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A cell-based small molecule screening method for identifying inhibitors of epithelial-mesenchymal transition in carcinoma.

Chua KN, Sim WJ, Racine V, Lee SY, Goh BC, Thiery JP - PLoS ONE (2012)

Bottom Line: The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics.Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target.In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling.

View Article: PubMed Central - PubMed

Affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.

ABSTRACT
Epithelial Mesenchymal Transition (EMT) is a crucial mechanism for carcinoma progression, as it provides routes for in situ carcinoma cells to dissociate and become motile, leading to localized invasion and metastatic spread. Targeting EMT therefore represents an important therapeutic strategy for cancer treatment. The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics. Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target. We have developed a novel screening assay that can lead to the identification of compounds that can inhibit EMT initiated by growth factor signaling. This assay is designed as a high-content screening assay where both cell growth and cell migration can be analyzed simultaneously via time-course imaging in multi-well plates. Using this assay, we have validated several compounds as viable EMT inhibitors. In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling. Overall, this EMT screening method provides a foundation for improving the therapeutic value of recently developed compounds in advanced stage carcinoma.

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Related in: MedlinePlus

Image processing procedure to determine the cell count and dispersion values of a well.(A) Colony nuclei image of each well was obtained by stitching four adjacent, non-overlapping fields together. The example here shows a primary cell colony surrounded by several cell outliers. (B) Nuclei segmentation, which consists of a wavelet transform and watershed algorithm steps, was applied to identify all nuclei in the well. (C) The nuclei segmentation mask was then dilated to generate merging region areas where distinct cell clusters could be isolated. In general, the largest region (yellow), representing the cell colony of interest, and other smaller regions (other colors), representing outlier cell clusters, were identified. (D) Nuclei within the colony of interest were kept for measurement. Cell count was determined by the total nuclei count within the colony. Cell dispersion was determined by applying the spreading coefficient formula. The blue arrow represents a vector centered on the colony center with distance equal to the spreading coefficient.
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pone-0033183-g002: Image processing procedure to determine the cell count and dispersion values of a well.(A) Colony nuclei image of each well was obtained by stitching four adjacent, non-overlapping fields together. The example here shows a primary cell colony surrounded by several cell outliers. (B) Nuclei segmentation, which consists of a wavelet transform and watershed algorithm steps, was applied to identify all nuclei in the well. (C) The nuclei segmentation mask was then dilated to generate merging region areas where distinct cell clusters could be isolated. In general, the largest region (yellow), representing the cell colony of interest, and other smaller regions (other colors), representing outlier cell clusters, were identified. (D) Nuclei within the colony of interest were kept for measurement. Cell count was determined by the total nuclei count within the colony. Cell dispersion was determined by applying the spreading coefficient formula. The blue arrow represents a vector centered on the colony center with distance equal to the spreading coefficient.

Mentions: The analysis routine developed for this screening assay is illustrated in Figure 2. For each well image, total cell numbers in the colony and a spreading coefficient value were measured through the image segmentation routine. The spreading coefficient is defined as the standard deviation of the cell positions in the colony relative to the center of the colony. By combining time-course images of T1 and T2, we obtained the derived measurements Cell Count Ratio (CCR) and Cell Dispersion Ratio (CDR), which correspond to the cell growth status and the cell migration/scattering status of each cell colony, respectively. The results generated from these two ratio parameters were used to determine the EMT inhibitory properties of the test compounds. We also analyzed the uniformity and robustness of the assay (positive and negative control well CDR uniformity plots shown in Figure S1). We validated that the CDR signal was robust in the screening assay, where intra-plate Z-factor was consistently above 0.5 between positive and negative control signals.


A cell-based small molecule screening method for identifying inhibitors of epithelial-mesenchymal transition in carcinoma.

Chua KN, Sim WJ, Racine V, Lee SY, Goh BC, Thiery JP - PLoS ONE (2012)

Image processing procedure to determine the cell count and dispersion values of a well.(A) Colony nuclei image of each well was obtained by stitching four adjacent, non-overlapping fields together. The example here shows a primary cell colony surrounded by several cell outliers. (B) Nuclei segmentation, which consists of a wavelet transform and watershed algorithm steps, was applied to identify all nuclei in the well. (C) The nuclei segmentation mask was then dilated to generate merging region areas where distinct cell clusters could be isolated. In general, the largest region (yellow), representing the cell colony of interest, and other smaller regions (other colors), representing outlier cell clusters, were identified. (D) Nuclei within the colony of interest were kept for measurement. Cell count was determined by the total nuclei count within the colony. Cell dispersion was determined by applying the spreading coefficient formula. The blue arrow represents a vector centered on the colony center with distance equal to the spreading coefficient.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303807&req=5

pone-0033183-g002: Image processing procedure to determine the cell count and dispersion values of a well.(A) Colony nuclei image of each well was obtained by stitching four adjacent, non-overlapping fields together. The example here shows a primary cell colony surrounded by several cell outliers. (B) Nuclei segmentation, which consists of a wavelet transform and watershed algorithm steps, was applied to identify all nuclei in the well. (C) The nuclei segmentation mask was then dilated to generate merging region areas where distinct cell clusters could be isolated. In general, the largest region (yellow), representing the cell colony of interest, and other smaller regions (other colors), representing outlier cell clusters, were identified. (D) Nuclei within the colony of interest were kept for measurement. Cell count was determined by the total nuclei count within the colony. Cell dispersion was determined by applying the spreading coefficient formula. The blue arrow represents a vector centered on the colony center with distance equal to the spreading coefficient.
Mentions: The analysis routine developed for this screening assay is illustrated in Figure 2. For each well image, total cell numbers in the colony and a spreading coefficient value were measured through the image segmentation routine. The spreading coefficient is defined as the standard deviation of the cell positions in the colony relative to the center of the colony. By combining time-course images of T1 and T2, we obtained the derived measurements Cell Count Ratio (CCR) and Cell Dispersion Ratio (CDR), which correspond to the cell growth status and the cell migration/scattering status of each cell colony, respectively. The results generated from these two ratio parameters were used to determine the EMT inhibitory properties of the test compounds. We also analyzed the uniformity and robustness of the assay (positive and negative control well CDR uniformity plots shown in Figure S1). We validated that the CDR signal was robust in the screening assay, where intra-plate Z-factor was consistently above 0.5 between positive and negative control signals.

Bottom Line: The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics.Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target.In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling.

View Article: PubMed Central - PubMed

Affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.

ABSTRACT
Epithelial Mesenchymal Transition (EMT) is a crucial mechanism for carcinoma progression, as it provides routes for in situ carcinoma cells to dissociate and become motile, leading to localized invasion and metastatic spread. Targeting EMT therefore represents an important therapeutic strategy for cancer treatment. The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics. Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target. We have developed a novel screening assay that can lead to the identification of compounds that can inhibit EMT initiated by growth factor signaling. This assay is designed as a high-content screening assay where both cell growth and cell migration can be analyzed simultaneously via time-course imaging in multi-well plates. Using this assay, we have validated several compounds as viable EMT inhibitors. In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling. Overall, this EMT screening method provides a foundation for improving the therapeutic value of recently developed compounds in advanced stage carcinoma.

Show MeSH
Related in: MedlinePlus