Abstract 5809: Fluorescence-guided spatiotemporal dynamics of epithelial-mesenchymal transition under inflammatory microenvironment during colorectal cancer progression
Authors: Ieda Takeshi , Tazawa Hiroshi , Kikuchi Satoru , Kuroda Shinji , Ohara Toshiaki , Noma Kazuhiro , Kishimoto Hiroyuki , Nagasaka Takeshi , Nishizaki Masahiko , Kagawa Shunsuke , Imamura Takeshi , Fujiwara Toshiyoshi 
Published: July 2017

Abstract
Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancer. However, the role of inflammatory microenvironment in the EMT-related tumor progression remains unclear. To explore the relationship between inflammatory microenvironment and EMT, a live imaging system for EMT is a promising strategy on the in vitro and in vivo experiments. In this study, we developed a fluorescence-guided live cell imaging system for the assessment of spatiotemporal dynamics of EMT, and investigated the potential of inflammatory microenvironment for the induction of EMT phenotype in human colorectal cancer.
Methods: Two human colorectal cancer cell lines, HCT116 and RKO, were stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. Both cell lines were treated with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or co-cultured with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis and q-PCR. EMT-induced HCT116 and RKO cells were treated with anti-inflammatory agents, aspirin (1mM) and salicylic acid (1 mM), for the suppression of EMT.
Results: Inflammatory cytokines (IL-1β and TNF-α) induced red fluorescence intensity and morphological change like mesenchymal phenotype in HCT116 and RKO cells. Removal of inflammatory cytokines attenuated red fluorescence intensity and morphological change in both cells. Inflammatory cytokines also induced the migration and invasion properties in association with EMT-related markers. Moreover, co-culture with LPS-stimulated inflammatory macrophages also induced red fluorescence intensity and morphological change as well as inflammatory cytokines. Anti-inflammatory agents significantly suppressed the fluorescence-related EMT phenotype under inflammatory microenvironment.
Conclusions: These results suggest that inflammatory microenvironment has a great potential for the induction of EMT process during colorectal cancer progression. This unique fluorescence-guided EMT imaging system is useful method for the exploration of inflammation-mediated tumor progression.
Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Takeshi Imamura, Toshiyoshi Fujiwara. Fluorescence-guided spatiotemporal dynamics of epithelial-mesenchymal transition under inflammatory microenvironment during colorectal cancer progression [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 5809. doi:10.1158/1538-7445.AM2017-5809