Patent Application
20070005263
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The present invention provides information technology based techniques including computer software in therapeutics or drug discovery. In an exemplary embodiment, the present invention provides software for determining information about properties of substances based upon information about structures of living, fixed or non-living cells, or cell fractions. Computer code according to the present invention enable researchers and scientists to identify promising candidates in the search for new and better medicines and medical techniques.
For a long time, researchers in the pharmaceutical field have sought for better ways of searching for substances possessing properties which make them suitable as medicines. In the early days, researches generally relied upon dyes, or extracts from plants, or microbiological broths for such substances. Examples of such substances include aspirin, and antibiotics.
Substances having desirable bio-active properties, however, are often difficult to isolate and identify. Advances in organic chemistry such as rapid chemical synthesis techniques have increased the number of compounds that researchers want to test for biological activity. Conventionally, substances were often tested on humans or animal subjects to determine biological activity. While results from such tests can be highly predictive, these types of tests can be time consuming, limiting the progress of the testing.
There have been some attempts to use image acquisition techniques to screen for large numbers of molecules based upon cell information. Conventional image acquisition systems exist that generally capture digitized images of discrete cells for identification purposes. Identification often occurs during the image capturing step, which is limiting. In most cases, these conventional techniques cannot comprehensively provide for complete cellular information but can only be used to identify a fairly limited set of information.
What is needed are techniques for collecting and managing information useful in finding the effects of manipulations on cell function, response or behavior.
According to the present invention, techniques for using information technology in therapeutics or drug discovery. In an exemplary embodiment, computer software for determining information about the properties of substances based upon information about a structure or the morphology of living, fixed or dead cells (e.g., elements, cell portions and cell fractions) exposed to substances are provided. Computer software according to the present invention enables researchers and/or scientists to identify promising candidates in the search for new and better medicines or treatments using, for example, a cellular informatics database.
According to the present invention, a computer program for identification and verification of biological properties of substances can include code that administers a sample of the substance to a cell. The code determines one or more features for two or more cell components, or markers, in the presence of the substance. The code can form one or more descriptors from the features. Descriptors can be formed by combining features of two or more cell components as identified using the markers. The code can then search one or more descriptors obtained from prior administered substances upon cells in order to locate descriptors having a relationship to the descriptors noted for the substance under study. The code predicts properties of the administered substance based upon the properties of the prior administered substances using the relationship between the descriptors. The code can provide for identifying properties of substances based upon effects on cell characteristics. Candidate drug mechanisms of action, potency, specificity, pharmacodynamic, and pharmacokinetic parameters, toxicity, and the like can be used as substance properties.
In another embodiment according to the present invention, computer programs for animal model selection, clinical trial design and patient management can be provided.
In a further embodiment according to the present invention, techniques for using cellular information in predictive methods for acquiring, analyzing and interpreting cellular data are incorporated into a computer program product including code. In one such embodiment, code for predicting properties of an unknown substance based upon information about effects of one or more known substances on a cell population is provided. The code performs a variety of tasks, such as populating a database with descriptors of cells subjected to known substances. Such descriptors can be determined from imaging the cell population. However, in some embodiments, descriptors can be derived by measurements and combinations of measurements and the like. The code determines descriptors for the unknown substance from imaging a second cell population. The second cell population has been treated with the unknown substance. Then, the code can determine a relationship between the descriptors determined from the unknown substance with the descriptors determined from the known substance. From this relationship, an inference can be made about the unknown substance.
In a yet further embodiment according to the present invention, a computer program for determining properties of a manipulation based upon effects of the manipulation on one or more cell fractions. The computer program includes code that can provide the manipulation to the cell fractions. The code can also determine one or more features of markers corresponding to cell components within the cell fractions in the presence of the manipulation. Code for forming descriptors from the features is also included. Code for searching in a database in order to locate descriptors based upon at least one of the descriptors obtained from the manipulation is also included. The computer program can include code for determining, based upon the descriptors located in the database, properties of the manipulation.
Moreover, the present invention provides computer software for mapping a manipulation of cells based upon a morphological characteristic. The computer software includes code for providing a plurality of cells, e.g., dead, live, cell fragments, cell components, cell substructures. The software also includes code for manipulating the plurality of cells, where manipulation occurs using a source(s) from one or a combination selected from an electrical source, a chemical source, a thermal source, a gravitational source, a nuclear source, a temporal source, and a biological source. The software code captures a morphological value from the plurality of cells. The morphological value can include one or any combination of characteristics such as a cell count, an area, a perimeter, a length, a breadth, a fiber length, a fiber breadth, a shape factor, an elliptical form factor, an inner radius, an outer radius, a mean radius, an equivalent radius, an equivalent sphere volume, an equivalent prolate volume, an equivalent oblate volume, an equivalent sphere surface area, an average gray value, a total gray value, and an optical density. The software code also assigns a degree of presence of the morphological value, and stores the morphological value from the plurality of cells. These values can be used for a statistical analysis to produce a statistical profile.
