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.

Fig 1. Epithelial to mesenchymal transition (EMT) on NanoCulture Plate (NCP).

(A) A549 cells were grown in 2D or 3D-NCP conditions with or without TGF-β2 for 4 days. The cells treated with TGF-β2 on 2D condition appeared to present fibroblastic spindle shape. In the 3D-NCP condition, TGF-β2-treated spheroid cells appeared to spread out from spheroids, and spheroid sizes were reduced. (B) Representative images of kinetic morphology change. Cells were pre-cultivated on NCP for 3 days and then treated with TGF-β2 for 4 days. Arrowheads indicate spheroids that fused into one spheroid. Arrow indicate spheroid collapse induced by the addition of TGF-β2. Scale bars, 200 μm. Live-cell imaging video is available in S1 and S2 Movies. (C) A549 cells were cultured with or without TGF-β2 only or TGF-β2 + SB431542 for 4 days. Representative images were shown. Scale bar, 200 μm. (D) mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8 were quantified with real-time qRT-PCR analyses. Values were normalized to TBP levels, and then were shown as the mean of fold change to vehicle control ± SD of three independent experiments

Fig 2. Hypoxia level of spheroids was positively correlated with E-cadherin level in pancreatic cancer cell lines.

(A) Representative phase contrast images of four types of pancreatic cancer cell lines cultured on 2D condition. Scale bar, 100 μm. (B) Western blotting analysis of E-cadherin in the 4 types of pancreatic cancer cell lines cultured on NCPs for 7 days. β-actin was examined as the loading control. Signal intensities were quantified, and relative values were shown on the graph. (C) Representative hypoxic images of pancreatic cancer cell lines cultured on NCPs. Hypoxia Probe was added to the culture medium and visualized under fluorescence microscopy, shown in red. The fluorescence and bright field images were merged. Scale bar, 200 μm. (D) Hypoxia levels among the cell lines. Integrated fluorescent intensities (IFI) of the Hypoxia Probe taken in spheroids were calculated as described in Materials and Methods. N = 3. Data are mean ± SD.

Fig 3. Hypoxia level of A549 spheroid was declined with TGF-β2 and elevated with SB431542.

(A) A549 spheroids cultured on NCP for 3 days were stimulated with TGF-β2 for 4 days in indicated concentrations. Hypoxia Probe was then added. Integrated fluorescent intensities (IFI) of Hypoxia Probe were reduced in a dose depending manner without affecting viability. (B) A549 spheroids were treated with TGF-β2 (5 ng/mL) and indicated concentration of SB431542 for 4 days. IFI of Hypoxia Probe were increased in a dose depending manner without affecting viability. N = 3. Data are mean ± SD. (C) Representative images of hypoxic spheroids restituted by SB431542. A549 spheroids were treated with 5 ng/mL TGF-β2 and SB431542 at indicated concentrations. Hypoxia levels were visualized with Hypoxia Probe in red color Scale bars, 200 μm.

Fig 4. Procedures for screening and validation of EMT inhibitors.

(A) A Scheme of EMT inhibitor screening using NCP and Hypoxia Probe. TGF-β2 (5 ng/mL) and drugs (1 μM) were added at day 3 after seeding of A549 cells. SB431542 was used as a positive control. Hypoxia Probe was added at day 6, and then cell viability were evaluated at day 7. (B) A schematic overview of EMT inhibitor screening of 1,330 compounds. We provided thresholds as more than 15.9% Cell viability and more than 71.4% SEMTIN activity. Nine compounds were over the thresholds. Of these, 4 compounds were selected for validation. Of these, 2 compounds showed the SEMTIN activity.

Fig 5. Pilot screening of EMT inhibitors from 1,330 pharmacologically active compounds.

Scatter plots was shown. The x-axis indicates the EMT inhibitory efficiency, which was calculated as described in Materials and Methods. These data were the mean value of the independent two experiments. SB-525334 (a potent ALK5/type I TGF-β-receptor kinase inhibitor), SU9516 (a cyclin-dependent kinase 2 (CDK2) inhibitor), GR 127935 (A selective and potent 5-HT1B/1D serotonin receptor antagonist) and Ebastine (a histamine H1-receptor antagonist) as indicated were selected for validation.

Fig 6. Dose dependency of SEMTIN activity of screened compounds.

(A) Representative images of spheroids that was altered by drugs. Spheroids were treated with 5 ng/mL TGF-β2 and each drug at indicated concentrations. Hypoxia levels of spheroids were measured and shown in red. Scale bars, 200 μm. (B) Dose-dependent SEMTIN activities of the compounds. SB431542 is a positive control. SEMTIN activity IC₅₀ of SB-525334, SU9516, and SB431542 were 0.31 μM, 1.21 μM, and 2.38 μM, respectively. N = 4. Data are mean ± SD. (C) Cellular viability under the exposure of compounds.

Fig 7. Validation of EMT inhibitory activities of screened compound, SU 9516.

(A) Representative images of immunofluorescence for E-cadherin of 3D cultured cells. A549 spheroids treated with or without TGF-β2 (5 ng/mL) and SU 9516 in indicated concentration were visualized by immunofluorescence for E-cadherin (green) and with DAPI (blue). (B) E-cadherin expression levels in the 3D culture system. E-cadherin signal intensity in a whole well was integrated and divided by integrated DAPI signal. The relative values were shown as E-cadherin expression change to the vehicle control ± SD of four wells. (C) Representative images of immunofluorescence for E-cadherin of 2D cultured cells. E-cadherin at the plasma membrane (green) and DAPI counter-stained nucleus (blue) were visualized by confocal microscopy. Scale bars, 50 μm.