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Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease with a 5-year survival rate of less than 10%. Although immune checkpoint blockade has recently emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of tumor-infiltrating T cells. Recently, oncolytic virotherapy has been shown to stimulate the immune system as an immunogenic antitumor therapy. Activation of p53 has been also known to enhance antitumor immunity. In this study, we investigated the potential of telomerases-specific p53-expressing oncolytic adenovirus (OBP-702, Pfifeteloxin) for inducing the immunogenic cell death in PDAC cells. Methods: OBP-702 (Pfifteloxin) is a telomerase-specific oncolytic adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter drives the expression of the viral E1 gene for tumor-specific replication, that expresses the wild-type p53 by inserting the human p53 cDNA at the deleted E3. OBP-301 (Telomelysin) is an original telomerase-specific adenovirus lacking p53. OBP-502 is an RGD mutant fiber-containing OBP-301. We used 4 human PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1) and mouse PDAC cell line (Pan02). In vitro antitumor effect of these viruses was evaluated using a XTT assay. The molecular mechanism of virus-mediated cell death was investigated by western blot analysis. The virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1) using ELISA assay. The comparative in vivo antitumor efficacy was also investigated using a syngeneic Pan02 subcutaneous tumor model. Results: Telomerase-specific oncolytic adenoviruses induced the antitumor effect in all PDAC cells in a dose-dependent manner. The antitumor effect of OBP-702 was superior to that of OBP-301 or OBP-502. OBP-301 and OBP-502 mainly induced autophagy, whereas OBP-702 induced autophagy and apoptosis at 72 h after infection. The concentration of extracellular ATP and HMGB1 was significantly increased in OBP-702-infected PDAC cells compared to OBP-301- or OBP-502-infected cells at 24 and 48 h after infection. In mice carrying subcutaneous Pan02 murine PDAC tumors, intratumoral injection of OBP-702 resulted in a significant inhibition of tumor growth. Moreover, the number of tumor-infiltrating CD8+ T cells was significantly increased in OBP-702-treated groups compared to mock-treated group. Conclusion: Our data suggest that telomerases-specific p53-expressing oncolytic adenovirus OBP-702 (Pfifteloxin) induces profound immunogenic cell death to boost the immune responses in PDAC. Clinical trials with OBP-702 and immune checkpoint inhibitors for PDAC are warranted. Citation Format: Hiroyuki Araki, Hiroshi Tazawa, Takuro Fushimi, Takeyoshi Nishiyama, Satotu Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Boosting immunity against pancreatic cancer by OBP-702 (Pfifteloxin), telomerase-specific replicative adenovirus armed with wild-type p53 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4852.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease due to early onset of local recurrence and distant metastasis. Invasive PDAC cells are the main causes of recurrence and metastasis after curative surgery. The precision medicine based on the genetic alterations in the KRAS, p53, CDKN2A, and SMAD4 genes has been recently expected as a promising strategy for the treatment of PDAC patients; however, therapeutic strategy for targeting these genetic alterations in PDAC has not been developed yet. To eliminate p53-inactivated malignant tumor cells, we have developed telomerase-specific replication-competent oncolytic adenovirus OBP-702 that expresses tumor suppressor p53. In this study, we investigated the in vitro and in vivo therapeutic potential of OBP-702 against PDAC cells. Methods: Four human PDAC cell lines (Capan-1, MIA PaCa-2, BxPC-3, Panc-1) with different invasive property were used. The therapeutic effect of OBP-702 and p53-nonexpressing OBP-301 was assessed in the proliferation, migration and invasion abilities of PDAC cells. The underlying mechanism of virus-mediated therapeutic effect was analyzed on the modulation of p53 signaling and KRAS-MAPK signaling. Subcutaneous and orthotopic BxPC-3 xenograft tumor models were used to evaluate the virus-mediated antitumor efficacy. Results: OBP-702 induced antitumor effect in association with autophagy and apoptosis more strongly compared to OBP-301 in PDAC cells through activation of p53 expression. OBP-702 inhibited the migration and invasion properties of PDAC cells more efficiently compared to OBP-301 through suppression of KRAS-ERK1/2 signaling, even when ERK signaling is enhanced by nerves and neurosecretory factors. Similar with OBP-702, treatment with ERK1/2 inhibitor or siRNA significantly reduced migration and invasion abilities. Moreover, OBP-702 significantly suppressed tumor growth in subcutaneous and orthotopic BxPC-3 xenograft tumor models. Conclusions: Our results suggest that OBP-702 is a promising antitumor reagent to eliminate invasive PDAC cells through p53 activation and ERK suppression. Further clinical study is warranted to evaluate the safety and feasibility of OBP-702 as a novel precision medicine based on genetic alterations in KRAS and p53 genes. Citation Format: Takuro Fushimi, Hiroshi Tazawa, Takeshi Koujima, Hiroyuki Araki, Takeyoshi Nishiyama, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Elimination of invasive pancreatic cancer cells by p53-activating oncolytic virotherapy as novel precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4823.

Background: The abscopal effect is a phenomenon that shrinkage of metastatic tumors occurs concurrently with shrinkage of primary tumors by local treatments such as radiotherapy, and is generally considered to be caused through activation of the immune system. Extracellular vesicles (EVs) are recent interesting topics in scientific fields and are known to play important roles in a variety of intercellular communication processes including immune response. We previously developed a telomerase specific oncolytic adenovirus, Telomelysin (OBP-301), which is currently being tested in clinical trials, and have sometimes observed therapeutic effects on metastatic tumors after local injection to primary tumors in immunodeficient mouse models. Here, we hypothesized that EVs derived from OBP-301-treated tumors may be associated with the abscopal effect of OBP-301. Methods: A human colon carcinoma cell line HCT116 and HCT116 expressing RFP (HCT116-RFP) were used in this study. EVs were isolated by ultracentrifugation from HCT116 cells treated with OBP-301 (Exo-301). Intercellular communication and cytotoxic activity via EVs after OBP-301 treatment were assessed in a non-contact co-culture system in vitro and a bilateral subcutaneous tumor mouse model in vivo. Results: Exo-301 showed potent cytotoxic effects on HCT116 cells and this cytotoxicity was significantly blocked in the presence of anti-CD9 antibody, which suggested that Exo-301, not OBP-301 itself, was involved in this cytotoxicity. In non-contact co-culture experiments, more RFP was taken up into HCT116 cells after OBP-301 treatment on HCT116-RFP cells than control treatments, which meant that EVs including RFP reached HCT116 cells through membrane after OBP-301 treatment on HCT116-RFP cells. In a HCT116 bilateral subcutaneous tumor model in BALB/c nude mice, intratumoral injection of OBP-301 significantly suppressed the growth of untreated tumors as well as treated tumors, and in a bilateral tumor model with HCT116 on one side and HCT116-RFP on the opposite side, immunohistochemical staining showed the presence of RFP in untreated HCT116 tumors after OBP-301 treatment on HCT116-RFP tumors. These findings suggested that EVs derived from OBP-301-treated tumors was possibly involved in some cytotoxic effects on the opposite untreated tumors. Conclusion: EVs may be an important factor in the abscopal effect brought by local treatment of OBP-301 besides immune activation. Citation Format: Yoshihiko Kakiuchi, Shinji Kuroda, Nobuhiko Kanaya, Kento Kumon, Tomoko Tsumura, Satoru Kikuchi, Masahiko Nishizaki, Shunsuke Kagawa, Hiroshi Tazawa, Yasuo Urata, Toshiyoshi Fujiwara. Involvement of extracellular vesicles in the abscopal effect induced by oncolytic adenovirotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4851.