Still further, the present invention provides a computer program product for populating a database with manipulated biological information, e.g., cellular enzymatic activities, cellular cascades, cellular promoters, transcription factors, translation factors, cell cycle stage and apoptosis. The computer program product includes code for providing a plurality of cells in various stages of the cell cycle, where the stages of the cell cycle may include at least one selected from interphase, G0 phase, G1 phase, S phase, G2 phase, M phase which itself includes prophase, prometaphase, metaphase, anaphase, and telophase. The computer program also includes code for manipulating each of the cells in the various stages of the cell cycle. The computer program includes code for capturing (e.g., image acquisition) an image of the plurality of manipulated cells where the code for capturing provides a morphometric characteristic of the manipulated cells. The computer program product also includes code for populating a database with the morphometric characteristic of the plurality of manipulated cells. Accordingly, the present invention provides software for populating a database, which can be queried.
Numerous benefits are achieved by way of the present invention over conventional techniques. The present invention can provide techniques for predictive cellular bioinformatics that can stream line a number of important decisions made in the drug discovery industry, medical diagnostics and biological research. The present invention can be implemented on conventional hardware including databases. In other aspects, the present invention can find useful information about substances as well as cells or portions of cells, especially morphology. Embodiments can provide a holistic approach to cell based drug discovery that enables the understanding of properties of substances based on their overall effects on cell biology. The properties include, among others, clinical uses and descriptors, human and veterinary diagnostic uses and tests, or human and veterinary prognostic uses and tests. Depending upon the embodiment, one or more of these advantages may be present. These and other benefits are described throughout the present specification.
A further understanding of the nature and advantages of the invention herein may be realized by reference to the remaining portions of the specification and the attached drawings.
According to the present invention, techniques for using information technology in therapeutics or drug discovery. In an exemplary embodiment, techniques for determining information about the properties of substances based upon information about the structure of living, fixed or non-living cells exposed to the substances are provided. Computer software according to the present invention enables researchers and/or scientists to identify promising candidates in the search for new and better medicines or treatments using, for example, a cellular informatics database. An embodiment according to the present invention is marketed under the name Cytometix™, which is not intended to be limiting.
In a particular embodiment according to the present invention, a cellular informatics database is provided. Embodiments according to the present invention can provide techniques for predicting candidate drug mechanisms of action, potency, specificity, structure, toxicity and the like. In some embodiments, substances or other manipulations can be used for target identification and validation. Embodiments can be useful in areas such as animal model selection, clinical trial design and patient management, including prognostics, drug response prediction and adverse effect prediction.
The system has other features. A network interface 148 may provide a direct connection to a remote server via a telephone link or to the Internet. Network interface 148 may also connect to a local area network (LAN) or other network interconnecting many computer systems. Many other devices or subsystems (not shown) may be connected in a similar manner. Also, it is not necessary for all of the devices shown in
Although the above has been described generally in terms of specific hardware, it would be readily apparent to one of ordinary skill in the art that many system types, configurations, and combinations of the above devices are suitable for use in light of the present disclosure. Of course, the types of system elements used depend highly upon the application. Other examples of system can be found in co-pending application U.S. application Ser. No. ______ (Attorney Docket No. 19681-000200), which has been noted above.
Cell components tracked in presently preferable embodiments can include proteins, protein modifications, genetically manipulated proteins, exogenous proteins, enzymatic activities, nucleic acids, lipids, carbohydrates, organic and inorganic ion concentrations, sub-cellular structures, organelles, plasma membrane, adhesion complex, ion channels, ion pumps, integral membrane proteins, cell surface receptors, G-protein coupled receptors, tyrosine kinase receptors, nuclear membrane receptors, ECM binding complexes, endocytotic machinery, exocytotic machinery, lysosomes, peroxisomes, vacuoles, mitochondria, golgi apparatus, cytoskeletal filament network, endoplasmic reticulum, nuclear membrane, proteosome apparatus, chromatin, nucleolus, cytoplasm, cytoplasmic signaling apparatus, microbe specializations and plant specializations.
The following table illustrates some markers and cell components commonly used by embodiments according to the present invention. Other markers can be used in various embodiments without departing from the scope of the invention.
