Abstract EMT (epithelial-mesenchymal transition) is a significant event in tumor metastasis and malignancy. Therefore, inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we introduce an innovative three-dimensional (3D) high-throughput (HTS) system that leads to an identification of EMT inhibitors. At first, we established 3D-HTS EMT model with NanoCulture Plate (NCP) that provided a gel-free micro-patterned scaffold for cells forming spheroids naturally. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs. Citation Format: Kazuya Arai, M Mamunur Rahman, Manabu Itoh, Norio Masuda, Takanori Eguchi, Tetsuya Nakatsura, Stuart K. Calderwood. A novel high-throughput 3D screening system for EMT inhibitors [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 846. doi:10.1158/1538-7445.AM2017-846