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Objectives To achieve ideal functional and aesthetic requirements, ridge augmentation is often required before dental implant placement. Bone augmentation (especially vertical), which normally consists of complex and invasive surgeries, still remains challenge. This study seeks to investigate the feasibility of an injectable in situ gel-forming system containing strontium hydroxyapatite (SrHA) and alginate for minimally invasive bone augmentation in a rat calvarial model. Material and methods SrHA–alginate solution was prepared by mixing SrHA powder with alginate solution (20 mg/mL) to the final concentration of 0.5% (w/v). Each animal received a 200-μL single subperiosteal injection of either SrHA–alginate solution or alginate solution. The new bone formation was assessed at 0, 4, and 8 weeks histologically and radiologically. Results The SrHA–alginate solution materials could form solid gel once injected. As such, no sutures were required to close the injection site. Significantly greater amount of new bone formation was observed in the SrHA–alginate group compared with the alginate group both by micro-CT and by histological section. The newly formed bone in the SrHA–alginate group originated both from the underlying original bone and from the elevated periosteum. A 2.3-fold increase of the vertical bone height was observed in the SrHA–alginate group compared with 1.3-fold increase in the alginate group. Conclusions Rat calvarial bone augmentation was achieved by a single subperiosteal injection of SrHA–alginate solution without any administration of stem cells or growth factors. The in situ gel-forming material may hold potential therapeutic benefits for local bone augmentation in a minimally invasive manner.

Background Molecular-based companion diagnostic tests are being used with increasing frequency to predict their clinical response to various drugs, particularly for molecularly targeted drugs. However, invasive procedures are typically required to obtain tissues for this analysis. Circulating tumour cells (CTCs) are novel biomarkers that can be used for the prediction of disease progression and are also important surrogate sources of cancer cells. Because current CTC detection strategies mainly depend on epithelial cell-surface markers, the presence of heterogeneous populations of CTCs with epithelial and/or mesenchymal characteristics may pose obstacles to the detection of CTCs. Methods We developed a new approach to capture live CTCs among millions of peripheral blood leukocytes using a green fluorescent protein (GFP)-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan). Results Our biological capturing system can image epithelial and mesenchymal tumour cells with telomerase activities as GFP-positive cells. After sorting, direct sequencing or mutation-specific PCR can precisely detect different mutations in KRAS, BRAF and KIT genes in epithelial, mesenchymal or epithelial–mesenchymal transition-induced CTCs, and in clinical blood samples from patients with colorectal cancer. Conclusions This fluorescence virus-guided viable CTC capturing method provides a non-invasive alternative to tissue biopsy or surgical resection of primary tumours for companion diagnostics.