Then, in a step 202, one or more samples of the manipulation can be provided to one or more of the cells or cell fractions. Manipulations can comprise chemical, biological, mechanical, thermal, electromagnetic, gravitational, nuclear, temporal factors, and the like. For example, manipulations could include exposure to chemical compounds, including compounds of known biological activity such as therapeutics or drugs, or also compounds of unknown biological activity. Or exposure to biologics that may or may not be used as drugs such as hormones, growth factors, antibodies, or extracellular matrix components. Or exposure to biologics such as infective materials such as viruses that may be naturally occurring viruses or viruses engineered to express exogenous genes at various levels. Bioengineered viruses are one example of manipulations via gene transfer. Other means of gene transfer are well known in the art and include but are not limited to electroporation, calcium phosphate precipitation, and lipid-based transfection. Manipulations could also include delivery of antisense polynucleotides by similar means as gene transfection. Other genetic manipulations include gene knock-outs gene over-expression or gene mutations. Manipulations also could include cell fusion. Physical manipulations could include exposing cells to shear stress under different rates of fluid flow, exposure of cells to different temperatures, exposure of cells to vacuum or positive pressure, or exposure of cells to sonication. Manipulations could also include applying centrifugal force. Manipulations could also include changes in gravitational force, including sub-gravitation. Manipulations could include application of a constant or pulsed electrical current. Manipulations could also include irradiation. Manipulations could also include photobleaching which in some embodiments may include prior addition of a substance that would specifically mark areas to be photobleached by subsequent light exposure. In addition, these types of manipulations may be varied as to time of exposure, or cells could be subjected to multiple manipulations in various combinations and orders of addition. Of course, the type of manipulation used depends upon the application.
Then, in a step 204, one or more descriptors of a state in the portions of the cells in the presence of the manipulation can be determined based upon the images collected by the imaging system. Descriptors can comprise scalar or vector values, representing quantities such as area, perimeter, dimensions, intensity, aspect ratios, and the like. Other types of descriptors include one or any combination of characteristics such as a cell count, an area, a perimeter, a length, a breadth, a fiber length, a fiber breadth, a shape factor, an elliptical form factor, an inner radius, an outer radius, a mean radius, an equivalent radius, an equivalent sphere volume, an equivalent prolate volume, an equivalent oblate volume, an equivalent sphere surface area, an average intensity, a total intensity and an optical density. In some embodiments, descriptors can include averages or standard deviation values, or frequency statistics from other descriptors collected across a population of cells. In some embodiments, descriptors can be reduced using techniques such as principal component analysis and the like. A presently preferable embodiment uses descriptors selected from the following table. Other descriptors can also be used without departing from the scope of the invention.
Then, in a step 205, a database of cell information can be provided. Next, in a step 206, a plurality of descriptors can be searched from a database of cell information in order to locate descriptors based upon one of the descriptors of the manipulation. Then, in a step 208, properties of the manipulation are determined based upon the properties of the located descriptors. Properties can comprise toxicity, specificity against a subset of tumors, mechanisms of chemical activity, mechanisms of biological activity, structure, adverse biological effects, biological pathways, clinical effects, cellular availability, pharmacological availability, pharmacodynamic properties, clinical uses and descriptors, pharmacological properties, such as absorption, excretion, distribution, metabolism and the like.
In a particular embodiment, step 206 comprises determining matching descriptors in the database corresponding to a prior administration of the manipulation to the descriptors of the present administration of the manipulation. In a particular embodiment according to the present invention, measurements of scalar values can provide predictive information. A database can be provided having one or more “cellular fingerprints” comprised of descriptors of cell-substance interactions of drugs having known mechanisms of action with cells. Such fingerprints can be analyzed, classified and/or compared using a plurality of techniques, such as statistical classification and clustering, heuristic classification techniques, a technique of creating “phylogenetic trees” based on various distance measures between cellular fingerprints from various drugs. In a present embodiment, scalar, numeric values can be converted into a nucleotide or amino acid letter. Once converted into a corresponding nucleotide representation, the fingerprints can be analyzed and compared using software and algorithms known in the art for genetic and peptide sequence comparisons, such as GCG, a product of Genetics Computer Group, with company headquarters in Madison Wis. In an alternative embodiment, numeric values for the fingerprints can be used by comparison techniques. A phylogenetic tree can be created that illustrates a statistical significance of the similarity between fingerprints for the drugs in the database. Because the drugs used to build the initial database are of known mechanism, it can be determined whether a particular scalar value in a fingerprint is statistically predictive. Finally, a compound fingerprint with no known mechanism of action can be queried against the database and be statistically compared and classified among the drugs in the database that the compound most resembles.
In a particular embodiment, relationships between measured morphological properties of images and physiological conditions can be determined. Relationships can include, for example, treatment of different cell lines with chemical compounds, or comparing cells from a patient with control cells, and the like. In a presently preferable embodiment, a clustering can be performed on acquired image feature vectors. Some embodiments can comprise statistical and neural network-based approaches to perform clustering and fingerprinting of various features. The foregoing is provided as merely an example, and is not intended to limit the scope of the present invention. Other techniques can be included for different types of data.
In some embodiments, clustering and fingerprinting can be performed on features extracted from cell images. In a presently preferable embodiment, procedures for comparisons and phylogenetic analysis of biological sequences can be applied to data obtained from imaging cells.