Background: OBP-301 (telomelysin) is an attenuated type-5 adenovirus with oncolytic potency that contains the human telomerase reverse transcriptase (hTERT) promoter to regulate viral replication. OBP-301 causes selective replication and lysis of a variety of cancer cells, and a phase I study has confirmed the safety and biological activity of OBP-301 alone in patients with advanced solid tumors in the US. We have also completed the preclinical study demonstrating that OBP-301 inhibits the repair of radiation-induced DNA double-strand breaks, leading to radiosensitization. To further determine the feasibility, efficacy, and pharmacokinetics of OBP-301 in combination with radiotherapy, a clinical trial was designed in esophageal cancer patients unfit for standard treatments such as in elderly.Methods: An open-label, phase I dose-escalation study of OBP-301 with radiotherapy (UMIN 000010158) was conducted in 13 histologically confirmed esophageal cancer patients who deemed unfit to receive standard surgery or chemotherapy. Study treatment consisted of intratumoral OBP-301 injections on days 1, 18, and 32 of treatments. Radiation therapy was administered concurrently over 6 weeks, beginning on day 4, to a total of 60 Gy. Virus administration was performed by intratumoral needle injection of the primary tumor through a flexible endoscope. Patients were also monitored for virus distribution and shedding into the body fluids. The primary and secondary end points were incidence of dose-limiting toxicities and objective response rate.Results: Of 13 patients, 7, 3, and 3 patients were treated in the cohorts with 10e10, 10e11, and 10e12 virus particles (vp) of OBP-301, respectively. The patients comprised 10 males and 3 females, with median age of 79.7 years (range, 53 to 92 years). Common grade 1 and 2 toxicities included fever, esophagitis, pneumonitis, anorexia, constipation, and gastroesophageal reflux. All patients developed a transient, self-limited lymphopenia. Distribution studies revealed that viral DNA was detected in 5 out of 6 patents received with10e11 and 10e12 vp of OBP-301, but rarely in the gargle, saliva, and urine. Eight patients had local complete response (CR); all of them exhibited pathologically no viable malignant cells in biopsy specimens, and 3 had partial response. The objective response rate was 84.6%. The clinical CR rate was 80.0% in stage I and 66.7% in stage II/III, respectively. Histopathologic examination in post-treatment specimens showed massive infiltration of CD8+ cells in 3 partially responded tumors.Conclusions: Multiple courses of endoscopic OBP-301 injection with radiotherapy were feasible and provided definite clinical benefits in patients with esophageal cancer, especially who are unfit for standard treatments. Citation Format: Toshiyoshi Fujiwara, Shunsuke Tanabe, Hiroshi Tazawa, Nobuhiko Kanaya, Kazuhiro Noma, Shunsuke Kagawa, Shinji Kuroda, Yasuo Urata, Yasuhiro Shirakawa. Phase I dose-escalation study of endoscopic intratumoral injection of OBP-301 (telomelysin) with radiotherapy in esophageal cancer patients unfit for standard treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT185.

Background: Peritoneal metastasis is the most frequent form of distant metastasis and recurrence in gastric cancer and its prognosis is extremely poor. A promising result in phase III trial using intraperitoneal (i.p.) paclitaxel (PTX) for the peritoneal metastasis of gastric cancer has been reported in recent year; however, it is not sufficiently effective to eradicate disseminated gastric cancer. In this study, we investigated whether novel i.p. oncolytic virotherapy could be synthetically lethal with PTX for the peritoneal metastasis of gastric cancer. Methods: OBP-401 (TelomeScan) is a green fluorescence protein (GFP)-expressing attenuated adenovirus with oncolytic potency that is driven by telomerase promoter. We assessed its synergistic effect with PTX using human gastric cancer cell lines (GCIY and KATO III) and xenograft peritoneal metastasis model. The mechanism underlying the synergistic antitumor effect was evaluated by in vitro viral replication, induction of mitotic catastrophe, and in vivo viral penetration into disseminated nodules. Results: OBP-401 synergistically suppressed the viability of human gastric cancer cells in combination with PTX. PTX was synthetically lethal with OBP-401 by enhancing viral replication ability in cancer cells and the combination therapy increased mitotic catastrophe induction, resulted in accelerating autophagy and apoptosis. Peritoneally disseminated nodules were selectively visualized as GFP-positive spots by the i.p. administration of OBP-401 in the orthotopic human gastric cancer peritoneal dissemination model. And, PTX enhanced the penetration of OBP-401 deeply into the disseminated nodules. Moreover, non-invasive in vivo imaging system (IVIS) demonstrated that the combination therapy of i.p. OBP-401 administration with PTX significantly inhibited tumor growth of peritoneal metastasis and the amount of malignant ascites. Conclusions: Intraperitoneal virotherapy with PTX may be a promising treatment strategy for the peritoneal metastasis of gastric cancer. Clinical trials to obtain the proof of concept are warranted in peritoneally disseminated gastric cancer patients. Citation Format: Toshihiro Ogawa, Satoru Kikuchi, Wataru Ishikawa, Hiroshi Tazawa, Motoyasu Tabuchi, Shinji Kuroda, Kazuhiro Noma, Masahiko Nishizaki, Shunsuke Kagawa, Yasuo Urata, Toshiyoshi Fujiwara. Boosting replication and penetration of telomerase-specific replicative virus by paclitaxel induces synthetic lethality in peritoneal metastasis of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2979.

While trastuzumab (Tmab), a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2), has greatly contributed to improve survival outcomes in metastatic gastric cancer, an issue of concern is that no current HER2-targeted therapeutic agent is effective against Tmab-resistant gastric cancer. Nanotechnology, which has progressed rapidly in recent years, greatly contributes to progress in medical fields including cancer therapy, and especially, gold nanoparticles (AuNPs) are attention-grabbing nanomaterials that are promising drug carriers with unique properties of high in vivo stability and a large surface area available for attachment of materials such as antibodies. Methods: We created HER2-targeted AuNPs by conjugating Tmab onto their surface (T-AuNPs) and examined their therapeutic efficacy and cytotoxic mechanisms using HER2-postive Tmab-resistant (MKN7) or Tmab-sensitive (NCI-N87) gastric cancer cell lines in vitro and in vivo. We also examined T-AuNPs on HER2-negative MKN74 gastric cancer cells with adenoviral vector-mediated overexpression of the HER2 extracellular domain (HER2-ECD). Results: The size of T-AuNPs was 85.39 ± 0.68 nm and the surface was negatively charged with 39.43 ± 0.85 mV. In in vitro assays, T-AuNPs showed 6-fold higher cytotoxic activity than Tmab against NCI-N87 cells. Then, T-AuNPs showed significantly stronger cytotoxic effects than controls against both MKN7 and NCI-N87 cells although Tmab had no effect on MKN7 cells. Autophagy was proven to play an important role in T-AuNPs cytotoxic mechanisms. This autophagy was considered to be induced by HER2-dependent internalization of T-AuNPs, some of which were interestingly located in the cytoplasm independent of lysosomal encapsulation. Although T-AuNPs were not cytotoxic towards MKN74 cells, they became cytotoxic following HER2-ECD overexpression. Finally, Intratumoral injection of T-AuNPs showed potent antitumor effects, which were mediated by autophagy, against NCI-N87 and MKN7 subcutaneous tumors in vivo mouse models. Conclusion: HER2-targeted AuNPs with conjugated Tmab can be a promising strategy for the development of novel therapeutic agents to overcome Tmab resistance in gastric cancer. Citation Format: Kento Kumon, Tetsushi Kubota, Shinji Kuroda, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Tomoko Tsumura, Satoru Kikuchi, Shunsuke Kagawa, Hiroshi Tazawa, Toshiyoshi Fujiwara. Trastuzumab-conjugated gold nanoparticles as novel HER2-targeted therapeutics against trastuzumab-resistant gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3617.

Although the neutrophil extracellular traps (NETs) are first recognized to function to trap invading microorganisms and eliminate them as a defense mechanism, the involvement of NETs in cancer metastasis is being elucidated. The mechanism of interaction between NET and cancer cells, however, remains to be fully understood. Here, we investigated the direct and indirect interaction between NETs and cancer. NETs were detected in surgically resected metastatic liver cancer tissue specimens. In direct co-culture, NETs actually attracted cancer cells with their web-like structure. Of note, indirect transwell co-culture of neutrophils and pancreatic cancer cells provoked NETs without the other stimuli. In addition, conditioned media derived from cancer cells also induced NETs. The co-culture of NETs and cancer cells dramatically accelerated their migration and invasion abilities. Further investigation revealed that NETs induced mesenchymal markers in cancer cells which was associated with upregulated epithelial to mesenchymal transition (EMT)-related transcriptional factors. Finally, the accelerated cancer migration and overexpression of mesenchymal markers induced via NETs were inhibited by pharmacological blockade of NETs including NADPH oxidase inhibitor, peptidylarginine deiminase inhibitor, and neutrophil elastase inhibitor. These results suggested that cancer cells and neutrophils reciprocally interact when they coexist and NETs drive malignant potentials via EMT, implicating NETs as a candidate therapeutic target. Citation Format: Hiroki Kajioka, Shunsuke Kagawa, Atene Ito, Kazuya Kuwada, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. The reciprocal interaction between neutrophil extracellular traps and cancer cells impacts on their malignant potentials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4519.