We report the electric-field modulation of magnetism of a hexagonal Cr1–δTe thin film. A gate voltage VG is ap-plied in the field effect capacitor (FEC) structure consisting of electric double-layer capacitor (EDLC) of an ion liquid and a 2nm-thick Cr1–δTe layer grown by molecular beam epitaxy (MBE) and the magnetization of the layer is directly measured using a superconducting quantum interference device (SQUID) magnetometer in the both configurations with magnetic fields perpendicular or parallel to the film plane. As a result, we observe a clear change in the magnetization vs. magnetic field (M-H) curves by applying VG at a low temperature of 15 K in the perpendicular field configuration; the magnetization increases and the coercivity decreases by applying either positive or negative gate voltage. When the temperature is increased up to 160K, slightly lower than the Curie temperature, or the magnetization was measured in the in-plane field configuration, the magnetization increases similarly by applying either positive or negative gate voltage, but the amount of the increase becomes much smaller. A possible mechanism of the electric-field modulation is discussed in relation to the Cr vacancies in the Cr1–δTe layer. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We investigate the structural and magnetic properties of Zn1–xFex Te with an aim of discerning the intrinsic magnetism of this ternary compounds. Thin films of Zn1–xFex Te with a high Fe composition up to x = 0.2 were grown by molecular beam epitaxy (MBE) in the excess of Te flux against Zn flux. The structural analyses using X-ray diffraction (XRD) and X-ray absorption fine structure (XAFS) measurements reveal that crystals of the pure diluted phase are grown at low Fe compositions less than x = 0.02, while precipitates of extrinsic phases start to be formed at higher Fe compositions. In the magnetization measurements, the films of x ≤ 0.02, which were confirmed to be of the pure diluted phase, exhibit paramagnetic behaviors down to 2 K. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Epithelial-mesenchymal transition (EMT) is a biological process by which epithelial cells acquire mesenchymal characteristics. In malignant tumors, EMT is crucial for acquisition of a mesenchymal phenotype with invasive and metastatic properties, leading to tumor progression. An inflammatory microenvironment is thought to be responsible for the development and progression of colorectal cancer (CRC); however, the precise role of inflammatory microenvironments in EMT-related CRC progression remains unclear. Here, we show the spatiotemporal visualization of CRC cells undergoing EMT using a fluorescence-guided EMT imaging system in which the mesenchymal vimentin promoter drives red fluorescent protein (RFP) expression. An inflammatory microenvironment including TNF-α, IL-1β, and cytokine-secreting inflammatory macrophages induced RFP expression in association with the EMT phenotype in CRC cells. In vivo experiments further demonstrated the distribution of RFP-positive CRC cells in rectal and metastatic tumors. Our data suggest that the EMT imaging system described here is a powerful tool for monitoring EMT in inflammatory microenvironment–CRC networks.

A solid tumor consists of cancer and stromal cells, which comprise the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are usually abundant in the TME, contributing to tumor progression. In cases of peritoneal dissemination of gastric cancer (GC), the contribution of intraperitoneal TAMs remains unclear. Macrophages from peritoneal washings of GC patients were analyzed, and the link between intraperitoneal TAMs and GC cells was investigated to clarify the interaction between them in peritoneal dissemination. Macrophages were predominant among leukocytes constituting the microenvironment of the peritoneal cavity. The proportion of CD163-positive TAMs was significantly higher in stage IV than in stage I GC. Co-culture with TAMs potentiated migration and invasion of GC. IL-6 was the most increased in the medium of in vitro co-culture of macrophages and GC, and IL-6 elevation was also observed in the peritoneal washes with peritoneal dissemination. An elevated concentration of intraperitoneal IL-6 was correlated with a poor prognosis in clinical cases. In conclusion, intraperitoneal TAMs are involved in promoting peritoneal dissemination of GC via secreted IL-6. TAM-derived IL-6 could be a potential therapeutic target for peritoneal dissemination of GC.

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.

While the importance of programmed death-ligand 1 (PD-L1), mutation burden caused by microsatellite instability (MSI), and CD8+ tumor infiltrating lymphocytes (TILs) has become evident, the significance of PD-L1 expression on prognosis still remains controversial. We evaluated the usefulness of combined markers of PD-L1 and MSI or CD8+ TILs as a prognostic biomarker in gastric cancer. A total of 283 patients with gastric cancer were reviewed retrospectively. PD-L1 expression on >5% tumor cells was defined as PD-L1-positive. PD-L1-positive rate was 15.5% (44/283). PD-L1 positivity was significantly correlated with invasive and advanced cancer and also significantly correlated with MSI, whereas no significance was observed with CD8+ TILs. Kaplan–Meier analysis showed that PD-L1 positivity significantly correlated with a poor prognosis (p = 0.0025). Multivariate analysis revealed that PD-L1 positivity was an independent poor prognostic factor (hazard ratio [HR]: 1.97, p = 0.0106) along with diffuse histological type and lymph node metastases. Combinations of PD-L1 and MSI (HR: 2.18) or CD8+ TILs (HR: 2.57) were stronger predictive factors for prognosis than PD-L1 alone. In conclusion, combined markers of PD-L1 and MSI or CD8+ TILs may be more useful prognostic biomarkers in gastric cancer, and better clarify the immune status of gastric cancer patients.

