Abstract 6289: Activated neutrophils induce robust mesenchymal migration of gastric cancer cells
Authors: Takahashi Kazuya , Ohzawa Hideyuki , Kaneko Yuki , Futoh Yurie , Tamura Kohei , Kimura Yuki , Saito Akira , Tojo Mineyuki , Kanamaru Rihito , Miyato Hideyo , Sata Naohiro , Kitayama Joji 
Published: June 2022

Background: Activated neutrophils can produce multiple substances including various enzymes such as neutrophil elastase, myeloperoxidase, as well as many cytokines and reactive oxygen species (ROS). In addition, in some circumstances, they produce neutrophil extracellular traps (NETs), which is unfolded DNA fibers showing such a web-like structure containing histone and various proteins. However, recent studies have suggested NETs can promote tumor metastasis. In fact, many neutrophils are infiltrating in tumor tissues which are called tumor associated neutrophils (TAN) and high TANs are associated with poor prognosis. However, the detailed effects of TAN on tumor cell behavior are still not fully understood.

Methods: Human peripheral polymorph neutrophils (PMN) collected from healthy donor were stimulated with 1μM PMA or 10μg/ml LPS for 15 min, washed extensively, and incubated for another 3 hours. Using trans-well system with culture inserts with 8μm pore membrane, we examined the migration of human gastric cancer cells NUGC-4 or MKN45 to 2% FBS placed in lower chamber with or without PMN. Random migration was also examined with time-lapsed analysis. Lamellipodia were detected with F-actin stained with phalloidin-488 and immunostaining to vinculin.

Results: PMN stimulated with either PMA or LPS produced large amounts of NETs stained with SYTOX. NUGC-4 and MKN45 showed little migration to unstimulated PMN, while many cells did migrate when PMA-activated PMN were present in lower chamber. Although NET-depleted supernatants (SN) also induced significant migration, the number of migrated cells was reduced to about 50%. When catalase (800U/ml) catalase was added, the migrations of NUGC-4 was significantly reduced (37%). Random migration was markedly enhanced by NET-depleted SN. Addition of the SN induced many lamellipodia in NUGC-4 within 30 min. The migration of NUGC-4 was also increased by PMN stimulated by LPS, but not by their NET-depleted SN.

Summary and Conclusion: PMA-stimulated neutrophils induce robust migration of cancer cells which is partly dependent on H2O2. LPS-stimulated neutrophils appear to produce less amounts of ROS, but can also enhance the NUGC-4 migration through NET dependent manner. Activated neutrophils in tumor microenvironment can stimulate migration of cancer cells through multiple mechanisms, and assist the initial step of metastatic process of primary tumor.

Citation Format: Kazuya Takahashi, Hideyuki Ohzawa, Yuki Kaneko, Yurie Futoh, Kohei Tamura, Yuki Kimura, Akira Saito, Mineyuki Tojo, Rihito Kanamaru, Hideyo Miyato, Naohiro Sata, Joji Kitayama. Activated neutrophils induce robust mesenchymal migration of gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6289.