Select embodiments comprising such approaches enable the use of a broad array of sophisticated algorithms to compare, analyze, and cluster gene and protein sequences. Many programs performing this task are known to those of ordinary skill in the art, such as for example, http://evolution.genetics.washington.edu/phylip.html, and http://evolution.genetics.washington.edu/phylip/software.html.
Embodiments can perform such analysis based upon factors such as numerical value, statistical properties, relationships with other values, and the like. In a particular embodiment, numbers in a numerical features vector can be substituted by one or more of nucleic acid or amino acid codes. Resulting “pseudo-sequences” can be subjected to analysis by a sequence comparison and clustering program. Depending upon the application, many different ways of using the database can be provided. Further details of a step of manipulation are noted more particular below.
In a preferred embodiment, cells can be placed onto a microscope, such as a Zeiss microscope, or its equivalent as known in the art. A starting point, named Site A1, is identified to the microscope. A plurality of exposure parameters can be optimized for automated image collection and analysis. The microscope can automatically move to a new well, automatically focus, collect one or more images, move to a next well, and repeat this process for designated wells in a multiple well plate. A file having a size and an intensity distribution measurement for each color and rank for each well can then be created for the images acquired. Based on this information, a user or a computer can revisit sites of interest to collect more data, if desired, or to verify automated analysis. In a presently preferred embodiment, image automatic focus and acquisition can be done using computer software controlling the internal Z-motor of the microscope. Images are taken using a 10×, 20×, or 40× air long working distance objectives. Sometimes multiple images are collected per well. Image exposure times can be optimized for each fluorescent marker and cell line. The same exposure time can be used for each cell line and fluorescent marker to acquire data.
In a step 246, the regions and objects determined by step 244 can be processed in view of a plurality of classification heuristics to determine cell state based upon selectable criteria. Further, in some embodiments, morphological criteria can be used based upon a cell type to determine cell state. Next, in a step 248, a plurality of region features can be determined. For example, in a representative embodiment, image features can include such quantities as area, perimeter, dimensions, intensity, gray level, aspect ratios, and the like.
In a particular embodiment according to the present invention, data analysis techniques for describing the fluorescence patterns of markers in multiple cell lines in the presence and absence of compounds are provided. Automated image analysis techniques can include determining one or more regions from around nuclei, individual cells, organelles, and the like, called “objects” using a thresholding function. Objects that reside on the edge of an image can be included or excluded in various embodiments. An average population information about an object can be determined and recorded into a database, which can comprise an Excel spreadsheet, for example. However, embodiments can use any recording means without departing from the scope of the present invention. Values measured can be compared to the visual image. One or more types of numerical descriptors can be generated from the values. For example, descriptors such as a Number of objects, an Average, a standard deviation of objects, a Histogram (number or percentage of objects per bin, average, standard deviation), and the like can be determined.
In a particular embodiment according to the present invention, data can be analyzed using morphometric values derived from any of a plurality of techniques commonly known in the art. Fluorescent images can be described by numerical values, such as for example, an area, a fluorescence intensity, a population count, a radial dispersion, a perimeter, a length, and the like. Further, other values can be derived from such measurements. For example, a shape factor can be derived according to a relationship 4π*area/perimeter. Other values can be used in various embodiments according to the present invention. Such values can be analyzed as average values and frequency distributions from a population of individual cells.
In a particular embodiment according to the present invention, techniques for the automatic identification of mitotic cells are provided. Image analysis techniques employing techniques such as multidimensional representations, frequency-based representations, multidimensional cluster analysis techniques and the like can be included in various embodiments without departing from the scope of the present invention. Techniques for performing such analyses are known in the art and include those embodied in MatLab software, produced by MathWorks, a company with headquarters in Natick, Mass.
Scalar values providing efficacious descriptors of cell images can be identified using the techniques of the present invention to perform predictive analysis of drug behavior. In a presently preferred embodiment, a plurality of heterogenous scalar values can be combined to provide predictive information about substance and cell interactions.
In step 288, a lookup table for all analyses is provided to database 287. The lookup table provides information about the analyses. In a step 289, a query of database 287 for process data is performed. The results are reformatted. Then in a step 290, the database 287 is queried. Next, in a step 291, features of the manipulations stored in the database are combined and reduced. Next, in a step 293, reduced features of step 291 can be compared. In a step 292, the results of step 293 are recorded in database 287. Then, in a step 294, a report of predictions based on comparisons performed in step 293 is generated.
In a decisional step 307, a determination is made whether any more optical channels in the well can be imaged. If this is so, then in a step 308 the next optical filter configuration is selected and an image is collected. Processing then continues with decisional step 303, as described above. Otherwise, if no further optical channels in the well can be imaged, then in a decisional step 309 a determination is made whether any wells remain to be imaged. If not all wells have been imaged, then in a step 310, the stage moves to the next well and processing continues with step 301, as described above. Otherwise, if all wells on the plate have been imaged, then in a decisional step 311, a determination is made whether any more plates can be processed. If this is so, then processing continues with step 300 as described above. Otherwise, in a step 312, the information is stored to a CD or other storage device as a backup.