We fabricated MnBi2Se4/(n − 1) quintuple layer (QL) Bi2Se3 magnetic topological heterostructures (n = 1–6) and measured the Dirac cone dispersion with angle-resolved photoemission spectroscopy. We observed a clear gap opening for films with n = 2–6 and the gap size decreased as the film thickness increased. From magnetization measurements using SQUID, all the heterostructures showed hysteresis loops at 4 K and at room temperature, confirming that MnBi2Se4 is a room temperature van der Waals ferromagnet. We were able to distinguish the contribution of the hybridization and magnetic effects on the observed Dirac-cone gap directly using a four-band model and its implications on the behavior of the expected quantum anomalous Hall effect is discussed.

130 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 also inhibits the repair of radiation-induced DNA double-strand breaks, leading to radiosensitization. We aimed to assess intratumoral injection of OBP-301 with radiotherapy in esophageal cancer patients unfit for standard treatments. Methods: An open-label, phase I dose-escalation study of OBP-301 with radiotherapy was conducted in 13 histologically confirmed esophageal cancer patients who deemed unfit to receive surgery or chemotherapy. Study treatment consisted of intratumoral OBP-301 injections 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 needle injection of the primary tumor through a flexible endoscope. The primary and secondary end points were incidence of dose-limiting toxicities and objective response rate. Results: Of 13 patients, seven, three, and three patients were treated in the cohorts with 1010, 1011, and 1012 virus particles 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. Eight patients had local complete response (CR); all of them exhibited pathologically no viable malignant cells in biopsy specimens, and three 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 three 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. Clinical trial information: 000010158.

Foraminifera carbonate exoskeleton was hydrothermally converted to biocompatible and biodegradable zinc-tricalcium phosphate (Zn-TCP) as an alternative biomimetic material for bone fracture repair. Zn-TCP samples implanted in a rat tibial defect model for eight weeks were compared with unfilled defect and beta-tricalcium phosphate showing accelerated bone regeneration compared with the control groups, with statistically significant bone mineral density and bone mineral content growth. CT images of the defect showed restoration of cancellous bone in Zn-TCP and only minimal growth in control group. Histological slices reveal bone in-growth within the pores and porous chamber of the material detailing good bone-material integration with the presence of blood vessels. These results exhibit the future potential of biomimetic Zn-TCP as bone grafts for bone fracture repair. © 2013 by the authors; licensee MDPI.

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

An aberrant left gastric vein (ALGV) directly entering the lateral segment of the liver is a rare variation in the portal vein system, whereas an aberrant left hepatic artery (ALHA) arising from the left gastric artery is observed relatively frequently. Here we report a case in which both ALGV and ALHA were encountered before laparoscopic distal gastrectomy with curative lymphadenectomy for gastric cancer. We accurately diagnosed these vessel anomalies preoperatively on abdominal contrast‐enhanced CT. During surgery, we divided the ALGV at the point of entry to the liver and preserved the ALHA by dividing the branches toward the stomach, in consideration of curability and safety. The postoperative course was uneventful overall, although temporary mild liver dysfunction was observed. This case highlights the importance of preoperative evaluation and preparation in a rare case of concurrent ALGV and ALHA.

Warunee Pluemsakunthai1), Shinji Kuroda2,*), Hitoyata Shimokawa3) and Shohei Kasugai1) 1)Oral Implantology and Regenerative Dental Medicine, Global Center of Excellence (G-COE) Program, Tokyo Medical and Dental University, Tokyo, Japan 2)Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan 3)Pediatric dentistry, Tokyo Medical and Dental University, Tokyo, Japan