Background: Telomelysin (OBP-301) is a telomerase-specific oncolytic adenovirus, in which the telomerase promoter controls its replication and induction of cell death in human tumor cells. A FDA-approved phase I study has confirmed the safety and biological activity of intratumoral administration of Telomelysin in patients with advanced solid tumors in the US. Anti-programmed death-1 antibody (PD-1 Ab) has dramatically improved clinical outcomes for patients with melanoma or lung cancer; however, the clinical benefit is limited to small population of patients with certain degree of tumor infiltrating lymphocytes (TILs). Therefore, more effective strategy to enhance the immunogenicity of non-immunogenic tumors is needed. Here, we show the potential of our oncolytic adenovirus as an immunogenic agent sensitizing non-immunogenic gastrointestinal tumors to PD-1 Ab. Methods: A murine colon cancer cell line CT26 (BALB/c) and a murine pancreatic cancer cell line PAN02 (C57BL/6) were used in this study. An OBP-301 variant (OBP-502) that expresses the RGD mutant fiber to infect murine tumor cells via interaction with integrin was used as an oncolytic adenovirus. PD-1 Ab (clone 4H2) was obtained from Ono Pharmaceutical Co. Ltd. As immunogenic cell death markers, extracellular ATP and high mobility group box 1 (HMGB1) levels were measured following OBP-502 infection. In vivo subcutaneous tumor models, CD8-positive TILs, vaccination efficiency of OBP-502-treated cells and combination therapy of OBP-502 with PD-1 Ab were evaluated. Results: OBP-502 efficinetly killed CT26 and PAN02 cells, and also significantly increased extracellular ATP and HMGB1 on both cell lines in vitro, suggesting that OBP-502-induced cell death might be immunogenic. Indeed, in the CT26 and PAN02 subcutaneous tumor models, OBP-502 induced massive accumulation of TILs compared to control. Vaccination of mice with OBP-502-infected CT26 cells rejected tumors in three of 10 vaccinated mice (30%) and also significantly suppressed the growth of remaining seven CT26 tumors. The combination therapy of OBP-502 and PD-1 Ab significantly suppressed the growth of CT26 subcutaneous tumors and successfully eradicated tumors in four of 12 mice (33%) while either single treatment failed to eradicate in most mice. When these 4 tumor-free mice treated with the combination therapy were re-challenged by inoculation of CT26 cells, two of 4 mice (50%) remained tumor-free. Similar antitumor effects of the combination therapy were observed in the PAN02 tumor model.H.E staining of major organs harvested after each treatment demonstrated no serious systemic toxicity that PD-1 Ab has been reported to cause sometimes in clinical practice. Conclusion: Our telomerase-specific oncolytic adenovirus has the potential to activate antitumor immunity, leading to sensitization of non-immunogenic gastrointestinal tumors to PD-1 Ab. Citation Format: Nobuhiko Kanaya, Shinji Kuroda, Toshiaki Morihiro, Yoshihiko Kakiuchi, Tetsushi Kubota, Satoru Kakiuchi, Masahiko Nishizaki, Yasuo Urata, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Telomelysin-induced immunogenic cell death synergizes with anti-PD-1 antibody in non-immunogenic gastrointestinal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2744.

Background: Scirrhous gastric cancer (SGC), which is characterized by poorly differentiated tumor cells that diffusely infiltrate the gastric wall, accounts for 10% of all gastric cancers, and often causes peritoneal metastasis. The prognosis of SGC patients is very poor, because no effective treatment to overcome peritoneal metastasis of SGC has been developed yet. In recent years, intraperitoneal (i.p.) administration of paclitaxel (PTX) has been focused against various cancers with peritoneal metastasis and has been also attempted to SGC, but regrettably its effect is not satisfactory. Therefore, a novel therapeutic strategy is required for the treatment of SGC with peritoneal metastasis. Methods: We previously developed a telomerase-specific replication-competent adenovirus, OBP-401 (TelomeScan), which can replicate only within the tumor cells selectively and express green fluorescent protein (GFP). OBP-401 infection also induces tumor-specific cell death in monotherapy and combination therapy with chemotherapy. In this study, we assessed the in vitro and in vivo antitumor effects of combination therapy with OBP-401 and chemotherapeutic agent, paclitaxel (PTX), in human SGC cell lines (GCIY and KATO III) and SGC xenograft peritoneal metastasis model, respectively. The molecular mechanism underlying synergistic effect of the combination therapy was evaluated in aspects of cell death and replication efficiency of OBP-401. Results: SGC cells were visualized as GFP-positive cells selectively by the infection of OBP-401, whereas normal fibroblast cells were not. OBP-401 synergistically suppressed the viability of human SGC cells in combination with PTX. OBP-401 monotherapy dose-dependently induced apoptosis and autophagy, although PTX monotherapy induced only apoptosis. The combination therapy induced autophagy at a lower concentration of OBP-401. In the time-lapse imaging of treatment, the combination therapy enhanced the replication efficiency of OBP-401 in tumor cells. Moreover, the combination therapy with i.p. administration of OBP-401 and PTX also significantly inhibited the tumor growth of peritoneal metastasis, as compared to monotherapy. Conclusion: These results suggest that OBP-401 has a promising potential to detect peritoneal micrometastasis of SGC intraoperatively and the combination therapy with i.p. induction of OBP-401 and PTX would be a novel theranostic strategy for the treatment of SGC with peritoneal metastasis. Citation Format: Wataru Ishikawa, Satoru Kikuchi, Hiroshi Tazawa, Toshiaki Ohara, Shinji Kuroda, Kazuhiro Noma, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Novel theranostic strategy against peritoneal metastasis of scirrhous gastric cancer: Combination with fluorescence oncolytic adenovirus and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4807.

Background: Epithelial-mesenchymal transition (EMT) is a biologic process by which epithelial cancer cells acquire mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancer (CRC). However, the precise role of inflammatory microenvironment in the EMT-related tumor progression remains unclear because EMT network is regulated by complex crosstalk between tumor and normal cells. In this study, we developed a fluorescence-guided live-cell imaging system for the spatiotemporal visualization of EMT state, and assessed the in vitro and in vivo visualization of EMT state in human CRC cells under the inflammatory microenvironment. Methods: We used two human CRC cell lines, HCT116 and RKO. The promoter of mesenchymal marker vimentin was used for inducing the EMT-dependent red fluorescence protein (RFP) expression in human CRC cells. The RFP expression was quantitatively analyzed to evaluate its association with EMT-related markers and malignant phenotype. Inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or co-culture with inflammatory mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide (LPS) (200 ng/mL) were used as an inflammatory microenvironment. Biodistribution of EMT cells was analyzed using in vivo experiments with primary and metastatic CRC xenograft tumors. Results: Inflammatory cytokines reversely induced the RFP expression in association with EMT-related malignant phenotype, such as morphologic change, EMT marker modulation, and invasion ability, in HCT116 and RKO cells. Co-culture with LPS-stimulated inflammatory macrophages also induced the RFP expression by secreting inflammatory cytokines. In vivo experiments revealed that EMT cells were detected within tumor tissues at colon, liver and peritoneum. Conclusions: Our results suggest that inflammatory microenvironment has a great potential to induce EMT program in CRC cells. This fluorescence-guided EMT imaging system is a useful tool for monitoring the EMT state in the inflammation microenvironment-CRC network in vitro and in vivo. Citation Format: Hiroshi Tazawa, Takeshi Ieda, Shuya Yano, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Masahiko Nishizaki, Shunsuke Kagawa, Takashi Saitou, Takeshi Imamura, Toshiyoshi Fujiwara. Visualization of epithelial-mesenchymal transition in inflammatory microenvironment-colorectal cancer network in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1101.

Although gastric cancer often develops peritoneal dissemination, the underlying mechanism of peritoneal metastasis is still unclear. Extracellular vesicles (EVs) are known to play roles in cell-cell communication. Here, we investigated the existence of EVs in peritoneal ascites of gastric cancer patients and explored their roles in the intraperitoneal cancer microenvironment. We found that EVs actually existed in peritoneal ascites of advanced gastric cancer patients. On the other hand, immune-fluorescent assay of the ascites by using antibodies against CD14 (Monocytes) and CD45 (Leukocytes) revealed that many macrophages existed along with the cancer cells in the peritoneal cavity. EVs purified from peritoneal ascites with cancer dissemination made peripheral blood mononuclear cells-derived macrophages M2-like phenotype which was confirmed by their morphology and expression of CD163/206 (M2 marker). Next, we purified EVs from the culture medium of gastric cancer cell lines. Gastric cancer-derived EVs induced the M2- differentiation in macrophages and the differentiated macrophages promoted migration ability of gastric cancer cells. Finally, we observed that gastric cancer-derived EVs induced apoptosis of cultured mesothelial cells in vitro. These results suggested that EVs in the ascites of gastric cancer patients are possibly derived from gastric cancer and play critical roles in the intraperitoneal cancer microenvironment by affecting the macrophages phenotype and inducing apoptosis in the mesothelial cells, which would be in part the underlying mechanism of peritoneal metastasis. Citation Format: Atene Ito, shunsuke kagawa, Shuichi Sakamoto, Kazuya Kuwada, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. Extracellular vesicles in the ascites from gastric cancer patients play critical roles to promote peritoneal dissemination in the intraperitoneal cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5200.

