Kazuya Kuwada

Abstract 5011: Tumor associated macrophages promote malignant phenotypes of disseminated human gastric cancer cells in intraperitoneal cancer immune microenvironment

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

Integrated fluorescent cytology with nano-biologics in peritoneally disseminated gastric cancer

Gastric cancer patients positive for peritoneal cytology are at increased risk of tumor recurrence, but although a certain proportion of cytology‐positive patients relapse rapidly with aggressive progression, others survive longer with conventional chemotherapies. This heterogeneity makes it difficult to stratify patients for more intensive therapy and poses a substantial challenge for the implementation of precision medicine. We developed a new approach to identify biologically malignant subpopulations in cytology‐positive gastric cancer patients, using a green fluorescent protein (GFP)‐expressing attenuated adenovirus in which the telomerase promoter regulates viral replication (TelomeScan, OBP‐401). The fluorescence emitted from TelomeScan‐positive cells was successfully quantified using a multi‐mode microplate reader. We then analyzed clinical peritoneal washes obtained from 68 gastric cancer patients and found that patients positive for TelomeScan had a significantly worse prognosis. In 21 cytology‐positive patients, the median survival time of those who were TelomeScan positive (235 days) was significantly shorter than that for those who were TelomeScan negative (671 days; P = 0.0062). This fluorescent virus‐guided cytology detects biologically malignant cancer cells from the peritoneal washes of gastric cancer patients and may thus be useful for both therapy stratification and precision medicine approaches based on genetic profiling of disseminated cells.

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.

Abstract 4156: Functional analysis of tumor-associated macrophage utilizing virus-guided fluorescent imaging of pancreatic cancer cells in the peritoneal cavity

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.

Abstract 3748: Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion

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-7029s 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.

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.