Accordingly, the present invention provides a novel database design. In a particular embodiment according to the present invention, a method for providing a database comprises measurement of a potentially large number of characteristics of one or more sub-cellular morphometric markers. Markers can be from any of a large variety of normal and transformed cell lines from sources such as for example, human beings, fungi, or other species. The markers can be chosen to cover many areas of cell biology, such as, for example markers comprising the cytoskeleton of a cell. The cytoskeleton is one of a plurality of components that determine a cell's architecture, or “cytoarchitecture”. A cytoarchitecture comprises structures that can mediate most cellular processes, such as cell growth and division, for example. Because the cytoskeleton is a dynamic structure, it provides a constant indication of the processes occurring within the cell. The cytoarchitecture of a cell can be quantified to produce a one or more scalar values corresponding to many possible cellular markers, such as cytoskeleton, organelles, signaling molecules, adhesion molecules and the like. Such quantification can be performed in the presence and absence of drugs, peptides, proteins, anti-sense oligonucleotides, antibodies, genetic alterations and the like. Scalar values obtained from such quantification can provide information about the ongoing cell biological function and physiologic state of the cell.
In a presently preferred embodiment, scalar values can comprise morphometric, frequency, multi-dimensional parameters and the like, extracted from one or more fluorescence images taken from a number of cellular markers from a population of cells. A vector of two or more such scalar values extracted from a plurality of cell lines and markers grown in the same condition together comprise a unique “fingerprint” that can be incorporated into a database. Such cellular fingerprints will change in the presence of drugs, peptides, proteins, antisense oligonucleotides, antibodies or genetic alterations. Such changes can be sufficiently unique to permit a correlation to be drawn between similar fingerprints. Such correlations can predict similar behaviors or characteristics with regard to mechanism of action, toxicity, animal model effectiveness, clinical trial effectiveness, patient responses and the like. In a presently preferred embodiment, a database can be built from a plurality of such fingerprints from different cell lines, cellular markers, and compounds having known mechanisms of action (or structure, or gene response, or toxicity).
The present invention also provides database and finger print comparisons according to other embodiments. In a particular embodiment according to the present invention, measurement of scalar values can provide predictive information. A database can be provided having one or more “cellular fingerprints” comprised of descriptors of cell substance interactions of drugs having known mechanisms of action with cells. Such fingerprints can be compared using a plurality of techniques, such as a technique of creating “phylogenetic trees” of a statistical similarity between the cellular fingerprints from various drugs. In a present embodiment, scalar, numeric values can be converted into a nucleotide or amino acid letter. Once converted into a corresponding nucleotide representation, the fingerprints can be analyzed and compared using software and algorithms known in the art for genetic and peptide sequence comparisons, such as GCG, a product of Genetics Computer Group, with company headquarters in Madison Wis. In an alternative embodiment, numeric values for the fingerprints can be used by comparison techniques. A phylogenetic tree can be created that illustrates a statistical significance of the similarity between fingerprints for the drugs in the database. Because the drugs used to build the initial database are of known mechanism, it can be determined whether a particular scalar value in a fingerprint is statistically predictive. Finally, a compound fingerprint with no known mechanism of action can be queried against the database and be statistically compared and classified among the drugs in the database that the compound most resembles.
In a particular embodiment, relationships between measured morphological properties of images and physiological conditions can be determined. Relationships can include, for example, treatment of different cell lines with chemical compounds, or comparing cells from a patient with control cells, and the like. In a presently preferable embodiment, a clustering can be performed on acquired image feature vectors. Some embodiments can comprise statistical and neural network-based approaches to perform clustering and fingerprinting of various features. The foregoing is provided as merely an example, and is not intended to limit the scope of the present invention. Other techniques can be included for different types of data.
In some embodiments, clustering and fingerprinting can be performed on features extracted from cell images. In a presently preferable embodiment, procedures for comparisons and phylogenetic analysis of biological sequences can be applied to data obtained from imaging cells.
Embodiments can perform such analysis based upon factors such as numerical value, statistical properties, relationships with other values, and the like. In a particular embodiment, numbers in a numerical features vector can be substituted by one or more of nucleic acid or amino acid codes. Resulting “pseudo-sequences” can be subjected to analysis by a sequence comparison and clustering program.
Other types of databases can also be provided according to other embodiments. The database includes details about the behavior of a plurality of standard drugs of known mechanism in the universal assay, called a morphometric cellular response. However, the comparative value need not be limited to drugs having a known mechanism of action. When the profile of a test compound is compared to the database, predictions about the test compound can be made against any known parameter of the other compounds in the database. For example, information about a compound in the database could include structure, mechanism of action, clinical side effects, toxicity, specificity, gene expression, affinity, kinetics, and the like. The fingerprint of a compound of unknown structure from a natural products library could be compared to the fingerprint of drugs with known structure and the structure could be deduced from such a comparison. Similarly, such information could lead to better approaches to compound analoging, pre-clinical animal modeling, clinical trial design (side effects, dose escalation, patient population) and the like.