Background: Gastric cancer is one of the most common cancers worldwide, especially in Eastern Asia. Scirrhous gastric cancer (SGC), which is characterized by poorly differentiated tumor cells that diffusely infiltrate the gastric wall, accounts for 10% of all gastric cancers, and often causes peritoneal metastasis. Peritoneal carcinomatosis shows the worst prognosis among various metastatic patterns. Since it is difficult to detect peritoneal micrometastasis preoperatively or intraoperatively, SGC patients with peritoneal micrometastasis show the recurrence and refractory to conventional systemic chemotherapy after surgery. In recent year, intraperitoneal (i.p.) administration of paclitaxel (PTX) has been shown to have therapeutic effects against ovarian cancer with peritoneal metastasis. However, the effective treatment option to overcome peritoneal metastasis of SGC has not been developed yet, although i.p. administration of some chemotherapeutic agents has been attempted to SGC. Therefore, a novel therapeutic strategy is required for the treatment of peritoneal metastasis of SGC. Methods: We previously developed a telomerase-dependent replication-selective adenovirus OBP-401 (TelomeScan), which can replicate within the tumor cells selectively and express green fluorescent protein (GFP). Moreover, OBP-401 infection also induces tumor-specific cell death in monotherapy or combination therapy with chemotherapy. In this study, we assessed synergistic effects in combination with novel theranostic agent OBP-401 and chemotherapeutic agent paclitaxel (PTX) using human SGC cell lines (GCIY and KATO III). In vitro experiment, SGC cells and normal human lung fibroblast (NHLF) cells were infected with OBP-401 at various doses. PTX was administrated after 48 hours of virus infection. Twenty-four hours after PTX administration, we evaluated the anti-tumor effect by XTT assay, and analyzed the synergistic effect by CalcuSyn Software. Results: SGC cells were visualized as GFP-positive cells selectively by the infection of OBP-401, whereas NHLF cells were not visualized with OBP-401. In both SGC cell lines, the suppression of cell viability dose-dependently by administration of OBP-401 or PTX alone was confirmed. OBP-401 synergistically suppressed the viability of SGC cells in combination with PTX as compared to monotherapy. Conclusions: These results suggest that OBP-401 has a promising potential to detect peritoneal micrometastasis of SGC intraoperatively and combination of OBP-401 and PTX would be a novel theranostic strategy for the treatment of peritoneal metastasis of SGC. Citation Format: Wataru Ishikawa, Satoru Kikuchi, Hiroshi Tazawa, Shinji Kuroda, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. Novel theranostic strategy against scirrhous gastric cancer; combination of chemotherapy and fluorescence oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1103. doi:10.1158/1538-7445.AM2017-1103

Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancer. However, the role of inflammatory microenvironment in the EMT-related tumor progression remains unclear. To explore the relationship between inflammatory microenvironment and EMT, a live imaging system for EMT is a promising strategy on the in vitro and in vivo experiments. In this study, we developed a fluorescence-guided live cell imaging system for the assessment of spatiotemporal dynamics of EMT, and investigated the potential of inflammatory microenvironment for the induction of EMT phenotype in human colorectal cancer. Methods: Two human colorectal cancer cell lines, HCT116 and RKO, were stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. Both cell lines were treated with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or co-cultured with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis and q-PCR. EMT-induced HCT116 and RKO cells were treated with anti-inflammatory agents, aspirin (1mM) and salicylic acid (1 mM), for the suppression of EMT. Results: Inflammatory cytokines (IL-1β and TNF-α) induced red fluorescence intensity and morphological change like mesenchymal phenotype in HCT116 and RKO cells. Removal of inflammatory cytokines attenuated red fluorescence intensity and morphological change in both cells. Inflammatory cytokines also induced the migration and invasion properties in association with EMT-related markers. Moreover, co-culture with LPS-stimulated inflammatory macrophages also induced red fluorescence intensity and morphological change as well as inflammatory cytokines. Anti-inflammatory agents significantly suppressed the fluorescence-related EMT phenotype under inflammatory microenvironment. Conclusions: These results suggest that inflammatory microenvironment has a great potential for the induction of EMT process during colorectal cancer progression. This unique fluorescence-guided EMT imaging system is useful method for the exploration of inflammation-mediated tumor progression. Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Takeshi Imamura, Toshiyoshi Fujiwara. Fluorescence-guided spatiotemporal dynamics of epithelial-mesenchymal transition under inflammatory microenvironment during colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5809. doi:10.1158/1538-7445.AM2017-5809

Interaction of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) induces functional impairment of antigen-specific T cells, which leads to immune evasion of tumors. Immune checkpoint therapy including PD-1/PD-L1 blockade is an emerging treatment strategy which brings great improvement in patient’s prognosis. Microsatellite instability (MSI) is a condition of genomic instability caused by the loss of DNA mismatch repair activity, which has recently received a lot of attention from the standpoint of cancer immunotherapy. While the TNM classification system is commonly used on various malignant tumors for prediction of prognosis and planning of treatment strategy, establishment of prognostic factors related to cancer immune system is considered important as cancer immunotherapy has recently become more and more popular. Thus, we investigated the usefulness of PD-L1 expression and MSI status in addition to histological type as prognostic factors in gastric cancer. In this study, 255 gastric cancer cases which had curative surgical resection at Okayama University Hospital between 2002 and 2009 were analyzed retrospectively. PD-L1 expression level was classified into 4 groups according to PD-L1 positive rate on tumors on immunohistochemical staining (0: < 1%, 1+: 1-5%, 2+: 5-10%, 3+: ≥10%), and 2+ and 3+ were defined as PD-L1 positive. MSI status was analyzed by PCR and classified into MSI-high (MSI-H), and MSI-low and Microsatellite stability (non-MSI). Histological type was divided into intestinal and diffuse according to the Lauren classification.PD-L1 positive rate was 15%, and PD-L1 positive was significantly correlated with advanced stage and invasive status. Overall survival (p=0.0109) and disease-free survival (p=0.0052) were significantly poor in PD-L1 positive group, and a multivariate analysis revealed that PD-L1 expression was an independent risk factor for recurrence after surgery (p=0.0160, odds ratio: 3.19). MSI-H was detected in 7.5%, and more observed in PD-L1 positive cases (p=0.0068). Although non-MSI, as a single factor, showed only a weak tendency to have poor prognosis compared to MSI-H (p=0.1965), combination of PD-L1 positive and non-MSI (PD-L1/non-MSI) showed significantly poor prognosis (p=0.0010). Although other combinations also showed the usefulness in detection of patients with poor prognosis, such as PD-L1 positive and diffuse type, and non-MSI and diffuse type, a multivariate analysis revealed that PD-L1/non-MSI was the most useful combination among them to predict poor prognosis (p=0.0009, hazard ratio: 3.88). In conclusion, while PD-L1 expression is a useful prognostic factor and predictive factor for recurrence after surgery as a single factor, combination with MSI status can be more useful and attractive prognostic factor in gastric cancer. Citation Format: Toshiaki Morihiro, Shinji Kuroda, Nobuhiko Kanaya, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Combination of PD-L1 expression and microsatellite instability status is a useful prognostic factor in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4717. doi:10.1158/1538-7445.AM2017-4717