Purpose: Low intensity pulsed ultrasound stimulation (LIPUS) has been clinically applied to promote bone fracture healing in the orthopedic field. Thus, it is likely that LIPUS also stimulates bone regeneration in bone defects in the cranial-maxillofacial area. However, this has not been clearly proved. Furthermore, optimal time point and period of the application after the surgery has not been reported. The purpose of the present study was to evaluate the effect of LIPUS on bone regeneration in the rat parietal bone defects especially focusing on time and period of the application. Materials and Methods: Eighteen Wistar rats (14 weeks old) were divided into 6 groups: 5 experimental groups and a control group. Bone defect of 5 mm diameter was prepared on each side of the parietal bone and customized gelatin membranes were placed over the bone defects. LIPUS (160 mW/cm2, 15 min/day) was applied to the defect area with an active transducer externally in the experimental groups according to the schedules of the applications: Group 1 (day 6 - 12), group 2 (day 13 - 19), group 3 (day 20 - 26), group 4 (day 6 - 19) and group 5 (day 6 - 26). All the animals were sacrificed at 28 days. The defects were analyzed with micro CT and then histologically. Results: In Group 1, new bone formation was significantly promoted and the newly-formed bone was thick and matured compared to the one of the control group. In other experimental groups there were tendencies of stimulation of new bone formation; however, they were not statistically significant. Discussion and Conclusion: The present study demonstrated that amount of new bone formation in the bone defect depended on the time and period of LIPUS application. It has been suggested that application of LIPUS at an early healing period, the second week after the surgery, effectively accelerated new bone formation.

ウイルスは本来,ヒトの細胞に感染し構造蛋白質を 産生することで複製・増殖し,その細胞を様々な機序 により破壊する.そのウイルス増殖機能に選択性を付 加することにより,ウイルスを癌細胞のみを標識し, 傷害する作用を発揮させることができる. ヒトのきわめて多くの癌細胞でテロメラーゼという 酵素活性が上昇していることが明らかになってい る.その詳細な生化学的作用についてはここでは割 愛するが,癌細胞の無制限増殖能に深く関わっている とされている.テロメラーゼは3つのサブユニットか ら構成されており,その中でも hTERTと呼ばれる部 分の発現レベルがテロメラーゼ活性に強く関わってい ることがわかっている.そこでわれわれはアデノウ イルス5型の増殖に必要なE1A遺伝子とE1B遺伝子 癌微小リンパ節転移を標的とするテロメラーゼ特異的制限増 殖型ウイルス製剤の開発 児 島 亨a*,渡 邉 雄 一b ,橋 本 悠 里a,c,黒 田 新 士a,山 崎 泰 源a,矢 野 修 也a, 田 澤 大a,c,宇 野 太a,c,香 川 俊 輔a,c,田 中 紀 章a,浦 田 泰 生b,藤 原 俊 義a,c

Trastuzumab(Tmab)is a humanized monoclonalantibody that binds to the human epidermalgrowth factor receptor 2 (HER2). It is clinically used for HER2-positive breast and gastric cancers; however, the use of Tmab is restricted to tumors expressing high levels of HER2(accounting for only 20%of tumors), and Tmab cannot be used for tumors resistant to Tmab. Although novel HER2-targeted agents have been developed to treat Tmab-resistant tumors, none of these have shown clinical efficacy in gastric cancer patients. Recent developments in nanotechnology have had a significant impact on the field of medicine. Gold nanoparticles(AuNPs), which show characteristics such as in vivo stability and ease of surface functionalization, have been developed as therapeutic and contrast agents for medical applications. Previous studies show that AuNPs exert cytotoxic effects through autophagy and apoptosis; therefore, AuNPs in combination with tumor-targeting antibodies are attractive therapeutic agents. In this study, we developed HER2-targeted AuNPs(Tmab-AuNPs)and showed that they had a potent antitumor effect on Tmab-resistant cell lines. In addition, Tmab-AuNPs were effective against HER2-negative gastric cancer cell lines when HER2 was artificially overexpressed. Thus, our results indicate that Tmab-AuNPs may overcome the shortcomings of Tmab-based therapy.

