy of Bevacizumab combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2323. doi:1538-7445.AM2012-2323

Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunocytochemistry. We recently generated a green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401, which induces ectopic GFP expression in tumor cells, but not in normal cells. In this study, we evaluated whether induction of GFP expression by OBP-401 infection is associated with CAR expression in tumor cells. OBP-401-mediated GFP induction was further examined in xenograft tumor tissues that have different levels of CAR expression and in surrounding normal tissues. We used six human sarcoma cell lines that have different levels of CAR expression and CAR-positive normal human lung fibroblasts (NHLF). Flow cytometry analysis confirmed that cells of four of the sarcoma cell lines (OST, U2OS, NOS-10 and MNNG/HOS) as well as the NHLF cells showed detectable CAR expression, whereas cells of the NMFH-1 and OUMS-27 sarcoma cell lines had no detectable CAR expression. To determine suitable conditions for OBP-401 infection, all tumor and normal cells was infected with OBP-401 at multiplicity of infections of 1, 10 and 100 plaque forming units/cell. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 hours after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. We further examined the potential of the OBP-401-mediated method for the detection of CAR expression in tumor and normal tissues, we used this method to analyze CAR expression of human xenograft tumor tissues. OBP-401 infection induced GFP expression in OST tumor tissues, but not in OUMS-27 tumor tissues or in normal muscle tissue. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4065. doi:1538-7445.AM2012-4065

Background: Epithelial-mesenchymal transition (EMT) is a tumor progression-related process by which epithelial cells lose their epithelial characteristics and acquire mesenchymal properties. EMT promotes the malignant phenotypes including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Therefore, to prevent tumor progression, EMT targeted therapy is required. Oncolytic virotherapy is a promising strategy for anticancer therapy. Although a variety of oncolytic adenoviruses have been developed to induce tumor-selective cell death, the effect of oncolytic adenovirus on the EMT-induced tumor progression remains unclear. In this study, we investigated the biological effect of oncolytic adenovirus on EMT in human cancer cells. Methods: We previously generated a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. Transforming growth factor-β (TGF-β) was used to induce EMT in human lung (A549) and pancreatic (Panc-1) cancer cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT in these cells, western blot and real-time PCR analysis for EMT-related markers were performed. We also examined cell mobility using transwell migration and invasion assays. In vitro antitumor effect of OBP-301 and chemotherapeutic agents in TGF-β-treated cancer cells were assessed by trypan blue exclusion assay. Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-like cell morphology, down-regulation of E-cadherin, up-regulation of vimentin and high motility, in A549 and Panc-1 cells. When TGF-β-treated cancer cells were infected with OBP-301, suppression of E-cadherin expression was attenuated. Conversely, the expression levels of mesenchymal markers, vimentin and N-cadherin, and EMT-inducing transcription factors, Snail, Slug and ZEB1, were reduced. OBP-301 infection also inhibited TGF-β-induced enhancement of migration and invasion in cancer cells. Moreover, OBP-301 efficiently killed TGF-β-treated cancer cells, whereas TGF-β-treated cancer cells were resistant to cisplatin and docetaxel. Conclusion: Our results suggest that telomerase-specific oncolytic adenovirus OBP-301 inhibits TGF-β-induced EMT in human cancer cells. OBP-301 has therapeutic potential against cancer cells that have undergone EMT. Therefore, OBP-301 might be a promising anticancer agent to suppress EMT-mediated cancer progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 343. doi:1538-7445.AM2012-343

We have previously reported a “true” orthotopic rectal model in which tumors start forming in the rectal mucosal tissue from which tumors actually arise. The model was generated by intrarectal transplantation of mouse rectal cancer cells stably expressing fluorescent protein, followed by disrupting the epithelial cell layer of the rectal mucosa by soaking with an acetic acid solution. Early stage tumor was detected on the rectal mucosa from as early as 6 days after transplantation, which then became invasive into the submucosal tissue. The tumor incidence on the rectal mucosa was 100%, and it was proved that this rectal tumor model reflects the natural course of rectal cancer accurately. Spontaneous lymph node metastasis and lung metastasis were observed 4 weeks after transplantation in over 90% of mice. In the present study, we will report whether this model is useful for evaluating therapeutic response of surgical local resection of tumor with combination of anticancer drugs such as a telomerase-specific replicating adenovirus designed for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2442. doi:1538-7445.AM2012-2442