Gastric cancer is the third leading cause of cancer-related death, and peritoneal dissemination is one of the most frequent site of recurrence. Although the role of tumor microenvironment is recognized, it is still unclear how intraperitoneal cancer-immune microenvironment contributes to peritoneal metastasis. Thus, we investigated the interaction between intraperitoneal cancer and immune cells, especially focusing on tumor-associated macrophages (TAM) in the peritoneal cavity. The peritoneal wash from gastric cancer patients was subjected to conventional cytology, flow cytometry, and immuno-fluorescent assay using antibodies against CD45 (leukocytes), CD14 (monocytes), CD80/163 (M1/M2 macrophages, respectively), in combination with genetically engineered cancer-imaging viral agent ‘TelomeScan’ which specifically expresses GFP in telomerase-positive cells. In cytology-positive samples, CD163-positive macrophages could be detected in approximately 70% of intraperitoneal macrophages with TelomeScan-positive cancer cells, while cytology negative samples contained few CD163-positive macrophages. In addition, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were induced to differentiate into CD163-positive M2 macrophages (TAM-like macrophages), and co-cultured with gastric cancer cells to examine the effect on their malignant phenotype such as migration and invasion. We found that PBMCs-derived CD163-positive M2 macrophages significantly potentiated the ability of migration and invasion in gastric cancer cells. In case of existence of free cancer cells in peritoneal cavity, intraperitoneal macrophages appeared to differentiate into TAM and they potentially accelerate malignant phenotypes of gastric cancer cells. These results suggested that intraperitoneal cancer-immune microenvironment may play an important role to promote peritoneal disseminations in gastric cancer. Citation Format: Shuichi Sakamoto, Shunsuke Kagawa, Kazuya Kuwada, Atene Ito, Tetsuya Kagawa, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. Tumor associated macrophages promote malignant phenotypes of disseminated human gastric cancer cells in intraperitoneal cancer immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2017-5011

Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. More than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis at the time of diagnosis. Moreover, even after curative surgery, PDAC patients still have poor prognosis due to local recurrence and systemic metastasis. Although activation of oncogenic KRAS is implicated in the initiation, promotion and progression of pancreatic cancer, the development of cancer treatment targeting KRAS remains unsuccessful. Moreover, tumor suppressor p53 is also frequently inactivated in PDAC patients. We recently developed two types of tumor-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702, which is modified OBP-301 to express tumor suppressor p53 protein. In this study, we investigated whether oncolytic adenoviruses inhibit cell viability, migration, invasion and KRAS-driven signaling pathway in PDAC cells. Methods: We used 4 PDAC cells (MIA PaCa-2, Capan-1, Panc-1, BxPC-3). The cell viability was examined by XTT assay. Migration and invasion properties were assessed using transwell chamber assay. The modulation of KRAS-driven signaling pathway was investigated by Western blot analysis. Results: OBP-301 induced moderate antitumor effect in MIA Paca-2, Capan-1 and BxPC-3 cells and strong antitumor effect in Panc-1 cells in a dose-dependent manner, whereas OBP-702 induced profound antitumor effect in all human PDAC cells, suggesting the broad spectrum of OBP-702's efficacy. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-301 and OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells, and the inhibitory effect of OBP-702 was stronger than that of OBP-301. We also found that oncolytic adenoviruses inhibited the expression of KRAS and KRAS-downstream target MEK/ERK proteins in high-invasive type PDAC cells. Conclusions: Our results suggest that oncolytic adenoviruses, OBP-301 and OBP-702, inhibit the survival and invasive phenotype of pancreatic cancer by suppressing KRAS-driven signaling pathway and reactivating p53-driven signaling pathway. In vivo experiments are under way to investigate the anti-tumor and anti-invasive effects of oncolytic adenoviruses using BxPC-3 xenograft tumors. The clinical trial of intratumoral administration of oncolytic adenoviruses in patients with invasive PDAC should be also warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Takeshi Ieda, Kazuya Kuwada, Terutaka Tanimoto, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3748.

Interaction of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) induces functional impairment of antigen-specific T cells, which leads to immune evasion of tumors. Immune checkpoint therapy including PD-1/PD-L1 blockade is an emerging treatment strategy which brings great improvement in patient's prognosis. Many reports have mentioned that PD-L1 expression on tumors correlates with poor prognosis in various types of cancers. While there is a few report about correlation between PD-L1 expression and prognosis in gastric cancer, one of the most common cancers in the world especially in Asia, it still remains controversial. In addition, almost no report has referred to the correlation of PD-L1 expression with metastatic or recurrence pattern of cancers. Thus, we investigated the correlation of PD-L1 expression with prognosis and recurrence pattern in gastric cancer patients. In this study, 255 gastric cancer cases which had curative surgical resection at Okayama University Hospital between 2002 and 2009 were analyzed retrospectively in terms of correlation of PD-L1 expression on tumors with patient's prognosis and recurrence pattern in addition to clinicophathological features. When PD-L1 expression was classified into 4 groups according to PD-L1 positive rate on tumors on immunohistochemical staining (0: < 1%, 1+: 1-5%, 2+: 5-10%, 3+: ≥10%) and 2+ and 3+ were defined as PD-L1 positive, PD-L1 positive rate was 15% (37/255). PD-L1 positive cases had significant correlation with advanced stage (p = 0.0424), lymphatic invasion (p = 0.0028) and venous invasion (p = 0.0254), and tendency to be more observed in intestinal type than diffuse type (p = 0.0599) in terms of histological types of cancer. Overall survival (p = 0.0109) and disease-free survival (p = 0.0052) were significantly poor in PD-L1 positive group. Moreover, PD-L1 expression had significant negative correlation with peritoneal dissemination (p = 0.0081) and had tendency to cause hematogenous recurrence (p = 0.1002) in terms of recurrence pattern after curative surgical resection, which might be reflected by the correlation of PD-L1 expression and histological types of cancer. Multivariate analysis revealed that PD-L1 expression was an independent risk factor for recurrence (p = 0.0160, odds ratio: 3.19), along with diffuse type and advanced stage. In conclusion, PD-L1 expression on tumors strongly correlated with poor prognosis and negatively correlated with peritoneal dissemination in recurrence pattern in gastric cancer patients who had curative surgical resection. Citation Format: TOSHIAKI MORIHIRO, Shinji Kuroda, Tetsushi Kubota, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. PD-L1 expression as a predictive factor for recurrence pattern and prognosis in curatively resected gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2649.

Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire the mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. However, as EMT is a reversible process and depends on tumor microenvironment, the precise role of EMT in cancer progression remains unclear. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancers. To evaluate the implication of EMT in the inflammation-mediated colorectal cancer progression, a live imaging system for EMT is needed on the in vitro and in vivo experiments. In this study, we generated the EMT-detectable colorectal cancer cells by introducing mesenchymal cell marker promoter-driven fluorescence protein expression vector, and investigated whether inflammation-induced EMT is detectable in vitro. Methods: To generate the EMT-detectable colorectal cancer cells HCT116-VIM635, human colorectal cancer cell line HCT116 was stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. EMT was induced in HCT116-VIM635 cells by treatment with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or by co-culture with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis. Results: Administration of IL-1β or TNF-α induced the TurboFP635 expression in consistent with morphological change like mesenchymal phenotype in HCT116-VIM635 cells. Removal of these inflammatory cytokines attenuated the TurboFP635 expression and morphological change in HCT116-VIM635 cells, suggesting the detection of reverse EMT process in these cells. Inflammatory cytokines also induced the migration and invasion properties and the expression of EMT-related markers in HCT116-VIM635 cells. Moreover, co-culture with RAW264.7 cells stimulated with LPS also induced the TurboFP635 expression and morphological change as well as inflammatory cytokines in HCT116-VIM635 cells. Conclusions: Our results suggest that this unique live imaging system for EMT has a great potential to detect reversible EMT process during inflammation-induced cancer progression. This system would be useful strategy to assess the role of EMT during inflammation-mediated cancer progression in vivo. Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel live imaging system for inflammation-induced epithelial-mesenchymal transition in colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1613.

Objectives: Postoperative recurrence occurs in approximately 80% of pancreatic cancer, and the peritoneum is the second most frequent metastatic site next to the liver. Recent evidence suggests that cancer microenvironment plays key roles in metastasis. To investigate mechanism of intraperitoneal metastasis in pancreatic cancer, we tried to detect cancer cell in the peritoneal wash from pancreatic cancer patients and analyze intraperitoneal cancer microenvironment. A genetically engineered adenovirus, TelomeScan, which replicates and expresses GFP only in telomerase-active cancer cells, was employed to detect cancer cells, and it enabled us to distinguish cancer cell from co-existence cells in the abdominal cavity. We explored the role of tumor-associated macrophages (TAMs) by using this virus-guided fluorescent imaging system. Methods: Peritoneal wash was obtained from 20 pancreatic cancer patients during operation. The cells in the wash were infected with TelomeScan for 24 hours. Samples from cases with TelomeScan-expressing GFP-positive cells were further subjected to immunofluorescence assay for analysis of microenvironment. The antibodies against CD45, CD14, and CD204 were used in immunostaining as markers of leukocytes, monocytes and TAMs, respectively. To investigate the effect of TAMs on pancreatic cancer, Panc1 and BxPC-3 cell lines were co-cultured with PMA (phorbol 12-myristate 13-acetate)-treated monocytes and analyzed their changes. Results: The three out of 20 cases were positive in cytology, and GFP positive cells were detected after TelomeScan infection in those cases. In the wash, pancreatic cancer cells existed together with many leukocytes including macrophages. Further analysis demonstrated that those macrophages were TAMs. After Panc1 or BxPC-3 cells were co-cultured with TAMs, E-cadherin was decreased in Panc1, α-SMA and Vimentin was increased in BxPC-3. These results suggested that Epithelial to Mesenchymal Transition (EMT) was induced in pancreatic cancer cells by TAMs. Conclusion: Pancreatic cancer cells and TAMs were detected in the peritoneal wash using TelomeScan and immunostaining. The results suggested that EMT induced by TAMs may promote intraperitoneal metastasis of pancreatic cancer. Citation Format: Kazuya Kuwada, Shunsuke Kagawa, Megumi Watanabe, Shuichi Sakamoto, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Tetuya Kagawa, Toshiyoshi Fujiwara. Functional analysis of tumor-associated macrophage utilizing virus-guided fluorescent imaging of pancreatic cancer cells in the peritoneal cavity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4156.

