Abstract 5525: Development of novel high-throughput screening assay system for exploring EMT inhibitors.

Abstract

Abstract Background: EMT (Epitherial-Mesenchymal transition) is a significant event in tumor metastasis and malignancy. Cancer cells are stimulated by EMT-inducible agonists from the surrounding stromal cells, and cause transformation such as weakening of cell-cell adhesion and the accompanying acceleration of cell migration and invasion. Inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we developed a high-throughput assay system of EMT inhibitor screen in 3D cell culture. Method: We adopted 3D cell culture method on NanoCulture® Plate (NCP), a scaffold type spheroid culture plate, as a culture method which can confirm spheroid morphologies. TGF-β and its inhibitor, SB431542, were used as an EMT inducer and as a positive control for EMT inhibitor, respectively. Expressions of E-cadherin, N-cadherin, vimentin and zeb-1, represent EMT maker genes, were evaluated by qRT-PCR. As hypoxia inside the spheroids reflected the spheroid size and cell-cell adhesion strength, we visualized the intra-spheroid hypoxia, and used it as an indicator for EMT induction and inhibition. Result: A549 cell spheroids gradually collapsed, by the weakening of cell-cell adhesion, and cells migrating out from the spheroids, by treating with TGF-β. This collapse of the spheroids was inhibited by SB431542 treatment. SB431542 treatment also accompanied inhibition of E-cadherin down-regulation, and N-cadherin, vimentin and zeb-1 up-regulation. These results indicate that this method is clearly reproducing EMT and its inhibition. And we elucidated that intra-spheroid hypoxicity was closely correlated with spheroid morphology. From this, it is demonstrated that this method is simple but high-precision assay. Conclusion: We developed a high-throughput assay system for EMT inhibitor screen, by evaluating intra-spheroid hypoxicity as an indicator for alternation of the spheroid morphology. This method is an unique method which enables to evaluate EMT condition, by monitoring the collapse of spheroids directly. We are now running a screen for EMT inhibitor candidate compounds, by using this assay system. Citation Format: Kazuya Arai, Ruriko Sakamoto, Manabu Itoh, Hiromi Miura, Manami Shimomura, Tetsuya Nakatsura. Development of novel high-throughput screening assay system for exploring EMT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2013-5525