Background: Malignant tumor cells often develop metastatic nodules at distant organs through circulation in blood flow and result in poor prognosis. Detection of circulating tumor cells (CTCs) is clinically an important issue to evaluate the metastatic potential of primary tumors in cancer patients. Furthermore, confirmation of genetic alterations in CTCs provides us the critical information to clarify the molecular mechanism in the acquisition of metastatic capability of tumor cells and to treat tumor metastasis by molecular targeted therapy. We recently developed a telomerase-specific replication-selective adenovirus (OBP-401; TelomeScan) expressing the green fluorescence protein (GFP). OBP-401-mediated GFP induction method was a simple and highly sensitive imaging system to detect a small population of viable CTCs in the peripheral blood. In this study, we assessed the potential of OBP-401-mediated CTC detection system to analyze the genetic alterations in CTCs. Methods: We used two types of human colon cancer cell lines SW480 and HCT116 that have the mutations in codon 12 and 13, respectively, of the K-ras gene. The detection method involved a 4-step procedure, including the lysis of red blood cells, the subsequent addition of OBP-401 to the cell pellets, the isolation of GFP-positive tumor cells using a fluorescence-activated cell sorting (FACS), and a confirmation of genetic mutations by direct sequencing. DNA was extracted from the captured cells using FACS and we investigated the mutation status in the K-ras gene by direct sequencing. Results: When only 10 cells of SW480 or HCT116 cells were added in a 5 ml of blood obtained from healthy volunteer, a total of 2-12 GFP-positive cells were captured from OBP-401-infected blood samples using FACS Aria. We confirmed the mutations in codon 12 and 13 of the K-ras gene in the FACS-captured cells from blood samples containing SW480 and HCT116 cells, respectively, by direct sequencing. Conclusion: These results suggest that OBP-401-mediated GFP induction and FACS-based capture system are useful methods to isolate small population of CTCs from peripheral blood samples and to evaluate the genetic alterations in CTCs. The combination method using OBP-401 and FACS would be a reliable strategy for the surveillance of genetic alterations in CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2385. doi:1538-7445.AM2012-2385

[Introduction] Lung cancer is one of the most aggressive malignancies with poor prognosis. Many kinds of new medicine for chemotherapy have been developed and introduced in clinical practice, whereas the mortality has not been satisfied. Furthermore, development of new medicine for chemotherapy costs a vast sum of money, which leads expensive treatment. Therefore, It's expected that development of new strategy extremely enhances the efficacy of ordinary therapeutic strategy. Iron metabolism and its relationship with cancer cell have been studied for a long time. Iron overload is known to induce some kinds of cancer, which suggests that prevention of iron overload could be a therapeutic strategy of cancer prevention. In fact, iron deficiency is also known to suppress the tumor growth in vivo study. However the efficacy is not superior to the ordinary chemotherapy, it's not a standard therapeutic strategy of cancer. In this study, we demonstrated that iron deficiency induced the suppression of the tumor growth and unwillingly up-regulation of angiogenesis in vitro and in vivo studies. Moreover using Bevacizumab together strongly suppressed the tumor growth in NSCLC. This result suggests that Iron controlled treatment can be novel therapeutic agent and biomarker of Bevacizumab. [Materials and Methods] A549, H1299 NSCLC cell lines were used. In vivo study, Normal diet and Iron deficient diet are prepared. Normal diet and Iron deficient diet were fed for 3 weeks before implantation of A549 cells in athymic male nude mice(n=8/group). In Bevacizumab administration study, as shown above, Bevaicuzmab(5 mg/kg) was administrated twice a week in each diet group. In vitro study, Iron chelator deferasirox was used in vitro study. Cell viability was determined by WST-1 assay. Cell cycle analysis was measured by flow cytometry. To determine angiogenesis via hypoxia, western blotting analysis and VEGF ELISA assay were done. [Results] Tumor growth was suppressed in iron deficient diet group (p=0.0037). Bevacizumab revealed to have an additive effect to inhibit the tumor growth. Deferasirox reduces A549 and H1299 cell proliferation in a concentration manner via cell cycle arrest. Deferasirox induces VEGF in A549 and H1299 supernatant via up-regulation of HIF1a. [Conclusion] Iron controlled treatment can be novel therapeutic agent and biomarker of Bevacizumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4247. doi:10.1158/1538-7445.AM2011-4247

