Patent Application
20150004620
The present invention generally relates to the field of drug screening. More particularly, the present invention relates to methods, devices or system for screening and identification of compounds that inhibit epithelial-mesenchymal transition in a proliferative disease such as cancer.
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 development of modern molecular targeted therapeutics. These small molecule inhibitors function by binding to the ATP-binding site of the dysregulated kinase oncogene, thereby inhibiting the phosphorylation and activation of its signal transduction cascade responsible for sustaining tumor growth.
Many preclinical studies have showed the effectiveness of targeted small molecule inhibitors in killing cancer cells or preventing tumor growth. Examples include Imatinib Mesylate for the treatment of chronic myeloid leukemia and Gefitinib for the treatment of non-small-cell lung cancer. Whilst originally identified and optimized for their anti-proliferative effects, evidence suggests that some of these targeted small molecule inhibitors may also inhibit EMT initiation or sustenance, since the EMT program is modulated by similar signaling pathways for which these molecules have been generated. For example, Ki26894, an ALK5 inhibitor, has recently been shown to decrease the invasiveness and EMT of scirrhous gastric cancer cells. However, an extensive screening effort to identify and quantify the relative effectiveness of existing targeted small molecule inhibitors in EMT modulation has not been methodically attempted. Thus, there is a need to provide a screening assay that can identify the EMT modulating properties of compounds.
According to a first aspect, there is provided a method of identifying inhibitors of epithelial-mesenchymal transition (EMT). The method can comprise comparing different sets of image data obtained from one or more cell colonies before (T1) and after (T2) exposure to a possible inhibitor of epithelial-mesenchymal transition. The method can further comprise
measuring the cell number and a spreading coefficient value in the one or more cell colonies for determining cell count ratio (CCR) and normalized cell dispersion ratio (CDR) for the one or more colonies. The possible inhibitor can then be identified to be an inhibitor of EMT if the determined CCR and CDR indicates that the possible inhibitor i) does not or marginally inhibit growth and inhibits EMT, or ii) inhibits growth and inhibits cell dispersion and optionally inhibits also EMT, or iii) is cytotoxic and inhibits cell dispersion and optionally inhibits also EMT.
In a second aspect, there is provided a device using the method as described herein for identifying inhibitors of epithelial-mesenchymal transition.
In a third aspect, there is provided a system comprising a device as described herein and a camera for recording an EMT time-lapse video.
The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.
As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a gene” includes a plurality of genes.
Epithelial Mesenchymal Transition (EMT) is a fundamental process driving embryonic development particularly during gastrulation and in morphogenesis of the heart primordium, neural crest and somites. Cells engaged in the EMT process undergo complex changes in cell architecture and behavior. In a typical epithelial layer, epithelial cells develop adhesive structures between adjacent cells, such as adherens junctions, desmosomes and tight junctions, to establish robust intercellular adhesions. Epithelial cells are apico-basal polarized, with the apical and basal surfaces serving different functions. Mesenchymal cells, on the other hand, do not have stable intercellular junctions and possess front-to-back leading edge polarity. These characteristics also increase the migratory capacity in mesenchymal cells, owing to the shift of weaker cell-cell adhesion and stronger cell-matrix adhesion. Thus, the EMT process describes a series of events during which epithelial cells lose many of their epithelial characteristics and take on properties that are typical of mesenchymal cells.
For more than a decade, EMT has been recognized as a potential mechanism for the progression of carcinoma. At the onset of tumor progression, dysregulation of the cell cycle machinery can result in proliferation of the normal epithelia to give rise to an adenoma, the adenoma, with additional genetic and epigenetic alterations, can later progress to a carcinoma in situ. The carcinoma in situ is believed to engage the EMT program at the micro-invasive stage, allowing individual carcinoma cells to migrate and intravasate into lymph and blood vessels and eventually disseminate and metastasize to distant organs.
Metastasis of the primary tumor is assisted by the release of cytokines and growth factors that are secreted by die surrounding stroma. Cancer patients are reported to have elevated serum levels of growth factors such as but not limited to hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-beta (TGF-β), insulin-like growth factor-1 (IGF-1) and the like. In addition, numerous carcinoma are found to have over-expression of either wild-type or mutated kinases. These kinase oncogenes play important roles in growth factor signal transduction regulation, and their dysregulation can lead to survival and excessive proliferation of cancer cells as well as the initiation and sustenance of the EMT program and tumor metastasis. These findings have generated great interest in understanding the role of oncogenes and their signaling cascades in tumor growth and the EMT program.