International audience; A Cr atom in a semiconductor host carries a localized spin with an intrinsic large spin to strain coupling, which is particularly promising for the development of hybrid spin-mechanical systems and coherent mechanical spin driving. We demonstrate here that the spin of an individual Cr atom inserted in a semiconductor quantum dot can be controlled optically. We first show that a Cr spin can be prepared by resonant optical pumping. Monitoring the time dependence of the intensity of the resonant fluorescence of the quantum dot during this process permits us to probe the dynamics of the optical initialization of the Cr spin. Using this initialization and readout technique we measured a Cr spin relaxation time at T=5 K in the microsecond range. We finally demonstrate that, under a resonant single-mode laser field, the energy of any spin state of an individual Cr atom can be independently tuned by using the optical Stark effect.

Laparoscopic and endoscopic cooperative surgery (LECS) is increasingly applied for gastric submucosal tumors (SMTs) such as gastrointestinal stromal tumors. However, the conventional LECS procedure has the potential risk that gastric contents and even tumor cells could spread into the abdominal cavity because the gastric wall has to be opened during the resection. To avoid this problem, we have developed a modified LECS procedure named “closed LECS.” Ten patients underwent closed LECS for the resection of gastric SMTs. Closed LECS consists of the following steps: endoscopic submucosal layer dissection around the tumor, laparoscopic marking of a resection line on the serosal surface along submucosal dissection line, seromuscular suturing with the marked lesion inverted into the inside of the stomach, endoscopic circumferential seromuscular dissection, and peroral retrieval. In three of the initial five cases, the closed LECS procedure was not completed as planned because of the tumor size and endoscopic inappropriate seromuscular dissection. After modification of the procedure, the entire procedure was successful in all five cases. The mean resected tumor diameter was 24.1 ± 7.6 mm. The mean operation time was 253 ± 45 min. One patient experienced an intra-abdominal abscess potentially related to delayed perforation as a postoperative complication. The closed LECS procedure for gastric SMTs can theoretically be applied without contamination and tumor cell dissemination into the abdominal cavity.

The external jugular vein (EJV) approach for totally implantable venous access devices (TIVADs) is safe. However, the success rate is unsatisfactory because of the difficulty in catheterization due to the acute angle between the EJV and the subclavian vein (SCV). A novel "shrug technique" to overcome this difficulty was developed, and its efficacy was assessed in a consecutive case series.TIVAD placement was performed via the EJV cut-down approach. "Shrug technique," a simple way to straighten the EJV-SVC angle by shrugging the patient's shoulder, was applied to facilitate the passage of the guidewire and sheath-introducer when there was acute angulation between the EJV and SCV.A total of 254 patients underwent TIVAD implantation by the EJV cut-down approach. The "shrug technique" was applied in 51 cases (20%), and catheterization was successful in all cases. Thus, TIVAD implantation was successfully completed in all 254 cases (100%) in a single operative setting. The median operating time was 38 [IQR 30-45] min. Eleven complications (4%) were observed, but none of them were EJV-specific.The "shrug technique" is simple but very useful to achieve a higher success rate and safer insertion of TIVADs from the EJV. J. Surg. Oncol. 2017;115:291-295. © 2016 Wiley Periodicals, Inc.

We report molecular beam epitaxial growth of a SnTe (111) layer on a CdTe template, fabricated by depositing it on a GaAs (111)A substrate, instead of BaF 2 which has been conventionally used as a substrate. By optimizing temperatures for the growth of both SnTe and CdTe layers and the SnTe growth rate, we could obtain SnTe layers of the single phase grown only in the (111) orientation and of much improved surface morphology from the viewpoint of the extension and the flatness of flat regions, compared to the layers grown on BaF 2 . In this optimal growth condition, we have also achieved a low hole density of the order of 10 17 cm −3 at 4 K, the lowest value ever reported for SnTe thin films without additional doping. In the magnetoresistance measurement on this optimized SnTe layer, we observe characteristic negative magneto-conductance which is attributed to the weak antilocalization effect of the two-dimensional transport in the topological surface state.

