Abstract 4002: Neutrophil extracellular traps (NETs) in abdominal cavity have supportive roles on postoperative development of peritoneal metastasis
Authors: Kanamaru Rihito , Tsukui Hidenori , Saito Shin , Kurashina Kentaro , Ui Takashi , Matsumoto Shiro , Yamaguchi Hironori , Sakuma Yasunaru , Horie Hisanaga , Hosoya Yoshinori , Sata Naohiro , Kitayama Joji 
Published: July 2017

Abstract
Background: Peritoneal lavages just after abdominal surgery contain many immune cells which consist mainly of granulocytes. Recent studies have demonstrated that neutrophil extracellular traps(NETs) play important roles not only on capture of pathogenic microbes but also on autoimmune disease, thrombus formation and tumor metastasis. Here, in this study, examined the NET forming capacity of granulocytes in postoperative peritoneal washings and assessed the possible role of the NETs on metastasis formation on peritoneum.
Method: In patients who underwent laparotomy due to digestive malignancies, abdominal cavity were washed with 1000 ml of normal saline at the timing of laparotomy (Preoperative) and just before wound closure (Postoperative). Cells recovered from the peritoneal washings were centrifuged on Ficoll-Hypaque solution and cultured at 37c in 5 percentage CO2 condition for 24 hour. Then, extracellular ds-DNA were stained with membrane impermeable dye, SYTOX. Also, the granulocytes were immunostained with mAbs against CD66b, histone, myeloperoxidase (MPO), neutrophil elastase (NE) and Matrix metalloproteinase (MMP)-9, and NETs structures were observed under fluorescein microscopy. In adhesion experiments, human gastric cancer cells were stained by PKH-26 and mixed with the granulocyte culture for 5 min and gently washed.
Results: 1. After Ficoll separation, CD66b (+) granulocytes were contained both in polymorophonuclear (PMN) and mononuclear cell (PBMC) layers, which are considered to be normo-density granulocytes (NDG) and low density granulocytes (LDG), respectively. The percentage of LDG in PBMC layer was markedly elevated in postoperative as compared preoperative lavages (Pre: M=0.71%, 0~8.9% vs Post: M=61.3%, 3~86.7% n=39, p&lt;0.001) The LDG in postoperative sample showed multinuclear morphology. 2. The short term culture of the postoperative peritoneal LDG, but not preoperative or peripheral blood granulocytes, many SYTOX (+) fibrous structures were observed, suggesting the vigorous NETs formation. The NETs appeared several min after the incubation, peaked at 2 hour and gradually decreased thereafter. Immunostaining showed that histone and MPO were co-localized on the NETs, while NE or MMP-9 were less clear on NETs. 3. After 5 min co-incubation, human gastric cancer cell, OCUM-1 and MKN45, were selectively attached on the NETs. 4. Similar SYTOX (+) NETs-like structure were clearly detected on the surface of human omental tissue resected by abdominal surgery.
Conclusion: After laparotomy, many LDG appear in human abdominal cavity which produce large amounts of NETs on peritoneal surface. The NETs efficiently trap the floating tumor cells, which may have positive roles on peritoneal recurrence in advanced cancer.
Citation Format: Rihito Kanamaru, Hidenori Tsukui, Shin Saito, Kentaro Kurashina, Takashi Ui, Shiro Matsumoto, Hironori Yamaguchi, Yasunaru Sakuma, Hisanaga Horie, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Neutrophil extracellular traps (NETs) in abdominal cavity have supportive roles on postoperative development of peritoneal metastasis [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 4002. doi:10.1158/1538-7445.AM2017-4002