According to the present invention, databases can be integrated with and complementary to existing genomic databases. Differential genomic expression strategies can be used for drug discovery using database technology. In one particular embodiment, cell data and morphometric cellular response data can be associated with a genetic expression profile assay to form a single assay. Live cells expressing fluorescence markers can be treated with a drug, imaged and analyzed for morphometry; and then analyzed for mRNA for expression. Such embodiments can provide rapid development of tools to link cellular behavior with genomics.
Database methods according to the present invention can be used to predict gene function and to assist in target validation. Databases that include genetic diversity, i.e., having cellular fingerprints from cells of differing genetic backgrounds (tumor, tissue specific, and gene knock out cell lines), can provide the capability to compare cells of unknown genetic background to those in the database. Similarly, fluorescent patterns of an unknown cellular marker in the presence of multiple drugs can be queried against the patterns of the known markers in the database. For example, if an unknown gene is tagged with Green Fluorescent Protein (GFP), the database may be used to identify the cellular structures for which that unknown gene encodes.
According to the present invention, target validation and cell-based assay screening can be performed using database system and methods to serve as a universal high-throughput cell-based assay that can evaluate the molecular mechanism of drug action. As new genes are isolated and identified, a large collection of available gene based knowledge is becoming available. From this large collection of new genes, potential targets can be identified using the genomic tools of sequence analysis and expression profiling. However, unless a gene mutation is tightly linked to a disease state, further validation of individual targets is a time consuming process, becoming a bottleneck in drug discovery. Furthermore, robotics and miniaturization are making “High Throughput Screening (HTS)” the industry standard, substantially reducing the time and cost of running a target-based biochemical assay. Therefore, it is now be possible to routinely screen large libraries and use a resulting “hit” to validate the target. In such approaches, a specialized cell-based assay would be developed to test hits for each target. Since this often involves the creation of cell lines expressing new markers, this stage may also become a bottleneck that cannot keep pace with HTS. In addition, these assays may not be amenable to high-throughput screening, making it difficult to test the increasing number of analogs arising from combinatorial chemistry.
In a particular embodiment according to the invention, a rapid characterization of large compound libraries for potential use as pharmaceutical products can be provided by predicting compound properties such as mechanisms of action. In many drug discovery situations, virtually millions of compounds can be passed through an HTS assay against one or two validated targets. These assays produce hundreds to thousands of potential hits that can be narrowed down to a selective few. These hits can then be subsequently screened by a pipeline of secondary and tertiary screens to further characterize their specificity, often time completely missing non-specific interactions with other proteins. Techniques according to the present invention can provide a replacement to such screening operations by providing information about cellular accessibility and mechanism of action for the substances usually placed on HTS systems. The cell information can be predictive of whether to continue into an animal model for each compound, and which animal model to pursue.
In some embodiments, techniques according to the present invention can provide tools for the later stages of drug development such as clinical trial design and patient management. The predictive value of the known drug trial and patient response information will be used in a similar fashion as the pre-clinical information. Because the human cell is the locus of drug action, a database containing cellular interactions can be able to provide predictive value for this aspect of drug development.
To prove the principle and demonstrate the objects of the present invention, experiments have been performed to determine the effects of manipulations on cell structure based upon imaging techniques applied to a variety of indications. These experiments were performed by growing multiple cell lines in the presence of multiple compounds, or substances. A fix and stain of cells using antibodies or labels to multiple cellular markers was performed. One or more images of the cells were then obtained using a digital camera. Indications were built by quantifying and/or qualifying patterns of each marker in the cell lines under study. A database was built from the indications.
As the database grows, it should be able to predict the mechanism of action and other compound properties of an unknown drug by comparing its effect with the effects of known compounds or to identify data clusters within large libraries of compounds.
In a first experiment, an automated method to count the number of cells and differentiate normal, mitotic, and apoptotic cells was created. Approximately 5,000 HeLa cells were plated per well in a 96 well plate and grown for 3.5 days. The cells were fixed with −20′ MEOH for 5 minutes, washed with TBS for 15 minutes, and then incubated in 5 mg/ml Hoechst 33342 in TBS for 15 minutes. Then, 72 images were collected with a 40× objective and 75 ms exposure time.
The analysis was performed on objects that met a certain size criteria that was based on 1) measuring the size of objects in the image that were clearly not cells and 2) excluding the first peak of the area histogram.
Histograms of the individual object data were generated for each type of information.