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. Thus, there is a need to provide a method that can extensively screen small molecule inhibitors to identify and quantify their effectiveness in modulating EMT.
Accordingly, the present disclosure relates to the design and development of epithelial-mesenchymal transition (EMT) inhibition drug screening assay. In particular, the present invention relates to a method of identifying inhibitors of epithelial-mesenchymal transition (EMT). The method as described herein may comprise: comparing different sets of image data obtained from one or more cell colonies before (T1) and after (T2) exposure to a possible inhibitor of epithelial-mesenchymal transition. The method as described herein may further comprise
measuring the cell number and a spreading coefficient value in the one or more cell colonies for determining cell count ratio (CCR) and normalized cell dispersion ratio (CDR) for the one or more colonies. Thus, a possible inhibitor may be identified to be an inhibitor of EMT if the determined CCR and CDR indicates that the possible inhibitor i) does not or marginally inhibit growth and inhibits EMT, or inhibits growth and inhibits cell dispersion and optionally inhibits also EMT, or iii) may be cytotoxic and inhibits cell dispersion and optionally inhibits also EMT. In one example, the EMT may be the process wherein epithelial cells lose their epithelial characteristics and transform to typical mesenchymal cells.
In the method as described herein, “image data” refers to images of one or more cell colonies that may be obtained by multiple adjacent field images with or without interstice in between. In particular, the image data may be obtained by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more adjacent field images without interstice in between. The image data may be obtained or provided as different sets of image data obtained from one, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cell colonies. Rather than using representative snapshots, the image data obtained for the method as described herein may encompass all cells in the cell receiving container or vessel. This eliminates the issue of sampling bias, a common problem for high-content image analysis especially when cell distribution is not uniform. In addition, when the entire cell population is analyzed, ratiometric analysis (i.e. comparing T1 and T2 images) may be employed to describe the growth of the cell colony (Cell Count Ratio/CCR) as well as to derive the colony dispersion over time (Cell Dispersion Ratio/CDR).
In one example, each set of image data may be positioned or segmented independently of the presence of nuclei. In particular, the nuclei may be positioned or segmented independently of other fields of images to prevent artifact at the field border. Nuclei positioning or segmentation as used herein may be achieved by the use of known algorithm for processing images, for example, but not limited to, wavelet transform and/or watershed algorithm. In particular, the combination of wavelet transform and watershed algorithm advantageously provides fast, accurate and robust noise and unhomogenous background analysis that is suitable for high-content screening. Furthermore, watershed algorithm allows the splitting of nuclei clusters observed in tightly formed colonies. Images from different nucleic positions or segmentations would then be combined together to provide a large image of the whole, cell colony. Nucleic positions within the cell colony may then be used to determine how much the one or more cell colonies have dispersed. The dispersion or spreading of the one or more cell colonies may then be calculated by a spreading coefficient.
Accordingly, a spreading coefficient is derived from nuclei positions within the cell colony for determining how much the one or more cell colonies have dispersed. As used herein, the “spreading coefficient” may be defined as the standard deviation of cell positions in the one or more cell colonies relative to the center of the one or more colonies. The spreading coefficient may be calculated according to the formula:
where Col indicates all cells of a colony, #Col is the total cell number, [Colx, Coly] is the average position of all nuclei in the colony, [cx, cy] is the position of the cell c.
In one example, a cell colony of the one or more cell colonies may be defined to be a cell colony by applying morphological dilation on the nucleus segmentation of a cell body forming part of a possible cell colony. That is, cell colonies or cell bodies may be estimated by applying a morphological dilation on the nucleus segmentation or position using a disk of about 10 pixels, about 20 pixels, about 30 pixels, about 40 pixels, about 50 pixels, about 60 pixels or more pixels in diameter. In particular, the disk may be of about 30 pixels (48 μm) in diameter. In some examples, cells may be contiguous, such as in a cell colony and nuclei segmentation or position dilation will result in the formation of continuous region areas with surrounding cells, which may then be identified as colonies. As would be apparent to the skilled person, a sample well for analysis may contain one or more large colonies that may correspond to the initial cell spot and one or more smaller colonies. Smaller colonies as provided herein includes, but not limited to, single cells, small clusters of cells, dust and contaminants. In one example, smaller colonies may be excluded from further analysis. That is, only images of nuclei contained in one or more large colonies may be retained for further analysis.
