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

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.

EMT: Present and future in clinical oncology

“…2). Moreover, high-throughput screenings aimed at identifying anti-EMT-related drugs have been recently developed (Arai et al, 2016;Carmody et al, 2012;Gupta et al, 2009). In one of them, 19 compounds were initially chosen as potential EMT and stemness inhibitors, uncovering a synthetic compound, ML245 (BRD-K59019422-001-01-3), that restrained cancer cell progression although additional studies are necessary to clarify the exact mechanism of action (Carmody et al, 2011).…”

“…Interestingly, two additional drugs, SB525334 and SU9516, targeting TGFbR1 (TGFb receptor 1) and CDK2 (cyclin dependent kinase 2), respectively (Fig. 2), were identified in another high-throughput screening showing EMT inhibitory activity in lung cancer cells (Arai et al, 2016).…”

Time-lapse imaging of HeLa spheroids in soft agar culture provides virtual inner proliferative activity

“…Cancer research models for screening have included the creation of spheroid microenvironments to test drug effects [1][2][3]. In one of the earlier studies using a spheroid-based screen, Friedrich et al [4] pointed out that using spheroid integrity and volume monitoring through phase-contrast imaging improved agent measurement when accompanied by the security of clonogenic survival.…”

Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3 and Reducing STAT3

“…Thus, a 3D culture system has enabled us to develop a tumoroid system in vitro, corresponding closely to the properties of native tumors in vivo [16,17]. Using the tumoroid system, we have identified a novel effect of a CDK2 inhibitor on suppressing epithelialmesenchymal transition (EMT) in cancer [18]. We have also developed an original 3D tumoroid-based multiplex assay system with MMP9 promoter-driven fluorescence reporter [2] and used this system in the present study for drug selection and for the evaluation of both tumoroid formation and progression.…”