Supplementary data are available at Bioinformatics online.
Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that “paints the cell” with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.
Quantitative microscopy has proven a versatile and powerful phenotypic screening technique. Recently, image-based profiling has shown promise as a means for broadly characterizing molecules’ effects on cells in several drug-discovery applications, including target-agnostic screening and predicting a compound’s mechanism of action (MOA). Several profiling methods have been proposed, but little is known about their comparative performance, impeding the wider adoption and further development of image-based profiling. We compared these methods by applying them to a widely applicable assay of cultured cells and measuring the ability of each method to predict the MOA of a compendium of drugs. A very simple method that is based on population means performed as well as methods designed to take advantage of the measurements of individual cells. This is surprising because many treatments induced a heterogeneous phenotypic response across the cell population in each sample. Another simple method, which performs factor analysis on the cellular measurements before averaging them, provided substantial improvement and was able to predict MOA correctly for 94% of the treatments in our ground-truth set. To facilitate the ready application and future development of image-based phenotypic profiling methods, we provide our complete ground-truth and test datasets, as well as open-source implementations of the various methods in a common software framework.
BackgroundLarge-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications.FindingsThis microscopy dataset includes 919 265 five-channel fields of view, representing 30 616 tested compounds, available at “The Cell Image Library” (CIL) repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments applied.ConclusionsBecause computational algorithms and methods for handling single-cell morphological measurements are not yet routine, the dataset serves as a useful resource for the wider scientific community applying morphological (image-based) profiling. The dataset can be mined for many purposes, including small-molecule library enrichment and chemical mechanism-of-action studies, such as target identification. Integration with genetically perturbed datasets could enable identification of small-molecule mimetics of particular disease- or gene-related phenotypes that could be useful as probes or potential starting points for development of future therapeutics.
High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cellbased and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.chemical diversity | biological performance diversity | biological activity | chemical similarity P rofiling small molecules based on multiple biological activity measurements can illuminate mechanisms of action by comparing profiles with compounds whose mechanisms of action are known (1-5). Here, we describe a previously unidentified use of small-molecule profiling-enabling the creation of activityenriched and performance-diverse compound libraries for smallmolecule probe and drug discovery.Biochemical and cell-based high-throughput screening (HTS) is routinely used to discover novel bioactive molecules through unbiased testing of up to several million compounds per screen (6). However, despite ongoing advances in throughput, compound libraries will always represent only a small fraction of all relevant compounds theoretically accessible through chemical synthesis (a concept often referred to as "chemical space") (7). Library composition therefore presents a strong source of bias and potential limitation for any screening endeavor.There is little dissent about the notion that a good screening collection should yield many high-quality hits for a wide range of biological targets or phenotypes. In other words, it should be enriched for bioactive compounds and have high biological performance diversity. A high percentage of compounds lacking any activity will contribute to high cost and low performance of a high-throughput screen. A practical example is a compound collection containing a high percentage of compounds that fail to penetrate cell membranes-such a library will be unlikely to perform effectively in a cell-based HTS exploring an intracellular process. Similarly, a screening collection of compounds with highly redundant biological activities will be less efficient than an equally sized library with diverse performance (Fig. 1). A systematic path to reach these goals, however, remains elusive. One common practice is analyzing structural features of compounds to maximize chemical structural diversity. Ho...
We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available via the open-source CellProfiler project and enables objective scoring of whole-animal high-throughput image-based assays of C. elegans for the study of diverse biological pathways relevant to human disease.
Summary: CellProfiler Analyst allows the exploration and visualization of image-based data, together with the classification of complex biological phenotypes, via an interactive user interface designed for biologists and data scientists. CellProfiler Analyst 2.0, completely rewritten in Python, builds on these features and adds enhanced supervised machine learning capabilities (Classifier), as well as visualization tools to overview an experiment (Plate Viewer and Image Gallery). Availability and Implementation: CellProfiler Analyst 2.0 is free and open source, available at http://www.cellprofiler.org and from GitHub (https://github.com/CellProfiler/CellProfiler-Analyst) under the BSD license. It is available as a packaged application for Mac OS X and Microsoft Windows and can be compiled for Linux. We implemented an automatic build process that supports nightly updates and regular release cycles for the software. Contact: anne@broadinstitute.org Supplementary information: Supplementary data are available at Bioinformatics online.
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