In one example, cell numbers and spreading coefficient value in the one or more cell colonies may be measured to determine cell count ratio (CCR) and normalized cell dispersion ratio (CDR). CCR and CDR values may be determined by combining calculated data obtained from image data obtained from before (T1) or after (T2) exposure to a possible inhibitor.
In one example, CDR threshold may be set suitably between positive controls' average CDR and negative controls' average CDR. Exemplary CDR threshold may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or about 80% of positive controls' average CDR and negative controls' average CDR. In one example, the CDR threshold may be set at about 50% between positive controls' average CDR and negative controls' average CDR.
In one example, negative controls may be determined by treating cells/cell colonies with carriers or solutions used to dilute the compound of interest. Cells/cell colonies in negative control population may be untreated cells/cell colonies. A suitable carrier or solution for negative control includes DMSO and the like.
In one example, positive controls may be determined by treating cells/cell colonies with compounds known to have EMT inhibitory effect. Examples of positive controls include, but not limited to, AG1478, JNJ-38S77605 and BMS-536924.
In another example, CCR threshold may be set at a growth rate that, corresponds to normal growth rate of cells/cell colonies during the T1-T2 time period, in the absence of an EMT inducer. Exemplary CCR threshold is about 1.0, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8 or about 1.9 of growth rate in the absence of an EMT inducer. In the presence of an EMT inducer, the CCR of cells/cell colonies may reach from about 2 to about 4 of growth rate. In particular, CCR threshold may be set at about 1.5 growth rate, which corresponds to normal growth rate of cells during the T1-T2 time period, in the absence of an EMT inducer.
In one example, cytotoxicity in cells/cell colonies may be determined by observing the CCR value. CCR value of less than about 1.5, about 1.4, about 1.3, about 1.2, about 1.1 or about 1.0 may correspond to cytotoxicity. In one example, CCR value of less than 1.0 may correspond to cytotoxicity as this would correlate to fewer cells in T2 than in T1. In one example, to validate cytotoxicity observed, cytotoxicity in cells/cell colonies may optionally be performed with a separate cell viability assay such as MTS assay.
The term “marginally” as used herein refers to at least less than about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% growth inhibition. In particular, marginal growth inhibition as used herein refers to close to at least less than about 1% or barely no growth inhibition observed. The term “marginally” as used herein may be interchangeably used with “substantially no growth” or “essentially no growth”. In one example, the term “marginally inhibit growth” may refer to CCR value of between about 1.1 to about 1.9, about 1.2 to about 1.8, about 1.3 to about 1.7 or about 1.4 to about 1.6. In one example, inhibition of growth may refer to CCR value of between about 0.9 to about 1.5, 1.0 to about 1.4 or 1.1 to about 1.3.
In one example, the inhibition of EMT may be measured and/or validated by assays commonly known by the person skilled in the art, for example Western Blots to detect changes in abundance of EMT-related proteins such as, but not limited to, E-cadherin, Plakoglobin, Vimentin, MMP-2, MMP-9, Snail, Slug, Twist, and the like).
In one example, the inhibition of cell dispersion may be measured by assays commonly known by the person skilled in the art, for example cell movement tracking, cell morphology assessment, transwell invasion assays, and the like.
The term “normalization” as used herein refers to CDR or CDR % value of each set of image data calculated by comparing the respective positive and negative controls for each induction condition as the boundary limits as follows:
where CDRpos and CDRneg are the average CDR values of the negative and positive control wells respectively in each test plate.
In one example, the time between obtaining the sets of image data obtained before (T1) and after (T2) the exposure to the possible inhibitor may be selected to be sufficient to allow the possible inhibitor to cause a reaction indicative for the activity of the possible inhibitor. The time between T1 and T2 may be optimized to allow for EMT and/or sufficient cell motility and/or dispersion to occur in the cell colonies without allowing significant cell proliferation response to occur as EMT quantification may be masked if the motility response is too slow. A non-limiting range of the time may be between 1 to 36 hours, or between 1 to 24 hours, or between 5 to 36 hours, or between 5 to 24 hours, or between 10 to 24 hours, or between 15 to 24 hours, or between 20 to 24 hours, about 24 hours, or the like. In one example, the method as disclosed herein may further comprise continuous recording of the one or more colony after addition of a growth factor.