International audience; We demonstrate the optical addressability of the spin of an individual chromium atom (Cr) embedded in a semiconductor quantum dot. The emission of Cr-doped quantum dots and their evolution in magnetic field reveal a large magnetic anisotropy of the Cr spin induced by local strain. This results in the zero field splitting of the 0, ±1, and ±2 Cr spin states and in a thermalization on the magnetic ground states 0 and ±1. The observed strong spin to strain coupling of Cr is of particular interest for the development of hybrid spin-mechanical devices where coherent mechanical driving of an individual spin in an oscillator is needed. The magneto-optical properties of Cr-doped quantum dots are modeled by a spin Hamiltonian including the sensitivity of the Cr spin to the strain and the influence of the quantum dot symmetry on the carrier-Cr spin coupling.

Background Bochdalek hernia (BH) is generally congenital, presenting with respiratory distress. However, this pathology is rarely detected in adults. Some adult cases of BH present with symptoms attributed to the hernia, but incidental detection of BH is increasing among asymptomatic adults due to advances in imaging modalities. This report presents the management of incidental BH patients detected in the preoperative period of gastric cancer. Case presentation An asymptomatic 76-year-old woman was diagnosed with advanced gastric cancer during follow-up after radiotherapy for uterine cervical cancer. Computed tomography (CT) was performed to exclude metastatic gastric cancer, incidentally detecting right-sided BH. We planned distal gastrectomy with lymph node dissection for gastric cancer and simultaneous repair of BH using a laparoscopic approach. We performed laparoscopic gastrectomy for gastric cancer and investigated the right-sided BH to assess whether repair during surgery was warranted. Herniation of the liver into the right hemithorax was observed, but was followed-up without surgical repair because the right hepatic lobe was adherent to the remnant right anterior hemidiaphragm and covered the huge defect in the right hemidiaphragm. No intra- or postoperative pneumothorax was observed during pneumoperitoneum. Conclusion Regardless of symptoms, repair of adult BH is generally recommended to prevent visceral incarceration. However, BH in asymptomatic adults appears to be more common than previously reported in the literature. Surgeons need to consider the management of incidental BH encountered during thoracic or abdominal surgery.

Background It is important to visualize the phenomenon which occurs in actual clinical cases to decide the timing of implant placement subsequently after sinus graft. Although several clinical cases of bone augmentation using xenograft have been evaluated, the number of those reports which have described bone remodeling by alloplasty are not sufficient. Findings In the present report, to investigate bone remodeling histologically after maxillary sinus augmentation with porous hydroxyapatite alloplast, bone cores from the sinus floor of three female nonsmoking patients (aged 64–73 years) were collected 6 months after sinus lift surgery, embedded in methyl methacrylate resin, and prepared by conventional methods. Bone architecture and graft residues were evaluated by micro-computed tomography of the same specimens. Hematoxylin–eosin and Villanueva–Goldner staining revealed mature osteoblasts and multinucleated osteoclasts on the grafted sinus floor and surface of residual hydroxyapatite particles. The particulate interspace was partially filled with osteoid and calcified tissue and showed active vascularization. Conclusion The results suggested that bone regeneration and angiogenesis within and between porous hydroxyapatite particles were sufficiently found after 6 month histologically in the grafted sinus floor. Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-1885-2) contains supplementary material, which is available to authorized users.

We studied the spin dynamics of a Cr atom incorporated in a II-VI semiconductor quantum dot using photon correlation techniques. We used recently developed singly Cr-doped CdTe/ZnTe quantum dots (A. Lafuente-Sampietro {\it et al.}, [1]) to access the spin of an individual magnetic atom. Auto-correlation of the photons emitted by the quantum dot under continuous wave optical excitation reveals fluctuations of the localized spin with a timescale in the 10 ns range. Cross-correlation gives quantitative transfer time between Cr spin states. A calculation of the time dependence of the spin levels population in Cr-doped quantum dots shows that the observed spin dynamics is controlled by the exciton-Cr interaction. These measurements also provide a lower bound in the 20 ns range for the intrinsic Cr spin relaxation time.

