Can flow cytometry play a part in cell based high‐content screening?

In parallel to gene therapy and cellular therapy it is clear that the more traditional pharmacological approaches still have a major role to play in the treatment of most diseases. Two drug-discovery strategies are widely used in the pharmaceutical industry: the so-called rational drug design and the most recent screening-based strategy. Rational drug design requires usually an excellent knowledge of the biomolecular target: molecular mechanisms of action, three-dimensional structure, etc. It is scientifically sound and often efficient but rather time consuming. To cope with an exponentially increasing number of potentially interesting targets derived from genomic and proteomic studies, the industry has developed the screening-based approach since the 1990s. It has proven to be very efficient to afford original hit compounds that may open the way to drug development. During the last decades there has been a rapid development of assays and screening methods to identify promising drug candidates. Using cell-based disease models will improve the predictive value of drug discovery (1). When compared with a classical drug design approach (based on a single target protein involved in a given biological event) the use of a cell-based essay is dramatically efficient for drug discovery and development. Pace has been increased via combinatorial chemistry, genomics knowledge, proteomics technologies, new target validation, methodologies, miniaturization, and high-throughput screening as some of these issues were recently profiled by Smith et al. for the cytometry community (2). Once you are convinced that understanding how drugs affect cellular processes and cellular pathways in situ is the most biologically reliable route to discover effective new drugs; high-content screening (HCS) will be your technology of choice. HCS is based on multiparameter analysis of intact cells by coupling the power of fluorescent trackers to bioinformatics and robotics. Typically the system is based on epifluorescence microscopy and combines automated cell field focusing and multichannel image capture with sophisticated proprietary algorithms to extract high biological content information from fluorescently labeled cells. Recent technological breakthroughs in automated microcapillary cytometry allowed avoiding expensive and time-consuming image analysis apparatus and replace them in most cases by faster FACS analyses (FCS, SCS, and today up to four fluorescence channels). For example, microcapillary cytometry HCS includes the following: receptor internalization, multiparameter apoptosis, mitotic index, cell cycle, mitochondrial activity and of course cell viability, cell tracking, and real-time cellular concentration. At present, a substantial variety of automated single-cell measurements can be assessed, such as the metabolic state of viable cells by flow cytometry or laser scanning cytometry (3, 4). Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. Even though there is no exact definition of HCS, it typically refers to a cytometric technique based on automated fluorescence microscopy. By connecting the imaging of cells in microtiter plates with powerful image analysis algorithms and data visualization software one can acquire knowledge on multiple biochemical or morphological pathways at the single-cell level (5). A recent review by Paul Lang et al. describes how cellular imaging technologies contribute to the drug discovery process (6). They concluded that indeed several factors limit the extensive use of cellular imaging in primary screening (high cost of reagents, complex assay development, and data-management issues) and that a combination of higher-resolution imaging systems should greatly facilitate the use of cellular imaging in primary screening. High-content screening by cellular imaging will always be the first choice in our western culture where the image is omnipresent in all media. In an equivalent approach, flow cytometry is a sensitive and quantitative platform for the measurement of particle fluorescence, and is capable of high-content characterization of diverse compound effects at the single cell level. Thousands of different assays have been successfully performed on cells or particles, ranging from single-parameter endpoint assays to complex assays involving multiple cell populations or simultaneous analysis of multiple responses. Flow cytometry has been historically used in a largely manual low-throughput mode working with individual samples (7). In 2004, Gasparetto et al. (8) were the first to use the term FLOW HCS when they described the identification of compounds that enhance the antilymphoma activity of rituximab®. Their classical assay is based on staining nuclei with fluorescent dye and simultaneous measurement of incorporation of bromo-deoxyuridine (BrdU) into cells passing the S phase of the cell cycle. The measurement of DNA content allows, together with BrdU incorporation, analysis of the percentage of cells in different cell cycle phases as well as detection of apoptotic cells measured as reduced DNA content. Such applications were predicted in 1999 by Burchiel et al. (9) when they claimed that flow cytometry is readily adaptable to high-throughput screening, and that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries even when at that time immunotoxicity testing was the only application on hand. More recently, Perez et al. describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity (10). They claim that multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that are too few in number for conventional biochemical analysis. Edwards et al. declared in 1999 that the tools for high-throughput flow cytometry have been limited, until then in part because of the requirement that the samples must flow under pressure (11). They described in 2001 an automated system with a novel patented fluidics-based pharmacology platform. They coupled to a flow cytometer a plug flow-coupling valve technology (12). They concluded that their added tool to flow cytometry represents a powerful high-throughput multifactorial approach to increase the efficiency with which novel bioresponse-modifying drugs may be identified and characterized. These authors advocate their machine as an innovation, which allows up to 30-fold faster serial processing of bulk cell samples. Recently, they