Targeted therapies utilizing monoclonal antibodies for malignant tumors has been increasingly developed and applied to clinical practice. Trastuzumab (Tmab) is a humanized monoclonal antibody which binds to the human epidermal growth receptor 2 (HER2), which leads to strong antitumor effects by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and inhibiting HER2 signaling pathway. While Tmab is clinically applied to HER2-positive breast and gastric cancers, there still remain some issues such as low HER2 expression (only 20%) and resistance to Tmab. Recent progress in nanotechnology has brought significant benefits to medical fields. Gold nanoparticles (AuNPs), marked by the in vivo stability and easy surface functionalization, have been developed as therapeutic and contrast agents in the medical field, and some AuNPs reportedly show some cytotoxicity by inducing autophagy and apoptosis, which indicates that AuNPs could be attractive therapeutic agents in combination with tumor-targeting antibodies. In this study, we created Tmab-conjugated AuNPs and evaluated the antitumor effects on HER2-positive but Tmab-resistant gastric cancer cells (MKN7) and HER2-negative gastric cancer cells (MKN74) in addition to HER2-positive and Tmab-sensitive esophagogastric cancer cells (NCI-N87, TE4, OE19) in vitro and in vivo. IC50 dose of Tmab-AuNPs was 6.5 times smaller than free Tmab on NCI-N87 cells. Tmab-AuNPs exhibited stronger antitumor effects on MKN7, NCI-N87, TE4 and OE19, HER2-positive cells, than the other controls on XTT assay, but not such strong effects on MKN74 and NHLF, HER2-negative cells. Notably, Tmab-AuNPs showed potent antitumor effects even on MKN7, HER2-positive but Tmab-resistant cells. Moreover, once HER2 was overexpressed on MKN74 cells by Ad-HER2/ECD, Tmab-AuNPs became effective on artificially-HER2-overexpressed MKN74 cells. More effective intracellular uptake of Tmab-AuNPs was observed by dark field microscopy and transmission electron microscopy on HER2-positive cells, which seemed to cause stronger induction of oxidative stress, autophagy and apoptosis. Finally, stronger antitumor effects of Tmab-AuNPs were also confirmed in mouse subcutaneous xenograft models using HER2-positive cells. In conclusion, Tmab-AuNPs demonstrated potent antitumor effects on Tmab-resistant cells as well as Tmab-sensitive cells, and if combined with HER2/ECD overexpression, Tmab-AuNPs became effective even on HER2-negative cells. These findings suggest that Tmab-AuNPs could be promising HER2-targeted nanodrugs which were able to overcome shortcomings in Tmab-based therapy. Citation Format: Tetsushi Kubota, Shinji Kuroda, Toshiaki Morihiro, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Novel HER2-targeted gold nanoparticles; integration of antibody therapy and nanotechnology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4747.

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. Cancer Res; 70(22); 9339–48. ©2010 AACR.

Objectives: In patients with gastric cancer, peritoneal dissemination is the most common metastasis. To predict future peritoneal recurrences, peritoneal lavage cytology is performed during operation. But even cytology-negative patients sometimes develop peritoneal recurrences. Thus an additional method to detect intraperitoneal free gastric cancer cells is necessary. We have developed a genetically engineered adenovirus, TelomeScan, which replicates and expresses GFP only in telomerase-activated cancer cells. Here we detected intraperitoneal free gastric cancer cells using TelomeScan, and investigated the correlation between the number of GFP-positive cells and patient prognosis. Methods: Peritoneal wash was obtained from 69 gastric cancer patients during operation. The cells in the wash were infected with TelomeScan for 24 hours. Finally, GFP-positive cells were counted under a fluorescence microscope. In some GFP-positive cases immunofluorescence assay was added. Clinicopathological data were obtained from medical records. Then we examined different cut-off values (the number of GFP-positive cells which indicates TelomeScan-positive) and estimated survival curves using the Kaplan-Meier method, and compared using the Wilcoxon test. Results: For a cut-off value of 10, 25 of the 69 cases were TelomeScan-positive (10 or more GFP-positive cells). And these 25 cases showed the most significant worse prognosis when compared to the 44 TelomeScan-negative cases (p = 0.0040). In addition, 17 of the 69 cases were conventional cytology-positive. Of these 17 cases, 9 were TelomeScan-positive, and these 9 cases showed significantly worse prognosis than the 8 TelomeScan-negative conventional cytology-positive cases (p = 0.0017, MST 195 days). Under fluorescence microscope we observed that GFP-positive cells sometimes formed cell clusters with GFP-negative cells. Immunofluorescence assay showed that these GFP-negative cells expressed CD45, which means these cells were leukocytes. Conclusion: We have successfully detected cancer cells in peritoneal wash using TelomeScan. The presence of GFP-positive cells in peritoneal wash was associated with worse prognosis. TelomeScan-positive patients, especially in conventional cytology-positive cases, showed remarkably worse prognosis than TelomeScan-negative conventional cytology-positive patients. Our data suggest that TelomeScan-guided cytological detection may have clinical implications as a prognostic biomarker in gastric cancer. Citation Format: Megumi Watanabe, Shunsuke Kagawa, Kazuya Kuwata, Michihiro Ishida, Yuuri Hashimoto, Naoto Hori, Satoru Kikuchi, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Hiroshi Tazawa, Yasuo Urata, Toshiyoshi Fujiwara. Virus-guided fluorescence imaging of intraperitoneal free gastric cancer cells: a preliminary clinical study as a potential clinical biomarker. [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 3412. doi:10.1158/1538-7445.AM2015-3412

Background: Iron overload is known to cause cancer in animal models. Several studies have shown that iron deprivation treatment has a strong anti-cancer effect. However, it is unclear whether iron deprivation treatment suppresses cancer stem cells or not. A cancer stem cell model, miPS-LLCcm, was recently epigenetically established from murine induced pluripotent stem cells (miPS cells) in Okayama University. In this model, the green fluorescent protein (GFP) and the puromycin resistant gene were inserted into the 59-bp untranslated region of the Nanog gene of miPS cells. By this procedure, undifferentiated cancer progenitor cells are recognized as GFP positive cells. We then examined the iron dependency of these cancer stem cells both in in vitro and in vivo studies and we used this model to examine the possibility of cancer stem cells by iron deprivation. Materials and Methods: In in vitro studies, the miPS-LLCcm cells were used as cancer stem cells and colon26 and 4T1 cells were used as differentiated murine cancer cells. Puromycin was used to purify the cancer stem cells before seeding of the miPS-LLCcm cells. The dependency on iron for cell proliferation was examined following transferrin addition. Transferrin (Holo) was used to simulate an iron rich condition. Iron free medium and the iron chelators, Deferasirox and Deferoxamine, plus 1% fetal bovine serum (FBS) were used to simulate iron depletion conditions. Cell proliferation assays and flow cytometric analyses were performed 48 hours after adjustment of the iron concentration level. In in vivo studies, an iron depleted diet was used to simulate iron depleted conditions. Nude mice were divided into normal diet and iron depleted diet groups. The mice were fed with these diets for three weeks and then a suspension of miPS-LLCcm cells was injected into the backs of the nude mice. Tumor size was measured and the tumors were immunohistologically examined. Results: In the in vitro studies, transferrin strongly promoted the proliferation of cancer stem cells under iron depletion conditions compared to no transferrin(p Conclusions: Iron is a key element for the proliferation and differentiation of cancer stem cells. Iron controlling therapy including iron chelators is a novel therapeutic target of cancer stem cells. Citation Format: Takayuki Ninomiya, Toshiaki Ohara, Hajime Kashima, Ryoichi Katsube, Kazuhiro Noma, Yasuko Tomono, Akifumi Mizutani, Tomonari Kasai, Masaharu Seno, Shinji Kuroda, Hiroyuki Kishimoto, Hiroshi Tazawa, Yasuhiro Shirakawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Iron control is a novel therapeutic target of cancer stem cells. [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 4243. doi:10.1158/1538-7445.AM2015-4243