Each image was thresholded to a level of 20. A standard area value was set at 9500 pixels. Automated information gathering about all of the objects was done and collected into an Excel spreadsheet (for more information see, section on imaging system). The following information was recorded:
The following results were obtained:
In sum, out of 917 single objects, the analysis correctly identified 106 out of 130 mitotic objects, or (81% predictive, 91% of identified mitotics). Out of 917 single objects, the analysis incorrectly identified only 10 non-mitotics as mitotics (1% total, 8% of identified mitotics); 14 mitotics as interphase (1.4% total, 1% interphase).
The next step is to develop an automated classification system which will automatically assign values to each object using these or other measurement parameters.
In a second experiment, the effects of Taxol on MDCK cells and the different types of morphological effects were observed. A plurality of MDCK cells grown in 96 well plates were treated with Taxol for 4.5 hours at different concentrations (10 uM-1 pM). They were then fixed, labeled with Hoechst, and imaged.
This experiment used a labeling protocol comprising: MEOH fix at −20°, Wash in PBS, Block in PBS/BSA/Serum/Triton-X 100, Incubate with 5 μg/ml Hoechst 10 minutes, and Wash.
The results of the experiment are that cells were inspected for different morphologies and manually counted at each different drug concentration in one well.
The next step is to test the automated Hoechst analysis of the first experiment with multiple drugs.
In a third experiment, the purpose is to determine whether the automated analysis methods developed in the first experiment can detect differences in Hoechst morphology in the presence of 6 known compounds at one concentration and exposure time in one cell line. In this experiment, HeLa cells were treated with 6 compounds with known mechanism of action. The quantitative methods described in the first experiment were applied to the Hoechst images.
Approximately 5,000 HeLa cells per well were plated in a Costar black walled 96 well tissue culture treated plate and left to recover in the incubator for 24 hours. After this time, 10 ug/ml of cytochalasin D (CD), Taxol, hydroxyurea, vinblastine, and nocodazole, and staurosporne was added to different wells at a 1:100 addition in DMSO. The cells were incubated in the presence of drug for 24 more hours. After 24 hours, the cells were removed and fixed as in the first experiment. Then, 9 images per well were collected of the Hoechst staining using a 10× objective.
The results of this experiment were that the low magnification images taken of Hoechst were run through the automated image analysis method described in the first experiment. Plots of the average gray value and area were made of each compound.
Based upon the results of this experiment, it can be concluded that these initial attempts at automatically identifying changes in cellular morphology demonstrate that the effects of different compounds can be distinguished. This method can also be used to count adherent cells.
The next steps that can be taken based upon the results of this experiment are to develop clustering algorithms that will assign statistically meaningful values to the representative two dimensional data shown in
In a fourth experiment was performed to obtain high magnification images of two markers in the presence of drugs. In this experiment, HeLa cells were treated with 80 generic compounds with known mechanism of action. The quantitative methods described in the first experiment were applied to the Hoechst images.
Approximately 5,000 HeLa cells per well were plated in a Costar black walled 96 well tissue culture treated plate and left to recover in the incubator for 24 hours. After this time, 10 ug/mL of each compound from the Killer Plate from Microsource Discovery Systems (Gaylordsville, Conn.) was added to different wells at a 1:100 addition in DMSO. The cells were incubated in the presence of drug for 24 more hours. After 24 hours, the cells were removed and fixed as in the first experiment. In addition to being labeled with Hoechst 33342 (against chromatin), cells were also labeled with 1 unit of rhodamine-conjugated phalloidin (against actin) for 30 minutes.
The 96 well plate was imaged twice. Once, 9 images per well were collected of the Hoechst staining using a 10× objective. After this, one image per well of both the phalloidin and Hoechst staining was collected using a 40× objective.
The resulting high magnification images were analyzed qualitatively and distinct pattern differences were detected in both the Hoechst and phalloidin images.
The low magnification images were analyzed as described in the first experiment, and different patterns were seen in both the average gray value vs. area plots, and in the number of cells per well (data not shown). Based upon the results of this experiment, it can be concluded that the fact that changes in patterns of a marker that is “down-stream” from the mechanism of action of a compound are detectable illustrates the efficacy of this approach.
The next step based upon the results of this experiment is to develop automated image analysis protocols for actin and other markers.
A fifth experiment was performed to test quadruple labeling of 9 different cell lines grown in normal conditions. In this experiment, NC1—H460, A549, MDA-MD-231, MCF-7, SK-OV-3, OVCAR-3, A498, U-2 OS, and HeLa cells were plated. Then, the cells were fixed and stained for DNA, tubulin, actin, and Golgi markers.
The following table summarizes the procedures for this experiment:
Cells were plated out at different densities for 48 hours. Cells were fixed and labeled by the above method. Cells were imaged using an automated imaging macro that collected 9 images from each marker using a 10× objective. Higher magnification images were collected of a few cells for demonstration purposes.
In this experiment, each cell line demonstrated different morphological patterns as determined by phase. For example, A549 cells are much more compacted than OVCAR-3 cells as determined by phase contract imaging (data not shown). The different fluorescent markers showed even bigger differences between different cell lines.
