Tetsushi Kubota

Establishment of a Non-Invasive Semi-Quantitative Bioluminescent Imaging Method for Monitoring of an Orthotopic Esophageal Cancer Mouse Model.

Orthotopic models of various types of tumors are widely used in anti-tumor therapeutic experiments in preclinical studies. However, there are few ways to appropriately monitor therapeutic effect in orthotopic tumor models, especially for tumors invisible from the outside. In this study we aimed to establish a non-invasive semi-quantitative bioluminescent imaging method of monitoring an orthotopic esophageal cancer mouse model. We confirmed that the TE8 esophageal cancer cell line implanted orthotopically into the abdominal esophagus of nu/nu mice (n = 5) developed not only a main tumor at the implanted site, but also local lymph node metastases and peritoneal disseminations within 6 weeks after inoculation. We established a TE8 cell line that stably expressed the firefly luciferase gene (TE8-Luc). We showed that TE8-Luc cells implanted subcutaneously into nu/nu mice (n = 5) grew over time until 5 weeks after inoculation. Tumor volume was strongly correlated with luminescent intensity emitted from the tumor, which was quantified using the IVIS imaging system. We then showed that TE8-Luc cells implanted orthotopically into the mouse abdominal esophagus (n = 8) also formed a tumor and that the luminescent intensity of such a tumor, as detected by IVIS, increased over time until 7 weeks after inoculation and was therefore likely to reflect tumor progression. We therefore propose that this orthotopic esophageal cancer model, monitored using the non-invasive semi-quantitative IVIS imaging system, will be useful for in vivo therapeutic experiments against esophageal cancer. This experimental setting is expected to contribute to the development of novel therapeutic technologies for esophageal cancer in preclinical studies.

Abstract 4747: Novel HER2-targeted gold nanoparticles; integration of antibody therapy and nanotechnology

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.

HER2-targeted gold nanoparticles potentially overcome resistance to trastuzumab in gastric cancer

An issue of concern is that no current HER2-targeted therapeutic agent is effective against Trastuzumab (Tmab)-resistant gastric cancer. Gold nanoparticles (AuNPs) are promising drug carriers with unique characteristics of a large surface area available for attachment of materials such as antibodies. Here, we created HER2-targeted AuNPs (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, T-AuNPs showed stronger cytotoxic effects than controls against MKN7 and NCI-N87 cells although Tmab had no effect on MKN7 cells. Autophagy played an important role in T-AuNP cytotoxic mechanisms, which was considered to be driven by internalization of T-AuNPs. Finally, T-AuNPs displayed potent antitumor effects against NCI-N87 and MKN7 subcutaneous tumors in in vivo mouse models. In conclusion, HER2-targeted AuNPs with conjugated Tmab is a promising strategy for the development of novel therapeutic agents to overcome Tmab resistance in gastric cancer.

Training system for laparoscopy-assisted distal gastrectomy

PurposeLaparoscopy-assisted distal gastrectomy (LADG) is likely to become a standard procedure for gastric cancer, which highlights the importance of establishing a training system in which even inexperienced surgeons can perform this procedure safely. This study assesses our training system for LADG based on short-term surgical outcomes.MethodsWe evaluated retrospectively the short-term outcomes of 100 consecutive LADGs with curative D1/D1+ lymph node dissection. Our training system was assessed based on the learning curve of trainees, and factors related to achieving good-quality operations were analyzed statistically.ResultsOverall, postoperative complications developed in 10 patients (10%), and included one case of anastomotic leakage (1%) and one case of pancreatic fistula (1%). The learning curve of the trainees plateaued after 10 operator cases in terms of operation time. The importance of the trainer’s position was also confirmed by the result that the operation time was significantly longer when trainees with ≤10 operator cases performed LADG with a trainer as scopist vs. a trainer as the first assistant. Univariate and multivariate analyses revealed that >10 operator cases were the most important factor for achieving good-quality operations.ConclusionThese results show that our current LADG procedure and training system are appropriate and effective.

Liposome-encapsulated plasmid DNA of telomerase-specific oncolytic adenovirus with stealth effect on the immune system

Oncolytic virotherapy has the disadvantage of being unsuitable for systemic delivery due to immune elimination. Liposomal encapsulation is well-recognized to reduce immune elimination and enhance the stability of drugs in the bloodstream. In the present study, the potential of liposome-encapsulated plasmid DNA of telomerase-specific oncolytic adenovirus (TelomeScan) expressing GFP (Lipo-pTS) as an oncolytic adenoviral agent suitable for systemic delivery was investigated. Lipo-pTS, which has a diameter of 40–50 nm, showed potent antitumor effects on HCT116 colon carcinoma cells in vitro and in vivo. Tumor selectivity of Lipo-pTS was independent of coxsackie and adenovirus receptor (CAR). Importantly, Lipo-pTS reduced production of adenovirus-neutralizing antibodies (AdNAbs) after intravenous administration into immune-competent mice compared to TelomeScan, and even in the presence of AdNAbs, Lipo-pTS maintained strong cytotoxicity. In conclusion, Lipo-pTS has the potential to become an oncolytic adenoviral agent suitable for systemic delivery with the characteristics of CAR-independent antitumor activity and a stealth effect on the immune system.

Abstract 2649: PD-L1 expression as a predictive factor for recurrence pattern and prognosis in curatively resected gastric cancer

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 patient9s 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 patient9s 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: 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.

Abstract 5520: HER2-targeted gold nanoparticles produce potent antitumor effects on human gastric cancer cells

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

Strategic approach to concurrent aberrant left gastric vein and aberrant left hepatic artery in laparoscopic distal gastrectomy for early gastric cancer: A case report.

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.