Oncolytic viruses engineered to replicate in tumour cells but not in normal cells could be used as tumour-specific vectors carrying the therapeutic genes. We previously developed a telomerase-specific oncolytic adenovirus, OBP-301, that causes cell death in human cancer cells with telomerase activities. Here, we further modified OBP-301 to express the wild-type p53 tumour suppressor gene (OBP-702), and investigated whether OBP-702 induces stronger antitumour activity than OBP-301. The antitumour effect of OBP-702 was compared to that of OBP-301 on OBP-301-sensitive (H358 and H460) and OBP-301-resistant (T.Tn and HSC4) human cancer cells. OBP-702 suppressed the viability of both OBP-301-sensitive and OBP-301-resistant cancer cells more efficiently than OBP-301. OBP-702 caused increased apoptosis compared to OBP-301 or a replication-deficient adenovirus expressing the p53 gene (Ad-p53) in H358 and T.Tn cells. Adenovirus E1A-mediated p21 and MDM2 downregulation was involved in the apoptosis caused by OBP-702. Moreover, OBP-702 significantly suppressed tumour growth in subcutaneous tumour xenograft models compared to monotherapy with OBP-301 or Ad-p53. Our data demonstrated that OBP-702 infection expressed adenovirus E1A and then inhibited p21 and MDM2 expression, which in turn efficiently induced apoptotic cell death. This novel apoptotic mechanism suggests that the p53-expressing OBP-702 is a promising antitumour reagent for human cancer and could improve the clinical outcome.

Autophagy is known to have a cytoprotective role under various cellular stresses; however, it also results in robust cell death as an important safeguard mechanism that protects the organism against invading pathogens and unwanted cancer cells. Autophagy is regulated by cell signalling including microRNA (miRNA), a post-transcriptional regulator of gene expression. Here, we show that genetically engineered telomerase-specific oncolytic adenovirus induced miR-7 expression, which is significantly associated with its cytopathic activity in human cancer cells. Virus-mediated miR-7 upregulation depended on enhanced expression of the E2F1 protein. Ectopic expression of miR-7 suppressed cell viability and induced autophagy by inhibiting epidermal growth factor receptor (EGFR) expression. Our results suggest that oncolytic adenovirus induces autophagic cell death through an E2F1-miR-7-EGFR pathway in human cancer cells, providing a novel insight into the molecular mechanism of an anticancer virotherapy.

Cholesterol-bearing pullulan (CHP) nanogel is a synthetic degradable biomaterial for drug delivery with high biocompatibility. Guided bone regeneration (GBR) is a bone augmentation technique in which a membrane is used to create and keep a secluded regenerative space. The purpose of the present study was to evaluate the effects of the novel CHP nanogel membrane in GBR. Thirty-six adult Wistar rats were used and bilaterally symmetrical full-thickness parietal bone defects of 5 mm diameter were created with a bone trephine burr. Each defect was covered with the collagen membrane or the CHP nanogel membrane or untreated without any membrane. The animals were sacrificed at 2, 4 and 8 weeks and analysed radiologically and histologically. Furthermore, after incubating human serum with CHP nanogel or collagen, the amount of PDGF in the serum was measured using ELISA. New bone formation in terms of bone volume was higher in the nanogel group than in the control or collagen groups at 2 and 4 weeks. At 8 weeks, both membrane groups showed higher bone volumes than the control group. Notably, the newly-formed bone in the bone defect in the nanogel group was uniform and histologically indistinguishable from the original bone, whereas in the collagen group the new bone showed an irregular structure that was completely different from the original bone. After incubating with CHP nanogel, the amount of PDGF in the serum decreased significantly. CHP nanogel GBR membrane favourably stimulated bone regeneration, in which a unique characteristic of CHP nanogel, the storage of endogenous growth factors, was likely implicated.