In one example, the one or more cells may be: cells grown at the surface of a container until colony formation before exposure to the possible inhibitor. In one example, the cells may be provided initially as cell suspension drop deposited onto a dry surface. To provide high-throughput, accurate and consistent deposition of cells, a multi-channel liquid-handling device or apparatus known in the art may be utilized. The cells would then be confined within the drop and will be kept confined until the one or more colonies formed. The confinement of cells within the drop advantageously allows fast attachments and growth of sparsely attached cells such that colonies are formed at a short period of time. The confinement of cells also ensures ease determination of the boundary of the initial epithelial colony. To encourage effective cell attachment, the drop of cell suspension may be cultured at 37° C. and 5% CO2 environment. In one example, the drop of cell suspension may be provided in small amount such as, but not limited to, about 0.1 μl, about 0.2 μl, about 0.3 μl, about 0.4 μl, about 0.5 μl, about 0.6 μl, about 0.7 μl, about 0.8 μl, about 0.9 μl, about 1.0 μl, about 1.5 μl, about 2.0 μl, or more. To prevent evaporation of the drop of suspension, drop of cells may be confined in sealed cell receiving container or vessel. Thus, in one example, the one or more cells may be comprised in a CO2 independent culture medium to compensate for the lack of circulating CO2 in the sealed container or vessel.
In one example, the one or more cells may be selected from cells that demonstrate epithelial-mesenchymal transition within its life cycle. The one or more cells may demonstrate fast epithelial-mesenchymal transition phenotypic response to known epithelial-mesenchymal transition stimuli, including, but not limited to at least one endogenous and/or at least one exogenous growth factors. The fast epithelial-mesenchymal transition advantageously allows for epithelial-mesenchymal transition quantification with minimal masking by cell proliferation response that may occur if the motility response is too slow. In one example, the epithelial-mesenchymal transition in the one or more cells may be initiated and maintained by exposure of the one or more cells to at least one exogenous and/or endogenous growth factor, for example epidermal growth factor (EGF), hepatocyte growth factor (HGF), Insulin like growth factor-1 (IGF-1), fibroblast growth factor (FGF), angiopoietin-1 (Ang1), enodostatin (Endo), interleukins-1, -4, -6, -8, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TGFα), stromal-derived factor-1 (SDF1), transforming growth factor beta (TGFβ), or a combination thereof. In one example, the epithelial-mesenchymal transition in the one or more cells may be initiated and maintained by exposure of the one or more cells to at least one exogenous growth factor.
In particular, the one or more cells may be selected from cells which react to the possible inhibitor of epithelial-mesenchymal transition. A non-limiting example of cells as disclosed herein includes but not limited to Nara Bladder Tumor No. 2 cells (NBT-II), A549 lung adenocarcinoma line and Madin-Darby Canine Kidney (MDCK) cell lines.
In one example, the cells may be exposed to the possible inhibitor at different concentrations. For example, the cells may be exposed to the possible inhibitor at about 0.1 μM, about 0.5 μM, about 1.0 μM, about 2.0 μM, about 2.5 μM, about 3.0 μM, about 4.0 μM, about 5.0 μM, about 6.0 μM, about 7.0 μM, about 8.0 μM, about 9.0 μM, about 10.0 μM, about 20.0 μM or more. The cells may be exposed to the possible inhibitor for about 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 12, 16, 24, 30, 36, 48, 72, 96 or more hours. In one example, the growth factor may be added to the one or more cells after the addition of the possible inhibitor. In particular, the growth factor may be added to the one or more cells between 1 to 36 hours, or between 1 to 24 hours, or between 5 to 36 hours, or between 5 to 24 hours, or between 10 to 24 hours, or between 15 to 24 hours, or between 20 to 24 hours, or about 24 hours after addition of the possible inhibitor. In another example, the possible inhibitor may be added at the same time the cell colonies are formed or after the cell colonies are formed.
In one example, the possible inhibitor may be small molecule compounds.
In one example, the one or more cells as described herein may be stably or transiently transfected with a reporter gene. In one example, the reporter gene may be an optically detectable reporter gene that may be detected for imaging purposes that allows for the tracking of cells. The optically detectable reporter gene may encode for a fluorescent or luminescent protein that may be detected using any known imaging microscopy that allows live-cell imaging, for example, an epifluorescent/confocal microscope, an epifluorescent/confocal microplate imager and the likes.
In one example, the possible inhibitor may be identified to be an inhibitor of EMT if the determined CCR and normalized CDR indicate that the possible inhibitor inhibits EMT and does not or marginally inhibit growth. The inhibition of epithelial-mesenchymal transition by a possible inhibitor as identified by the method as described herein may be indicative of an anti-cancer drug or a drug that may be used in cancer treatment.