[Introduction] Epithelial-Mesenchymal-transition(EMT) plays a great roles in cancer metastasis and invasion.Mecanism of EMT remains controversial, but it is recognized that some protein change their expression, including E-cadherin and N-cadherin.It is reported that downregulation of N-cadherin reduce cancer metastasis and invasiveness. Iron metabolism and its relationship with cancer cell were studied for a long time. Iron overload is known to induce some kinds of cancer, which suggests that prevention of iron overload is a therapeutic strategy of cancer prevention. Iron deficiency is also known to suppress the tumor growth in vivo study. But the efficacy is not superior to the ordinary chemotherapy, it's not a standard therapeutic strategy of cancer. Recently, it is reported that iro metabolism is essential for EMT, and iron defficiency downregulate EMT change. In this study, we demonstrated that iron deficiency downregulate the expression of the EMT protein, N-cadherin, and suppressed the ability of metastasis and invasiveness. This result suggests that Iron controlled treatment can be novel therapeutic agent for cancer under EMT. [Matherials and Methods] TE-1,TE-4,TE-8 and TE-10 esophageal squamous cell carcinoma, OE19 and OE33 esophageal adenocarcinoma cell lines were used in this study. In vitro study, cell viability was determined by XTT assay. Iron chelator deferasirox was prepared in vitro study to make iron deficient condition. To determine that iron deficiency suppresses EMT and invasiveness of cancer cell line, western blotting analysis, the three-dimensional spheroid model analysis and migration assay were done. In vivo study, Normal diet and Iron deficient diet were fed for 3 weeks before implantation of TE-4 cells in athymic male nude mice(n=8/group). After implantation, each diet was fed continually. [Results]Deferasirox reduces proliferation of all cell line in a concentration manner. According to western blotting analysis, deferasirox induces reduction of N-cadherin protein in TE-4 and TE-10 cell lines. N-cadherin downregulation declines invasiveness of TE-4 and TE-10 cell lines in the three-dimensional spheroid model analysis and migration assay. In vivo study, tumor growth was suppressed in iron deficient diet group (tumor volume: normal diet vs iron deficient diet = 30.7±1.1 vs 10.9±2.2) [Conclusion]Iron controlled treatment can be novel therapeutic agent for invasiveness of cancer under EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2011-3363