Higher magnification images were taken of the OVCAR3 cells.
These data exemplify the differences in morphology seen between different cell types. Thus the automated image analysis software will have to be customized for each marker in each cell type. Different drugs should effect these morphologies differentially.
The next steps based upon the results of this experiment are to customize and develop an automated quantification for each marker and cell line.
A sixth experiment was conducted with a more sophisticated software package and to develop more flexible image recognition algorithms. In this experiment, prototype image features extraction was performed using Matlab programming language with image toolbox and SDC morphology toolboxes. Algorithms are being developed that will automatically identify objects on images and to measure various morphological and intensity parameters of these objects. Since at present it is not known which of the measurements will be most useful for subsequent clustering, many different measurements for each of the cellular markers were acquired.
An example of a MatLab program called “AnalyseDNA” that takes as an input an unlimited number of images, identifies individual objects in these images based on either their intensities, or based on edge-detection algorithms, and extracts a number of morphological and intensity characteristics of these objects is provided in a particular embodiment according to the present invention. It will be understood that other programs could also be used in other embodiments without departing from the scope of the present invention. Thus, the copy of this program reproduced below is intended to be representative but not limiting:
%=========end MaskDNA1=================================
Given the list of image files or montages of images as an input, this program creates an individual file for each image that contains the following quantitative measurements for all objects identified in the image:
A fragment of an output for a single file, containing 9 images of cells stained for DNA and acquired with a 10× lens, is provided in a particular embodiment according to the present invention. It will be understood that other results could also be obtained in other embodiments without departing from the scope of the present invention. Thus, the copy of this example output file reproduced in Appendix A is intended to be representative but not limiting.
A montage image that was used as a source to generate data in Appendix A is presented in
An example of summary output obtained by running AnalyseDNA against 10 montage files is provided in a particular embodiment according to the present invention. It will be understood that other results could also be obtained in other embodiments without departing from the scope of the present invention. Thus, the copy of this example output file reproduced in Appendix B is intended to be representative but not limiting.
A seventh experiment was conducted in order to use sequence analysis algorithms to analyze features of cell images. In this experiment, HeLa cells were treated for 24 hour with several different compounds, fixed, and stained with a fluorescent DNA dye. One image of these cells was acquired for each of the treatments and following morphometric parameters were measured:
Resulting measurements were arranged into a string of numbers and reduced to a pseudo-nucleic acid sequence using following rules: At any given position in the sequence a number was substituted by “t” (a code for thymidine) if its value is among highest 25% of the values at the corresponding position in the data set, “g” if it is between 50% and 25%, “c” if it is between 75% and 50%, and “a” if it belongs to lowest 25% of values. Thus one sequence was generated per treatment as illustrated in
Resulting sequences were clustered using an AlignX module commercial software package Vector NTI (http://informaxinc.com), which uses a Neighbor Joining algorithm for sequence clustering.
Resulting dendrogram is presented in
Although the above has generally described the present invention according to specific computer based software and systems, the present invention has a much broader range of applicability. In particular, the present invention is not limited to a particular kind of data about a cell, but can be applied to virtually any cellular data where an understanding about the workings of the cell is desired. Thus, in some embodiments, the techniques of the present invention could provide information about many different types of cells, substances, and genetic processes of all kinds. Of course, one of ordinary skill in the art would recognize other variations, modifications, and alternatives.
Data derived from select embodiments has been attached as a paper appendix, the entire contents of which is incorporated herein by reference for all purposes
The following commonly-owned co-pending applications, including this one, are being filed concurrently and the others are hereby incorporated by reference in their entirety for all purposes: 1. U.S. patent application Ser. No. ______, James H. Sabry, et al., titled, “A DATABASE METHOD FOR PREDICTIVE CELLULAR BIOINFORMANCS,” (Attorney Docket Number 19681-000100US); 2. U.S. patent application Ser. No. ______, James H. Sabry, et al., titled, “A DATABASE SYSTEM FOR PREDICTIVE CELLULAR BIOINFORMATICS,” (Attorney Docket Number 19681-000200US); 3. U.S. patent application Ser. No. ______, Cynthia L. Adams, et. al., titled, “A DATABASE SYSTEM AND USER INTERFACE FOR PREDICTIVE CELLULAR BIOINFORMANCS,” (Attorney Docket Number 19681-000300US); and 4. U.S. patent application Ser. No. ______, Eugeni A. Vaisberg, et al., titled, “A DATABASE SYSTEM INCLUDING COMPUTER CODE FOR PREDICTIVE CELLULAR BIOINFORMANCS,” (Attorney Docket Number 19681-000400US)
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09718685 | Nov 2000 | US |
| Child | 11436977 | May 2006 | US |
| Parent | 09311996 | May 1999 | US |
| Child | 09718685 | Nov 2000 | US |