Colorectal cancers harboring KRAS or BRAF mutations often show resistance to anti-cancer drugs, and are poor prognostic disease that causes metastasis and recurrence compared to those with wild type KRAS and BRAF. KRAS/BRAF-wild type colorectal cancers are sensitive to both cetuximab and panitumumab that are epidermal growth factor receptor (EGFR)-targeting agents. However, KRAS/BRAF-mutant colorectal cancers are resistant to EGFR-targeting agents because of constitutive activation of the EGFR-downstream RAS/RAF/MEK signaling pathway. Therefore, the development of novel therapeutic strategy is required to improve the clinical outcome in patients with KRAS/BRAF-mutant colorectal cancers. We developed two types of telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702 armed with the wild-type p53 tumor suppressor gene. In this study, we established the companion diagnostics-based selection algorithm for our oncolytic viruses. We compared the antitumor effects of OBP-301 and OBP-702 in human colorectal cancer cells with wild type KRAS/BRAF (SW48, Colo320DM), mutated KRAS (DLD-1, SW620) and mutated BRAF (RKO, HT29). We evaluated the antitumor effect of OBP-301 and OBP-702 in human colorectal cancer cells using XTT assay. Oncolytic adenovirus-mediated induction of apoptosis- and autophagy-related cell death was analyzed by Western blot analysis. KRAS-mutant colorectal cancer cells were sensitive to both OBP-301 and OBP-702 as well as KRAS/BRAF-wild type cells. In contrast, BRAF-mutant colorectal cancer cells were sensitive to OBP-702 rather than OBP-301. Western blot analysis showed that OBP-301 induced autophagy, whereas OBP-702 induced both autophagy and apoptosis. Taken together, our data suggest that oncolytic viruses should be used according to the genetic background of the RAS/RAF/MEK signaling pathway, especially in patients with colorectal cancer. Citation Format: Shuta Tamura, Hiroshi Tazawa, Naoto Hori, Takeshi Koujima, Satoru Kikuchi, Shinji Kuroda, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Companion diagnostics-based telomerase-specific oncolytic virotherapy: preclinical evaluation in human colorectal cancer cell lines differentially affected in the RAS/RAF/MEK signaling pathway. [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 3531. doi:10.1158/1538-7445.AM2015-3531

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

Pancreatic cancer is one of the worst prognosis disease all over the world and has an overall 5-year survival rate of less than 5%. At the time of diagnosis, more than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis. Moreover, even after curative surgery, pancreatic cancers still show poor prognosis due to local recurrence and systemic metastasis. Gemcitabine is the standard chemotherapeutic agent to treat advanced pancreatic cancers, but median overall survival ranged from 5.0 to 7.2 months, suggesting that standard chemotherapy is less effective to most of pancreatic cancer patients. Therefore, the development of novel therapeutic strategy is needed for the treatment of pancreatic cancers. We developed a novel oncolytic adenovirus, OBP-702, in which a human telomerase reverse transcriptase gene promoter drives viral E1 gene for virus replication, and armed with the wild-type tumor suppressor p53 gene. Precise antitumor effects of OBP-702 have been evaluated in human pancreatic cancer cells. We used four pancreatic cancer cell lines with different invasion ability, including non-invasive type (MIA PaCa-2, Panc-1) and invasive type (BxPC-3, Capan-1). The antitumor effect of OBP-702 for each cell line was assessed using XTT assay. OBP-702 induced profound anti-tumor effect in all human pancreatic cancer cells, whereas OBP-301 lacking the p53 tumor suppressor gene showed differential effects among the cell types. OBP-301 induced moderate anti-tumor effect in MIA Paca-2, BxPC-3 and Capan-1 cells and strong anti-tumor effect in Panc-1 cells in a dose-dependent manner, suggesting the broad spectrum of OBP-702's efficacy. The molecular mechanism of anti-tumor effect was assessed by Western blot analysis. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. These results suggest that an oncolytic adenovirus, OBP-702, is a promising antitumor agent to induce profound cell death in invasive human pancreatic cancer cells. The clinical trial of intratumoral administration of OBP-702 in patients with invasive pancreatic cancer is warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Naoto Hori, Shuta Tamura, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel tumor-specific oncolytic biological therapy against invasive pancreatic cancer. [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 3535. doi:10.1158/1538-7445.AM2015-3535

Background: Antibody-targeted therapy for malignant tumors has been increasingly developed and applied to clinical practice in recent years. Trastuzumab (Tmab) is a humanized monoclonal antibody that binds to the human epidermal growth receptor 2 (HER2) and interferes with its signal transduction leading to tumor progression, and is currently used for patients with breast cancer and gastric cancer. In inoperable recurrent or metastatic gastric cancers, HER2 is reportedly overexpressed in approximately 20% of cases, and Tmab contributes to prolongation of survival for these patients at some level. However, there still remain some issues to be overcome such as limited application of Tmab (only 20%) and limited treatment options for these advanced gastric cancers. Recent progress in nanotechnology is remarkable and various nanomaterials have been developed for medical applications. Gold nanoparticles (AuNPs) have been tested as therapeutic and contrast agents in the medical field, which are stable in vivo and easy to conjugate antibodies, proteins or peptides to the surface. Moreover, reportedly AuNPs show antitumor effect by induction to autophagy and apoptosis. These characteristics make AuNPs more attractive as molecular targeted agents. In this study, we created HER2-targeted AuNPs by conjugating Tmab on the surface of AuNPs, and examined antitumor effects on HER2-positive or negative gastric cancer cells in vitro. Methods: HER2-positive human gastric cancer cell lines (MKN7, NBI-N87), HER2-negative human gastric cancer cell lines (MKN45, MKN74) and normal human lung fibroblast (NHLF) were used in this study. The antitumor effects of Tmab-conjugated AuNPs (Tmab-AuNPs), PEGylated AuNPs (PEG-AuNPs), Tmab only (antibody amount is presumably equivalent to antibody attached on the surface of Tmab-AuNPs), mixture of PEG-AuNPs and Tmab (PEG-AuNPs + Tmab), and PBS were evaluated by XTT assay. Cellular uptake of AuNPs was observed on dark field microscopy and electron microscopy. HER2 expression was examined by western blotting and flow cytometry. Results: Dark field microscopy and electron microscopy showed that Tmab-AuNPs were taken up into cells more effectively on MKN7 and NBI-N87 cells (HER2-positive) than PEG-AuNPs and PEG-AuNPs + Tmab. XTT assay showed that Tmab-AuNPs exhibited a stronger antitumor effect on MKN7 and NBI-N87 cells (HER2-positive) than the other control treatments, but not such a strong effect on MKN45 and MKN74 cells (HER2-negative) and NHLF. The antitumor effect of Tmab-AuNPs on NBI-N87 cells was achieved by more than twice as much as Tmab, which means that Tmab-AuNPs enables to reduce the amount of Tmab to less than half. Conclusions: Tmab-AuNPs proved potent and HER2-dependent antitumor effects on human gastric cancer cells in vitro. Tmab-AuNPs could have the potential to provide more effective and less toxic treatment option for HER2-positive gastric cancer patients. Citation Format: Tetsushi Kubota, Shinji Kuroda, Katsuyuki Aoyama, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. HER2-targeted gold nanoparticles produce potent antitumor effects on human gastric cancer cells. [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 5520. doi:10.1158/1538-7445.AM2015-5520

