Abstract 3830: The enhanced formation of neutrophil extracellular traps (NETs) contributes to peritoneal and hepatic metastasis in diabetic conditions
Authors: Rei Takahashi, Hideyo Miyato, Akira Saito, Eri Suizu, Misaki Matsumiya, Hirofumi Sonoda, Hideyuki Ohzawa, Hironori Yamaguchi, Naohiro Sata, Joji Kitayama
Journal: Cancer Research (2025)

Abstract Background: The risk of cancer development and cancer-related mortality increase in diabetic patients. Recent studies have shown neutrophils in diabetic patients tend to form neutrophil extracellular traps (NETs). NETs have been reported to promote metastasis by capturing tumor cells in target organ. This study aims to clarify how diabetic conditions influence peritoneal and hepatic metastasis through NETs formation. Methods: Neutrophils were isolated from the bone marrow or peritoneal cavity of C57BL/JHamSlc-ob/ob (type 2 diabetes model) and wild-type mice using Ly6G-based immunomagnetic positive selection. After stimulation of neutrophils with 100nM PMA, the amount of NETs formation was evaluated with SYTOX green staining. YTN16P gastric cancer cells (5×105 cells) were injected into peritoneal cavity of ob/ob mice. 15000U/kg of DNase I or PBS were administered every day. After two weeks, the number of metastatic nodules on the mesentery was compared between the two groups. As for a hepatic metastasis model, YTN16H (5×104 cells) were intrasplenically injected. Metastatic tumors were immunohistochemically stained with anti-MPO antibody and anti-citrullinated histone 3 to detect NETs formation. In total of 1178 patients with gastric cancer who received curative gastrectomy from April 2010 to March 2021 in our institute, the prognosis was compared between diabetic and non-diabetic patients. Results: Neutrophils isolated from ob/ob mice produced higher amount of NETs than those from wild-type mice. NETs produced by neutrophils obtained from the peritoneal cavity of ob/ob mice effectively captured many YTN16P in vitro. The concentration of dsDNA in plasma of ob/ob mice was higher than that in wild mice (676±34.8ng/mL vs 590±32.6ng/mL, p=0.036, n=3). ob/ob mice had higher number of peritoneal metastasis than wild type mice (23.5 (9-104) vs 15.5 (0-31), n=10). The accelerated peritoneal metastasis was suppressed by the treatment with DNAse I (23.5 (1-23) vs 16.0 (9-34), p=0.034, n=8, 7). Regarding hepatic metastasis models, ob/ob mice had higher number of metastatic nodules on the surface of the liver than lean-type mice (123 (52-354) vs 1 (0-3), p=0.007, n=5). Immunohistochemistry showed NETs in omental and hepatic metastasis. Disease-free survival (DFS) (p=0.004, HR=1.65) and overall survival (OS) (p=0.035, HR=1.46) of 203 diabetic patients were significantly worse than those of 975 non-diabetic patients. DFS and OS were worth in the diabetic group in Stage I (DFS: p=0.020, HR=3.02, OS: p=0.056, HR=1.92). Regarding DFS, multivariate analysis showed that DM was an independent risk factor. Diabetic patients had a higher recurrence rate in the liver (5.9% vs 2.6%, p=0.026) than non-diabetic patients. Conclusion: Enhanced NETs formation in diabetic conditions may contribute to the development of metastasis formation in liver and peritoneum in gastric cancer. Citation Format: Rei Takahashi, Hideyo Miyato, Akira Saito, Eri Suizu, Misaki Matsumiya, Hirofumi Sonoda, Hideyuki Ohzawa, Hironori Yamaguchi, Naohiro Sata, Joji Kitayama. The enhanced formation of neutrophil extracellular traps (NETs) contributes to peritoneal and hepatic metastasis in diabetic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3830.