published successful HCS results obtained with the apparatus they now refer as HyperCyt® (13). HyperCyt® is an automated analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to a flow cytometer. The HyperCyt® basically interfaces a flow cytometer and an autosampler. As the sampling probe of the autosampler moves from one well to the next of a multiwell microplate, a peristaltic pump sequentially aspirates sample particle suspensions from each well. Between wells, the continuously running pump draws a bubble of air into the sample line. This results in the generation of a tandem series of bubble-separated samples for delivery to the flow cytometer. Sample and bubble volumes are determined by the time the autosampler probe is in a microplate well or above a well in taking air. In their latest article (14) the same authors describe the development and application of a screening approach to detect potential antiinflammatory compounds that block ligand binding to formylpeptide receptor family of G protein-coupled receptors. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (∼2,500 cells analyzed/sample) allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound from among a collection of 880 compounds in the Prestwick Chemical Library (Illkirch, France). The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format. The only limit today of such a fast and accurate methodology is that the HyperCyt platform now in use is a custom instrument built at the University of New Mexico and not yet commercially available. Some authors like to combine flow and image HCS. More recently developed techniques allowing for the simultaneous analysis of multiple targets in signaling pathways in T cells by flow cytometry and the use of HCS, which is based on automated, multicolor fluorescence imaging of arrays of treated cells, are of great interest for biomarker identification, target validation, and target and cell function research (15). Thus multicolor fluorescence flow cytometry can be used for multicolor HCS where cells can be analyzed for phenotype and phenotypic response to small molecule and biologic treatments. It is possible to screen the phenotypes of a million cells automatically, analyzing and archiving the data on morphometric parameters and up to eight fluorescence-based cellular parameters in less than 5 min. Specific cellular profile information can be collected based on stage of cell cycle, apoptosis, receptor binding, receptor internalization, organelle function, to name a few, in either fixed or living cells. Data mining of the large data sets from pathway mapping and target function studies from HCS flow cytometry will also yield important new information and knowledge of cell and cell constituent functions. In the last few years there has been a rapid development of cell-based assays and screening methods to identify promising apoptosis-inducing drug candidates for the treatment of cancer (5). Distinguishing between the complex processes involved in apoptosis and other forms of cell death requires information on both biochemical and morphological processes in the cell. Traditionally, many assays have been limited to measuring, for example, caspase activity using fluorogenic substrates (5). Our group acquired lately a benchtop 96-well cytometer based on capillary technology, a sheathless machine with no competition on the market today and so difficult to compare with other brands. We picked out this machine for its capability of direct cell count (cell counts cannot be determined on a conventional cytometer because the sheath fluid causes an uncontrolled dilution); second, for an efficient cell agitation device added to classical flux parameters (FCS, SCS, and four fluorescent colors) (16). Consequently, we were able to screen our in house chemical library (7,500 molecules) in less than 3 months for proapoptotic properties (Annexin V) and cytotoxicity (7-AAD), and this on two cell lines MCF7 (human breast adenocarcinoma) and CaCo2 (human colon adenocarcinoma) cell lines (confidential results due to biopharmaceutical company collaboration). On the same device, we accomplished cytokine secretion tests using different brands, that is, we tested BD™ cytometric bead array (Human Inflammation Kit and Th1/Th2 Cytokine Kit), Bender FlowCytomix Multiplex Kits multiplex (11 cytokines), or the MACS® cytokine secretion assay by Miltenyi. The results obtained (comparative example shown in Fig. 1) persuaded us that, when associated to new technological development, flow remains competitive for HCS and that this device will help us to develop and perform numerous tests by providing accessible, powerful, yet cost-effective cell analysis (17). Comparative run of multiplex assay on sheath and sheathless cytometer. This figure does not compare the same cell supernatant but is just an illustration to show that a somewhat equivalent resolution can be obtained on each machine with the “human inflammation BD™ Cytometric Bead Array.” (A) Sample run on a classical sheath flow cytometer (2,000 particles acquired with a 500 μl run) and (B) a different sample run on the sheathless cytometer (800 particles acquired with a 10 μl run). Compound screening throughput may be further enhanced by multiplexing of assays on color-coded bead or cell suspension arrays and by integrating computational techniques to create smaller, focused compound libraries. Novel bead-based assay systems allow studies of real-time interactions among solubilized receptors, ligands, and molecular signaling components that recapitulate and extend measurements in intact cells. These new developments, and its broad usage, position flow cytometry as an attractive analysis platform for high-throughput, high-content biological testing, and drug discovery. Thus, reasonably priced walk away automated benchtop 96-well cytometry will give a second chance to flow cytometry to be competitive in flow HCS when compared with the numerous techniques based on cellular imaging cytometry. Jean Peluso*, Helena Tabaka-Moreira*, Nathalie Taquet*, Serge Dumont*, Christian D. Muller*, Jean-Marie Reimund , * Faculty of Pharmacy, Institut Gilbert Laustriat, UMR 7175 CNRS, University Louis Pasteur-Strasbourg 74, Route du Rhin 67401, Illkirch Cedex, France, Department of Gastroenterology, Hepatology, and Nutrition, Faculty of Medicine, Caen University Hospital (Hôpital Côte de Nacre), Caen, France.