In one example, the method as described herein may further comprise conducting of downstream analysis of protein or gene expression of the cell colonies after incubation with, growth factor and inhibitor. Possible downstream assays that may be used include, but not limited to Western-Blot, Southern Blot, PCR, RT-PCR or the like. Thus, in one example, the method as described herein may further comprise a Western-Blot with cells of the one or more cell colonies after incubation with growth factors.
The method of screening of the present invention advantageously may be implemented or programmed into a robotic liquid handler to deposit consistent, reproducible cell colonies as confined spots onto multi-well plates.
For example, also disclosed is a device using the method as described herein for identifying inhibitors of epithelial-mesenchymal transition. The device may be a high-throughput screening device that comprises a multi-channel liquid-handling machine. This ensures the formation of consistent, reproducible cell colonies in well confined spots within the cell receiving container or vessel. In one example, the device may comprise a droplet dispenser unit to eject droplets of cell suspensions into a cell receiving container or vessel such as, but not limited to, a plate, well or flask. In one example, the cell receiving container may be a well. In particular, the cell receiving container may be a multi-well container, such as a 96 well plate. In one example, the container is sealable to avoid evaporation of cell culture medium after dispensing of the cell suspension into the cell receiving container.
Also disclosed is a system that may comprise a device as described herein and a camera for recording an EMT time-lapse video. In one example, the system may further comprise means to carry out downstream analysis, such as a Western blot, with cells of the one or more cell colonies.
In one example, a method for identifying or screening inhibitors of epithelial-mesenchymal transition in a proliferative disease is provided. The method may be carried out as conducted in Example 1. The concept behind the method as described herein is illustrated in
One possible arrangement of the method as described herein is illustrated in
In one example, the method as described herein comprise the step of capturing images of cells before (T1) and after (T2) the addition of the inhibitor.
In one example, the captured images of T1 and T2 may then be analysed for any changes in cell proliferation and cell dispersion pattern as compared to a positive and negative control. Cell proliferation and cell dispersion pattern may be analysed as illustrated in Example 2. Briefly, total cell numbers in a colony and a cell dispersion or spreading coefficient value may be measured through image segmentation routine as illustrated in
In one example, possible useful inhibitors are detected through analysis of cell dispersion ratio and cell count ratio. Any cells that displays cell dispersion ratio vs. cell count ratio above a threshold criteria would be considered to be a possible useful inhibitors (Example 3 and
In one example, as illustrated in Example 3 and Table 2, 25 shortlisted compounds were tested for their EMT inhibitory properties. An exemplary profile of an inhibitor (for example Gefitinib) that is effective against EMT by specific growth factors only is illustrated in
In one example, downstream assays as those described in Examples 4 and 5 may be incorporated in the method as described herein to provide for secondary validation. In particular, time-lapsed video may be used to observe cell colony behaviour after the addition of potential inhibitor. Alternatively, the expression of EMT markers such as E-cadherin and matrix metalloproteinase-13 may be used to confirm the phenotype of cells treated with potential inhibitor (
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
Test compounds are purchased from various vendors (Selleck Chemicals, Sigma Aldrich, SYN|thesis MedChem, and Tocris Bioscience). Compound stocks are assembled in 96-well V-bottom plates (Greiner). For screening studies, test compounds at both 0.25 mM and 1.0 mM concentrations in DMSO were prepared, with each occupying a single well in columns 2-11 of the stock plates. For dose response studies, test compounds were prepared in duplicate wells and serially diluted in DMSO, starting with a 1.0 mM concentration. Stock plates were stored at −20° C. and thawed to room temperature-before use.
Nara Bladder Tumor No. 2 (NBT-II) cells were purchased from American Type Culture Collection and were maintained for less than six months after receipt. The cell line was authenticated in Institute of Molecular and Cell Biology by desmoplakin and E-cadherin markers and by their morphology. The cells were tested Mycoplasma free prior to the experiments (MycoAlert, Lonza).
