A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.

Kazuya Arai,Manabu Itoh,Norio Masuda,Ruriko Sakamoto,M Mamunur Rahman,Ken-Ichi Kozaki,Stuart K Calderwood,Takanori Eguchi,Tetsuya Nakatsura,Michael Klymkowsky

doi:10.1371/journal.pone.0162394

Kazuya Arai, Manabu Itoh + Show 8 more

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https://doi.org/10.1371/journal.pone.0162394

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Abstract

Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. 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. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 μM and 1.21 μM, respectively, while IC50 of SB431542 was 2.38 μM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

Highlights

Cell-based assays for a development of anti-cancer drugs have been mostly performed in monolayer culture condition [1]
Kumar et al reported that mesenchymal transition of non-small cell lung carcinoma (NSCLC) cell lines were much more efficiently induced on 3D cell culture condition than 2D [6]
Chua et al reported a development of a method screening Epithelial-mesenchymal transition (EMT) inhibitor by calculating cell motility under 2D cell culture condition [35]

Summary

Introduction

Cell-based assays for a development of anti-cancer drugs have been mostly performed in monolayer culture condition [1]. Other researchers have reported that three-dimensional (3D) cell cultures can replicate the intratumor microenvironment [3,4,5]. These findings suggest the importance of 3D cell culture conditions for evaluation of the cellular phenotype and of chemical compounds and/or cytokines. Kumar et al reported that mesenchymal transition of non-small cell lung carcinoma (NSCLC) cell lines were much more efficiently induced on 3D cell culture condition than 2D [6]. These reported persuaded us to develop a novel 3D screening system

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