Human epidermal growth factor receptor 2 (HER2) is a member of a family of receptors associated with tumor cell proliferation, apoptosis, adhesion, migration, and differentiation. According to numerous preclinical and clinical studies, HER2 can be immunogenic and antibodies against this transmembrane receptor have become in use in individuals with HER2-overexpressing tumors. Trastuzumab, a monoclonal antibody that targets HER2, induces antibody-dependent cellular cytotoxicity (ADCC) and inhibits HER2-mediated signaling. Numerous randomized trials have confirmed the utility of trastuzumab in HER2-overexpressing breast cancer in various clinical scenarios; however, HER2-overexpression has been identified only in approximately 25-30% of primary breast cancers. Moreover, it has been reported that the majority of metastatic breast cancer patients treated with trastuzumab develop acquired resistance within a year. In this study, we analyzed the mechanism of resistance to trastuzumab and examined for molecular sensitization by exogenous expression of HER2 extracellular domain in human breast cancer cells. A long-term exposure to trastuzumab induced its resistance in HER2-positive breast cancer cells. We found that trastuzumab-resistant cells exhibited down-regulation of HER2 expression in cell surface and impaired ADCC activity, suggesting that trastuzumab-resistant cells could be re-sensitized to trastuzumab by exogenous induction of extracellular domain (ECD) of HER2.We constructed the expression vector with HER2-ECD and stably transfected into low HER2-expressing human breast cancer cell line, MCF7. HER2-ECD expression had no apparent effect on the growth and signaling pathway in MCF7 cells; however, ADCC activity was strongly enhanced in HER2-ECD-expressing MCF7 cells compared to parental MCF7 cells. These results suggest that exogenous expression of HER2-ECD might be an appropriate strategy to sensitize HER2-negative or trastuzumab-resistant human breast cancer cells to trastuzumab. The modification of the tumor-specific replication-competent adenovirus (OBP-301, Telomelysin) to contain the HER2-ECD gene for exogenous expression is currently under the investigation. We will further examine the antitumor effects of this virus in the combination with trastuzumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2011-720

Background: Circulating tumor cells (CTCs) in the peripheral blood are considered as a useful biomarker for disease outcome and treatment response because the presence of CTCs is associated with short survival. We previously established the biological imaging system to detect viable CTCs among millions of peripheral blood leukocytes using telomerase-specific replication-selective adenovirus expressing green fluorescent protein (GFP) (Kojima et al, J Clin Invest, 2010). In present study, we demonstrate the clinical potential of our method for monitoring the efficacy of treatment in patients with gastric cancer. Methods: We used a GFP-expressing adenovirus variant, in which the telomerase promoter regulates viral replication (OBP-401), to selectively label human tumor cell with fluorescence. Whole blood cells collected from 7.5ml of peripheral blood were infected OBP-401 after elimination of anti-adenovirus antibodies and incubated at 37 oC. Viruses were inactivated 24 hours after virus-infection and then red blood cells were lysed. The GFP-expressing cells were detected using fluorescence microscopy. Results: A total of 72 blood samples from 39 patients with histologically-confirmed gastric cancer (stage IA to IV) were analyzed. CTCs were detected in 32 of 39 patients (82%, range 1 to 40 Conclusion: Our data suggest that patients with early gastric cancer also have CTCs in peripheral blood and the dynamic change of CTCs correlate with treatment response. Although numerous samples and long-term outcome should be analyzed, the enumeration of CTC using this GFP-expressing virus-based method might be useful for monitoring the efficacy of treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2011-3160

18 Background: The presence of circulating tumor cells (CTC) in the peripheral blood is associated with short survival and, therefore, the detection of CTC is clinically useful as prognostic factors of disease outcome and/or surrogate markers of treatment response. Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made possible to detect a few CTC in the blood; however, there is no sensitive assay for detecting viable CTC. We developed a new approach to visually detect live CTC among millions of peripheral blood leukocytes using telomerase-specific replication-selective adenovirus expressing green fluorescent protein (GFP). Methods: We constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan). The detection method for viable human CTC in the peripheral blood involves a three-step procedure including the lysis of red blood cells, the subsequent addition of OBP-401 to the cell pellets, and the automated scan under the fluorescent microscope. We analyzed fresh blood samples collected from 37 patients with histologically confirmed gastric cancer. We further assessed the CTC dynamics in patients who were undergoing chemotherapy or surgery to demonstrate the clinical potential of our approach for monitoring treatment responses. Results: OBP-401 increased the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal can be amplified only in viable human tumor cells by viral replication. Although the CTC level varied widely, ranging from 0 to 47 in 5-ml samples, 26 gastric cancer patients (70.3%) had more than one CTC; there was, however, no apparent relationship between CTC counts and TNM stages. Patients who had a recurrence of gastric cancer had decreased CTC counts after systemic chemotherapy. In the patients who underwent surgery, the CTC level dropped after complete resection. Conclusions: This GFP-expressing virus-based method is simple and allows precise enumeration of CTC, which might be useful for monitoring the efficacy of local and systemic treatments. [Table: see text]

e13626 Background: Cancer stem cells (CSCs) are established firmly to play a key role in tumor formation, maintenance, and resistance to the conventional therapies. This resistance is reported to d...