NBT-II cells were stably transfected with mcherry-fluorescent H2B and maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Scientific), 1 μg/ml puromycin (Sigma) and 100 units/ml penicillin-streptomycin (1× pen-strep, Invitrogen). Cells were grown to 80% confluency in tissue culture flasks prior to plating. Cells were trypsinized and concentrated to a density of 5×106 cell/ml in CO2-independent medium (Invitrogen) supplemented with 10% FBS. The cell suspension was then evenly dispersed into the wells of two columns of a 96-well V-bottom plate. Using a robotic liquid-handling station (Bravo, Agilent Technologies), 0.5 μl of cell suspension was transferred from the two columns of the cell suspension-loaded plate and deposited into the center of the wells of two columns of a 96-well clear bottom, black assay plate (Corning). This process was repeated six times so that all 96 wells of the assay plate were deposited with a cell suspension spot. The plate was then sealed to minimize evaporation of the cell suspension spots and transferred to a 37° C., 5% CO2 incubator to allow for cells to attach to the culture surface. After 1 hour, the plate was gently washed with medium once to remove unattached cells, refreshed with 100 μl of assay medium (DMEM supplemented with 10% FBS and 1× pen-strep), and then further incubated to allow for cell-cell contacts to establish in the cell colonies.
After 4 hours of incubation, the cell colonies for each well were imaged using a confocal microplate imager (MetaXpress Ultra, Molecular Devices) with 10× Plan Fluor objective, 561 nm laser excitation and 593/40 nm emission filter configuration. Four tiled, non-overlapping images were acquired around the center of each well, and were then stitched together during image analysis to generate a montage covering an area of 3.2 mm×3.2 mm. These images (T1) represent the initial state of the cell colonies before EMT induction.
After the T1 images were acquired, 1 μl of test compounds were transferred from compound stock plates and added to the assay plates. Appropriate negative controls (1 μl DMSO) and positive controls (1 μl 1.0 mM compound in DMSO) were also added into columns 1 and 12 of each assay plate, respectively. The assay was optimized to use AG1478, JNJ-38S77605 and BMS-536924 as reference positive control compounds for EGF-, HGF- and IGF-1-induced EMT, respectively. The cultures were then further incubated overnight.
The next day, 50 μl of growth factor-containing medium was added to each well of the assay-plates. For each of the growth factor-induced EMT spot migration assays, the final growth factor concentrations in each well is optimized to be 20 ng/ml EGF (Sigma), 4 ng/ml HGF (Calbiochem) or 150 ng/ml IGF-1 (R&D Systems), respectively. The cultures were then incubated for another 24 hours, to allow for EMT and sufficient cell motility/dispersion to occur in the cell colonies.
Finally, the cell colonies were imaged again using the microplate imager, as described above. These images (T2) represent the final state of the cell colonies after compound treatment and EMT induction. The acquired T1 and T2 image sets for each assay plate were then sent for image analysis.
The acquisition of each well was obtained by four adjacent field images with no interstice in between. Each field image was loaded and nuclei were initially segmented independently of other fields to prevent artifact at the field border. Nuclei segmentation was achieved by combining a wavelet transform which is robust to noise and unhomogeneous background, and a watershed algorithm based on intensities to split nuclei clusters. Mask of nuclei segmentation of the different fields were then stitched together in order to obtain a large segmentation of the whole well. Cell bodies were estimated by applying a morphological dilation on the nucleus segmentation using a disk of 30 pixels (48 μm) in diameter. In cells that are contiguous, such as in a cell colony, nuclei segmentation dilation will result in the formation of continuous region areas with surrounding cells which can then be identified as colonies. In general, a well will contain one big colony (corresponding to the initial cell spot) and several much smaller colonies (corresponding to single cells, small cluster of cells, dust and contaminations. Only nuclei contained in the biggest colony were kept and subsequently analyzed. A spreading coefficient was derived from nuclei positions in order to measure how much the colony had dispersed. The spreading coefficient is defined as the standard deviation of the cell positions in the cell colony relative to the center of the colony:
where Col indicates all cells of a colony. #Col is the total cell number, [Colx, Coly] is the average position of all nuclei in the colony, [cx, cy] is the position of the cell c. The coefficient sp is homogenous with a distance and indicates the relative cell dispersion from the colony center.
Total cell number and spreading coefficient values for each well were then exported into an Excel sheet. The Cell Count Ratio (CCR) and Cell Dispersion Ratio (CDR) values were calculated by combining data calculated from T1 and T2 images for each well, while the normalized CDR or CDR % of each well calculated by taking the CDR values of negative and positive controls as the boundary limits:
where CDRpos and CDRneg are the average CDR values of the negative and positive control wells respectively in each test plate.
EMT inhibitory effects of selected compounds were validated by NBT-II epithelial colony time-lapse videoscopy. NBT-II cells were plated onto a 12-well plate (BD) at a low density of 2,000 cells per well in 1 ml of assay medium. Cells were allowed to grow and form epithelial colonies for a period of 72 hours. The cultures were then refreshed with assay medium containing test compounds and further incubated overnight. The next day, growth factor (EGF, HGF or IGF-1) was added prior to video imaging. Video imaging of individual cell colonies was performed using a video microscope incubator system (Axiovert-200M, Carl Zeiss). Time-lapse images were taken at 5 min intervals for 19 h.