Date Y, Yokoyama Y, Kondo H, Kuroda S, Ohya K, Ota MS, Iseki S, Kasugai S. Restricted expression of chromatin remodeling associated factor Chd3 during tooth root development. J Periodont Res 2012; 47: 180–187. © 2011 John Wiley & Sons A/S Background and Objective: The tooth root is one of the critical parts to maintain tooth function; however, the molecular mechanisms of root development remain unknown. We aimed to identify specific factors for root morphogenesis using a newly developed experimental system. Material and Methods: Tentative cementoblasts and periodontal ligament cells from mouse mandibular molars were isolated using laser capture microdissection. More than 500 cementoblasts and periodontal ligament cells were separately captured. After RNA extraction and amplification, mRNA expression in isolated cementoblasts was compared with that of periodontal ligament cells by cDNA microarray analysis. Then, putative cementoblast-specific genes were subjected to in situ hybridization analysis to confirm the results in mouse mandible. Results: Approximately 2000 genes were differentially expressed between these tissues. Among those genes, zinc finger helicase (ZFH), also termed chromodomain-helicase-DNA-binding protein 3 (Chd3), was one of the highly expressed transcripts in tentative cementoblasts. In situ hybridization revealed that ZFH/Chd3 was strongly expressed in Hertwig’s epithelial root sheath rather than in cementum. Moreover, its expression disappeared when root formation was advanced in the first molar. In contrast, Chd3 was continuously expressed in dental epithelial cells of the cervical loop, in which root extension is never terminated. Conclusion: These results suggest that ZFH/Chd3 might play an important role in tooth root development and subsequent cementogenesis.

A sputtering technique followed by a low temperature hydrothermal treatment has been demonstrated to produce a dense-and-bioactive hydroxyapatite thin film coating. The purpose of the present study was to investigate osteoblast and osteoclast responses to the hydroxyapatite coated plates and titanium plates with similar roughness. Rat bone marrow stromal cells were cultured on these plates to induce osteoblasts. The cells showed a significantly enhanced proliferation on the hydroxyapatite surface, accompanied by increase of osteoblastic phenotypes. The co-cultured osteoclasts exhibited the significantly different cell number and morphology between the hydroxyapatite and the titanium surfaces. A series of osteoclast marker genes were more stimulated on the hydroxyapatite and thirty two percent of the hydroxyapatite surface area could be resorbed by osteoclasts. The thin film sputtered hydroxyapatite could provide a favorable surface for both osteoblast and osteoclast formation and their function, indicating its good osteoconductivity and biodegradability.

DNA修復機能阻害は放射線感受性を増強させるため，DNA修復に関与する因子の阻害剤は放射線増感剤となり得る．我々の開発したテロメラーゼ依存的腫瘍融解アデノウイルス製剤OBP-301（テロメライシン）は，アデノウイルスE1B55kDaタンパクを介して細胞のDNA修復に重要な役割を果たすMRN複合体（Mre11，Rad50，NBS1）を分解する機能を有する．このMRN複合体の分解によりATM（ataxia-telangiectasia mutated）の活性化が抑制され結果的にDNA修復機構が阻害される．我々はOBP-301と放射線との併用が強力な相乗効果を生み出すことをマウスの皮下腫瘍モデルおよび食道癌同所性モデルにおいて証明した．これらの結果はOBP-301が将来有望な放射線増感剤となり得ることだけでなく，E1B55kDaタンパクを産生する腫瘍融解アデノウイルス製剤と放射線との併用が悪性腫瘍に対する有力な治療戦略となり得ることを示す．

We have investigated the exciton spin dynamics in an ultrathin CdTe/ZnTe single quantum well (QW) and self-assembled quantum dots (QDs) by observing time-resolved Kerr rotation (TRKR). At temperatures low enough to suppress the thermal escape of holes from the quantum-confinement structures, excitons dominate the TRKR signal. It shows characteristic dependence on the magnetic field, which is distinctly different from that of isolated electrons. From the data, we have estimated the electron–hole exchange energy to be 137 µeV in the QW, and larger than 190 µeV in the QDs.

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.

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

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli , and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V max value was 1.40 μmol min −1 mg −1 , and the K m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl- l -serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl- l -serine from l -alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl- l -serine was efficiently produced from l -alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl- l -serine from l -2-aminobutyric acid.

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