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. OBP-301 causes selective replication and lysis of a variety of human cancer cells, and also inhibits the repair of radiation-induced DNA double-strand breaks, leading to radiosensitization. A phase I study has confirmed the safety and biological activity of intratumoral administration of OBP-301 alone in patients with advanced solid tumors in the United States. To further determine the feasibility, efficacy, and pharmacokinetics of OBP-301 in combination with radiotherapy, a phase I/II study was designed in elderly patients with esophageal cancer. Methods: Patients with histologically confirmed esophageal cancer who were not eligible for standard treatments such as surgery and chemotherapy were enrolled into this study (UMIN000010158). Study treatment consisted of intratumoral needle injections of OBP-301 on days 1, 18, and 32 of treatment. Radiation therapy was administered concurrently over 6 weeks, beginning on day 4, to a total of 60 Gy. Virus administration was performed by intratumoral injection of the primary or metastatic tumor through a flexible endoscope. OBP-301 doses will be escalated initially in cohorts of two for the first 9 patients (1 × 10e10 and 1 × 10e11 virus particles [vp]). Six subsequent patients will receive the highest dose (1 × 10e12 vp). Virus shedding will be monitored in the saliva, sputum, urine, and plasma by a quantitative DNA-PCR assay. Results: Six patients were enrolled and treated in the cohort with 1 × 10e10 vp of OBP-301. The patients comprised 4 males and 2 females, with median age of 83.5 years (range, 68 to 92 years). Only two patients had prior platinum-based chemotherapy. By November 2014, 3 patients completed treatment. All patients developed a transient, self-limited lymphopenia. A 92-year-old female showed a grade 4 lymphopenia classified as being possibly related to the treatment, although it recovered by the interruption of radiation. No other virus-related toxicities were noted. Objective responses were complete response (CR) in 2 patients and partial response (PR) with tumor regression, resulting in reopening of the esophagus, in 1 patient. Pathological analysis in biopsy specimens obtained from completely responded patients demonstrated no viable malignant cells for 3 to 5 months after the treatment completion. Conclusions: Multiple courses of endoscopic OBP-301 injection in combination with locoregional radiotherapy were feasible and well tolerated in elderly patients with esophageal cancer, and appeared to provide clinical benefit. Citation Format: Shunsuke Tanabe, Hiroshi Tazawa, Shunsuke Kagawa, Kazuhiro Noma, Kiyoto Takehara, Takeshi Koujima, Hajime Kashima, Takuya Kato, Shinji Kuroda, Satoru Kikuchi, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Phase I/II trial of endoscopic intratumoral administration of OBP-301, a novel telomerase-specific oncolytic virus, with radiation in elderly esophageal cancer patients. [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 CT123. doi:10.1158/1538-7445.AM2015-CT123

Background: Oncolytic virotherapy is an emerging treatment strategy that allows to destroy cancer tissues only but not normal tissues. We previously developed a telomerase-specific oncolytic adenovirus (Telomelysin) whose replication is controlled by the human telomerase reverse tran¬scriptase (hTERT) promoter, and the clinical safety and pharmacokinetics of Telomelysin has been already evaluated in the phase I clinical trial conducted in the US. While oncolytic adenoviruses can produce promising treatment efficacy, their application is basically limited to locoregional administration because of nonspecific trapping in the liver and neutralization by immune response after systemic delivery. Nano-sized materials have been identified as effective and attractive drug carriers in the fields of cancer treatment and diagnosis. Liposome is one of the most popular and widely-used drug carriers. In this study, we investigated the possibility of systemic delivery of telomerase-specific oncolytic adenoviral plasmid DNA via lipid encapsulation as an alternative approach to overcome the shortcomings of oncolytic adenovirotherapy. Methods: TelomeScan, a modified Telomelysin expressing the green fluorescence protein (GFP), is used in this study. Cytotoxic activity of TelomeScan plasmid DNAs encapsulated by liposomes (Lipo-TelomeScan-pDNA) against various types of human cancer cell lines including HCT 116, a human colorectal carcinoma cell line, and MKN1, a human gastric carcinoma cell line, were evaluated by XTT assay and compared with liposome complexes of GFP plasmid DNA controlled by the cytomegalovirus (CMV) promoter (Lipo-GFP-pDNA) as a control agent. Results: Freeze-thaw procedure proved that viable oncolytic adenoviruses were successfully produced in H1299, a human lung cancer cell line, after Lipo-TelomeScan-pDNA transfection. Lipo-TelomeScan-pDNA showed cytotoxic activity on various types of cancer cell lines. Lipo-TelomeScan-pDNA killed HCT116 and MKN1 cells in a dose-dependent manner and produced more effective cytotoxic activity than Lipo-GFP-pDNA. Conclusions: Lipo-TelomeScan-pDNA proved potent cytotoxic effects on cancer cells by producing viable oncolytic adenoviruses. In vivo examination is needed for the next step. Citation Format: Katsuyuki Aoyama, Shinji Kuroda, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Development of systemically-deliverable telomerase-specific oncolytic adenovirus. [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 719. doi:10.1158/1538-7445.AM2014-719

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

Peritoneal dissemination is a common pattern of metastasis in patients with gastric cancer, and associated with worse prognosis. Peritoneal lavage cytology during the operation is an important factor in predicting future development of peritoneal metastasis and determining the treatment strategy, but its sensitivity and specificity are unsatisfactory. TelomeScan is an adenovirus engineered to replicate and express GFP only in telomerase-activating tumor cells, so that we can easily detect viable cancer cells even among numerous normal cells. We hypothesized that TelomeScan might be applicable to detecting free cancer cells in peritoneal wash. Methods: Peritoneal washes were obtained from 42 gastric cancer patients during operation. The number of GFP-positive cells was determined for each sample and compared with cytology results and clinicopathological data Results: Clinical stage was ranged from IA to IV, and thirteen cases were diagnosed as class IV or class V by peritoneal lavage cytology. More than 10 GFP-positive cells were detected in 12 out of 42 cases, and these cases showed a worse prognosis when compared to the other 30 cases. Conclusion: We were able to detect gastric cancer cells as GFP-positive cells in peritoneal wash using TelomeScan. Furthermore, the presence of GFP-positive cells in peritoneal wash was associated with worse prognosis. These results suggest that number of cancer cells detected by TelomeScan in the peritoneal wash may have important clinical implication as prognostic and therapeutic biomarkers in gastric cancer. Citation Format: Megumi Watanabe, Shunsuke Kagawa, Michihiro Ishida, Naoto Hori, Satoru Kikuchi, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Hiroshi Tazawa, Toshiyoshi Fujiwara. Virus-guided fluorescence imaging of intraperitoneal free gastric cancer cells as a potential clinical biomarker. [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 4726. doi:10.1158/1538-7445.AM2014-4726

Nanoparticles represent a novel class of agents for cancer treatment. In the present study, we examined the effects of epidermal growth factor receptor (EGFR)-targeted hybrid plasmonic magnetic nanoparticles (NP) on non-small cell lung cancer (NSCLC) cells. The nanoparticles (NP) consisted of a paramagnetic iron core that is surrounded by a gold layer and is functionalized with monoclonal anti-EGFR antibody (C225) or control rabbit IgG antibody (IgG). The nanoparticles were tested against a panel of human lung cancer cells that were mutant (HCC827, H1819), wild-type (H1299) or null (H520) for EGFR and compared to EGFR-positive normal fibroblasts (MRC9, WI38) and normal human bronchial epithelial (NHBE) cells. Treatment of tumor cells with C225-NP resulted in a strong and significant inhibitory effect of cell growth compared to normal cells. C225-NP-mediated inhibitory effect was observed only in EGFR-positive tumor cells (HCC827, H1819, H1299) but not in EGFR-null H520 cells. EGFR-mutant cells (HCC827) were the most sensitive to C225-NP. No significant growth inhibitory effects were observed in IgG-NP-treated cells when compared to untreated cells. Molecular analysis showed C225-NP selectively inhibited phosphorylated p-EGFR protein expression and EGFR-mediated signal transduction resulting in induction of autophagy followed by apoptosis. Autophagy occurred as early as twenty-four hours after treatment with an increase in LC3-II content and autophagic vacuoles observed by Western blotting and electron microscopy respectively. Apoptosis as determined by flow cytometry and Western blotting showed an increase in the number of sub-G1 population cells and cleavage of caspase-3 and PARP in C225-NP-treated cells. Optical images determined by dark-field microscopy showed an increase in the concentration of C225-NP bound to EGFR-expressing NSCLC cells but not EGFR-null NSCLC cells. C225-NP exhibited 2-7 fold higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody. Free C225 antibody did not induce autophagy in cells. Investigation into how C225-NP but not C225 antibody or NP-alone triggers autophagy and initiates apoptosis showed induction of a DNA damage response as evidenced by the formation of γH2AX foci in C225-NP-treated cells. Minimal to no γH2AX foci were observed in cells that were untreated, treated with C225 antibody or NP-alone or treated with IgG-NP. Our data show that EGFR specific NP selectively induced cytotoxic effects in EGFR-expressing lung cancer cells via a novel cell-death mechanism that is not characteristic of the free antibody thus increasing efficacy of therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3505.