NBT-II cells were treated with compounds at 0.5, 2 and 8 μM overnight and incubated with a growth factor for 24 h. Cells were lysed with protease/phosphatase inhibitor-containing RIPA buffer. Proteins are separated in 8% polyacrylamide gels and transferred to PDVF membranes. Membranes were blocked in 5% BSA and incubated at 4° C. overnight with MMP-13 (Millipore), E-cadherin (BD) and α-tubulin (Sigma) primary antibodies. Membranes were then developed with HRP-conjugated secondary antibody (Amersham) and ECL substrate (Millipore).
For western blot experiments with other cell lines, cells are treated either with AZD0530, such as 2 μM AZD0530 or DMSO, such as 0.02% DMSO control for 48 h. The samples are then processed using the same procedure as described above. The samples were probed with MMP-13, E-cadherin, plakoglobin (Cell Signaling), α-tubulin, and β-actin (Cell Signaling) antibodies.
Cells were maintained in RMPI supplemented with 10% fetal bovine serum and 1× pen-strep. Cells were grown to 80% confluency in tissue culture flasks prior to plating. Cells were trysinized and plated onto a 12-well plate (BD) at a low density of 2000 cells per well in 1 ml of RMPI. Each ovarian cell line was seeded into two wells of a 12 well tissue culture plate. For each ovarian cell line, one of the two wells was treated with 2 μM AZD0530 and the other well treated with 0.02% DMSO control for 48 hours. Cells were imaged using a time-lapse video microscope incubator system (Leica) controlled by Metamorph software (Molecular Devices). Time-lapse images were taken at 5 min intervals for 24 hours and saved as tiff format. Each individual tiff images were compiled into stack images using the stack creation function in Metamorph. Each cell was tracked using the particle tracking function in Metamorph software. The tracking function detected and recorded the x and y coordinates of the cell being tracked for each image in the stack. The x and y coordinates were used to plot the track of the individual cell in excel. The total distance travelled, displacement and average velocity of the tracked cell were then tabulated.
Error bars in CDR dose response plots represent standard deviation of replicate samples. CDR IC50 values were calculated through sigmoidal curve fitting of CDR dose response plots using GraphPad Prism software.
An overview of the EMT screening assay is illustrated in
In this study, the NBT-II reporter cell line was used. The cells were stably transfected with H2B-mcherry to label the nuclei, so that the migration of these cells could be tracked through live-cell fluorescent imaging. NBT-II is an ideal cell model for the study of EMT because of its fast EMT phenotypic response to several known EMT stimuli, such as EGF, HGF and IGF-1. As evident in
For compound screening, the spot migration assay to identify compounds that could inhibit EMT induced by EGF, HGF. or IGF-1 signaling was optimised. Appropriate compounds (AG1478, JNJ-38877605 and BMS-536924) were selected as reference positive controls for each EGF, HGF and IGF-1 EMT assay, respectively. The screen is conceptualized as a high-content imaging assay, whereby colony nuclei in each well are imaged and analyzed prior to compound treatment (T1 images), and 24 hours after EMT induction (T2 images). The effect of the screening compounds in this assay was grouped into three categories: [1] compounds that are cytotoxic or growth inhibitory to cells: [2] compounds that can inhibit EMT and are not growth inhibitory to cells; and [3]compounds that are not EMT or growth inhibitory. The cell colony examples shown in
The analysis routine developed for this screening assay, is illustrated in
A collection of 267 targeted inhibitor compounds was tested to assess if any of them could inhibit EGF-, HGF- or IGF-1-induced EMT in this screening assay (
To assess the EMT inhibition potency of the 25 shortlisted compounds, the compounds were retested at diluting concentrations starting from 6.67 μM, using the same EMT spot migration assay for EGF, HGF or IGF-1 signaling. CDR dose response plots were generated for each compound/growth factor combination and the CDR IC50 values corresponding to the EMT inhibition potency were determined. Compounds that were effective against EMT by specific growth factors only were identified (
aClinical trials information: ClinicalTrials.gov
One compound was selected from each target group and their EMT inhibitory response were validated via time lapse video. The videos confirmed that an EGFR, c-Met and IGF-1R inhibitor could specifically inhibit EGF-, HGF- or IGF-1-induced EMT, respectively, as expected (data not shown). ALK5, MEK, PI3K and SRC targeting compounds were also shown to inhibit EMT induced by all three growth factors (data not shown). This finding is surprising as these compounds are not the immediate and direct antagonists of the growth, factors linked to EMT signaling.