3572 Background: Telomerase activation is considered to be a critical step in carcinogenesis and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We constructed an adenovirus 5 vector (OBP-301 [Telomelysin]), in which the hTERT promoter drives expression of E1A and E1B genes linked with an IRES. OBP-301 causes selective replication and lysis of a variety of human cancer cells, and has shown preclinical efficacy against human tumor xenografts. A phase I study was designed to determine the feasibility and to characterize the pharmacokinetics of OBP-301 in patients with advanced solid tumors. Methods: Patients with histologically confirmed advanced solid tumors were enrolled into this phase I trial. Nine patients received escalating dose levels of OBP-301 (1 x 1010 to 1 x 1012 virus particles [vp]) as monotherapy. Patients were monitored over two months for safety and tolerability, vector distribution, antibody formation, and tumor response. Virus shedding wa...

Background: We previously reported that an attenuated adenovirus (OBP-301, 'Telomelysin'), in which the hTERT promoter element drives expression of E1A and E1B genes linked with an IRES, could replicate in cancer cells, and causes selective lysis of cancer cells. In this study, we further investigated the virus replication and antitumor activity of Telomelysin in various human cancer cell lines, and also established the assay to determine the concentration of Telomelysin in circulating blood. Methods: We designed the specific primer targeted for E1 gene or IRES sequence for real-time quantitative PCR (Q-PCR), and then analyzed its potent of quantity and sensitivity. We also analyzed the correlation between hTERT mRNA expression and viral replication of Telomelysin in human cancer cells. CAR expression was determined by flow cytometry, and cell killing effect of Telomelysin in 13 human cancer cell lines and normal human fibroblasts was examined by XTT assay. Results: By Q-PCR, the minimum detection limit was 100 plaque-forming units (PFU)/ml of Telomelysin in the plasma, using the primer for IRES. A positive correlation tendency was observed between hTERT mRNA expression and viral replication of Telomelysin in human cancer cells. Telomelysin killed all human cancer cell lines dose-dependently, but not normal human fibroblasts. Average of ID50 after three days of infection was 12.1 multiplicity of infection (MOI). Conclusion: Our data suggested that Telomelysin may be useful for treatment of various human cancer cells, and telomerase activity is a marker for Telomelysin replication in cancer cells.

Gene therapy was investigated as a promising treatment for cancer. However, low transduction efficiency limits the efficacy of gene therapy in clinics. We previously demonstrated that telomerase- specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase (hTERT) promotor element drives expression of E1A and E1B genes linked with an IRES, induced selective E1 expression and efficient antitumor effect in human cancer cells, but not normal human fibroblasts. In this study, we evaluated the anticancer effect of the combination therapy of E1-deleted replication-deficient adenovirus expressing the wild-type p53 tumor suppressor gene (ADVEXIN, Ad-p53) and OBP-301 in human H1299 non-small cell lung cancer cells and SW620 colon cancer cells by using cell proliferation assay, immunoblotting, and cell-cycle analysis. We found that E1-deficient Ad-p53 could replicate in human cancer cells in the presence of OBP-301 and kill infected cells more efficiently than Ad-p53 alone or OBP-301 alone. Western Blotting Analysis demonstrated that OBP-301 plus Ad-p53 induced high levels of p53 protein expression, leading to cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). In addition, flowcytometry showed that the sub-G1 population was dramatically increased with OBP-301 and Ad-p53 infection. The in vivo combination effect of intratumoral injection of Ad-p53 and OBP-301 in a human tumor xenograft model will be also reported. Our data demonstrate that OBP-301 in combination with Ad-p53 efficiently enhances antitumor activity, and provide the potential to affect the development of oncolytic virotherapy and gene therapy.