The possibilities that ALK5, MEK, PI3K and SRC targeting compounds could modulate the expression of EMT markers, such as E-cadherin and matrix metalloproteinase-13 (MMP-13) under EMT-activated conditions were also investigated (
The synergism effects between compound combinations in inhibiting the EMT phenotype was also investigated (Table 3). In this assay, EMT inhibition combination index (CI) values of ALK5 inhibitor A83-01 and c-Met inhibitor JNJ-38877605 combination against HGF-induced EMT was investigated. Cell dispersion ratio dose response profiles of A83-01 and JNJ-38877605 at fixed combinations ratios of 1:4, 1:2, 1:1 and 3:1 were generated using the spot migration assay. To determine if the EMT inhibitory effects obtained with different compound combinations were synergistic, the inhibition effect CI values were calculated according to the Chou0Talalay method using CalcuSyn software (Biosoft) (where CI>1.1, antagonism; CI=0.9-1.1, additive effect, CI=0.2-0.9, synergism; and CI<0.2 strong synergism). The results indicated that the combination treatment acted synergistically against HGF-induced EMT.
The development and implementation of an EMT inhibition screening assay adapted for high-throughput, high-content screening of small molecule compounds is described herein. In one example, a robotic liquid handler was programmed to deposit consistent, reproducible cell colonies as confined spots onto multi-well plates (
For image analysis, the wavelet transform and watershed segmentation methods were used because the resultant nuclei segmentation is fast and accurate, suitable for high-content screening (
The EMT spot migration assay has key advantages over traditionally described cell migration quantification methods, such as the Boyden chamber or the in vitro scratch techniques. In general, these techniques are prone to sampling bias because, for practicality reasons, only representative microscope views and not the entire well image are chosen for analysis. Another key strength of the spot migration assay is that cell proliferation and cell dispersion within each well may be quantitated simultaneously (
The assay of the present invention may be used to analyze whether targeted compounds previously selected and optimized to kill oncogene-addicted cells, can also be used to effectively inhibit EMT signaling.
The screening assay of the present invention may address the relative propensity and potency for small molecule compounds to block growth factor-induced EMT signaling. Through the primary screen and subsequent secondary assays, the druggable targets ALK5, MEK, SRC and to some extent PI3K were discovered by the method, of the present invention to play a more significant role in EMT modulation and cancer progression, as their associated targeted compounds are inhibitory to several EMT-inducing growth factors (Table 2). As would be appreciated by the person skilled in the art, the targeted compound library provided herein represents only a small subset of the targeted compounds that have been developed by universities and the pharmaceutical industry, and does not encompass all the druggable targets identified to date. Therefore, further extension of this EMT spot migration assay to include other diverse targeted compound libraries, such as the one assembled by Bamborough et al., may allow the discovery other potent EMT inhibitors and EMT modulating targets. The selected compounds may then be evaluated for their ability to revert the mesenchymal-like phenotype of cancer cells in vitro and immuno-compromised mice. The synergism effects between compound combinations in inhibiting the EMT phenotype were also investigated and disclosed herein (example in Table 3). The method of the present invention may facilitate in the design of new therapeutic modalities based on the EMT concept to interfere with tumor progression and to suppress resistance to chemotherapeutic agents.
The present invention may be used to screen for inhibitors of the EMT in a proliferative disease such as cancer.
Advantageously, the method of screening is robust and time efficient. The method of the present invention provides for a high-throughput and high-content screening method of screening small molecule compounds.
Advantageously, the present invention can be used to screen compound libraries for small molecule compounds that are effective in inhibiting EMT signaling in response to growth factor treatment. Thus, allowing the discovery of unknown potent EMT inhibitors and EMT modulating targets.
Advantageously, the present invention allows for simultaneous analysis of both cell growth and cell migration responses on the same test sample. The present invention also facilitates for dose titration studies to further characterize the EMT inhibition potency of screened compounds.
It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.
This application claims the benefit of priority of U.S. provisional application No. 61/582,276, filed 31 Dec. 2011, the contents of it being hereby incorporated by reference in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2012/000491 | 12/28/2012 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61582276 | Dec 2011 | US |
