High-Throughput Flow Cytometry in Drug Discovery

Flow cytometry has proven to be a powerful technology, enabling multiparametric analysis of single cells or particles, and widely used in both clinical and basic research. Flow cytometry has a broad range of applications, including quantification of cell surface and intracellular proteins, DNA analysis, cell proliferation, cell viability, cellular granularity, and cell size. ¹

There are a number of commercially available flow cytometry instruments, both cell sorters and analyzers (for review, see Picot et al. ² ). However, these traditional flow cytometry platforms are limited to low-throughput applications due to their slow sampling speed and requirement for large sample volumes. Therefore, increasing sampling speed and reducing sample volumes are critical improvements required to enable flow cytometry for high-throughput applications that require rapid, high-content analysis of large collections of biological samples. An added benefit of reducing sample volume is the reduction of cell numbers required for each sample.

High-throughput flow cytometry (HTFC) has been made possible by the introduction of novel sample handling and analysis technologies developed by academic and commercial groups (extensively reviewed by Edwards and Sklar, 2015 ³ ). The High-Throughput Sampler (HTS) system, commercialized by Becton Dickinson as an accessory for its high-end flow cytometers, enables the analysis of 96-well plates in as little as 15 min (~6–7 wells/min), a substantial improvement over the hour of time typically required using conventional automated tube carousel or plate sampling systems. HyperCyt technology, developed at the University of New Mexico (UNM) ⁴ and commercialized by Intellicyt Corp., delivers a continuous stream of air-gap-separated samples from plates directly into a flow cytometer to enable plate processing and analysis at even faster rates (~32–40 wells/min). Adapted for use as a front end for a variety of commercial flow cytometers at UNM and Intellicyt (also in a custom system described in this issue), ⁵ this technology is compatible with assay volumes as small as 5 µL and plates with up to 1536 wells.

There has been a recent groundswell of new HTFC applications reflected in the content of this special issue of SLAS Discovery. This special issue presents a range of research papers, application notes, and technical notes that reflect recent advances in this field, provide new insights and perspectives for HTFC method design, and highlight areas to be improved for a broad range of applications in drug discovery.

Screening Pipeline Development

Hardware

Robust and high-throughput sample preparation is another critical component of HTFC as cell treatment and staining for marker proteins are often required for HTFC applications. Several automation systems with or without HTFC integrated have been presented previously.^6–10 In this special issue are three papers that provide details on the upstream sample preparation automation systems and HTFC sample acquisition systems used at the Genomics Institute of the Novartis Research Foundation (GNF), AstraZeneca and Novartis Institute for Biomedical Research. The system presented by Joslin et al. ⁵ is a fully automated HTFC GNF system with sample preparation and HTFC sample acquisition integrated, enabling phenotypic drug discovery at GNF with a throughput of 50,000 wells/day using 384-well or 1536-well plates. ⁵ The paper presented by Wilson et al. ¹¹ describes a semiautomated workflow and a workflow for HTFC data analysis. Ding et al. ¹² present AstraZeneca’s current and future-looking HTFC automation platforms for sample preparation, acquisition, and HTFC data analysis workflow. The current HTFC automation systems used at AstraZeneca can successfully screen a 500,000-small-molecule-compound library using 384-well or 1536-well plates, as well as several smaller phenotypic screens using 384-well plates. The authors also provide their insights into the practical challenges encountered during the course of HTFC deployment at AstraZeneca.

Software

Along with sample acquisition, HTFC screens generate large amounts of data with multiparametric readouts. Available software products enabling easy HTFC data analysis, data visualizations, and follow-up processing of HTFC screening data are essential for broad use of HTFC. The papers presented by Joslin et al., Wilson et al., and Ding et al.^5,11,12 describe different software and data analysis workflows.

Drug and Biologics Discovery

Spurring the initial development of HTFC technology was the desire to implement high-content screening of small-molecule compound libraries in assays employing cell and bead suspensions. The multiparameter analysis capabilities of flow cytometry were particularly attractive in support of the shift in emphasis from target-centric to target-agnostic or mechanism-informed phenotypic drug discovery. ¹³ This is the emphasis of three articles in this issue. Bredemeyer et al. ¹⁴ describe an HTFC-based method to identify small molecules that inhibit DNA repair in cancer cells mediated via the mechanism of nonhomologous end joining (NHEJ) of double-strand breaks. Expression of green fluorescent protein (GFP) signals NHEJ inhibition, while light scatter profiles of cells help eliminate cytotoxic compounds and proliferation-associated false positives. A custom platform previously developed for HTFC screening in the 1536-well format ¹⁵ is modified to enable automated, parallel processing of four 384-well plates at a time (160 wells/min) and is used to screen the NeXT diversity set of 83,536 compounds from the U.S. National Cancer Institute. In the second paper, Buranda et al. ¹⁶ document development of an HTFC assay to identify drugs capable of inhibiting cell binding and infection by hantaviruses, pathogens that cause a cardiopulmonary syndrome with fatality rates of 30%–50%. ¹⁷ The assay assesses the binding interaction between UV-inactivated, fluorescently labeled virus particles and the binding site expressed on intact cells. Screening identifies antimycin, which is subsequently confirmed to inhibit infection by live virus as well as infection-associated integrin activation. The third paper, from Zhao et al., ¹⁸ describes a novel enhancement of an HTFC method for the discovery of small molecules capable of augmenting cluster of differentiation 3 (CD3)-triggered lytic granule exocytosis in cytotoxic T cells. ¹⁹ The original assay simultaneously distinguished binding of T cells to beads coated with CD3-activating antibodies, the number of beads bound per cell, and resulting expression levels of an exocytosis-linked membrane protein. ¹⁹ The new twist is to add another level of multiplexing by which to evaluate exocytosis response kinetics in each well at the primary screening stage, accomplished by adding fluorescently barcoded T cells to each assay well at three different time points prior to HTFC analysis. This enhancement minimizes false negatives by identifying active compounds that would be missed by screening using a single time point.

There is current widespread interest, and investment, in developing biologic drugs, especially novel antibodies. ²⁰ Three articles in this issue address novel HTFC applications in this field. Wang et al. ²¹ describe an HTFC sample preparation and readout workflow that optimizes antibody discovery by multiplexing multiple cell lines of interest in one well to simultaneously quantify antibody concentration and on-target and nonspecific activity. O’Rourke and Liu ²² describe the development of a no-wash, 384-well HTFC screening approach for other aspects of antibody discovery. This is a multiplexed cell- and bead-based competition assay capable of simultaneously differentiating between monoclonal and polyclonal wells, determining immunoglobulin G (IgG) quantity for downstream functional assays, providing antibody isotype information, and monitoring cell proliferation and viability. In addition to affinity and selectivity, the epitope targeted by an antibody can also be an important factor in therapeutic applications. Chan et al. ²³ describe a novel HTFC approach that uses multiplexed cells or beads to rapidly classify antibodies into epitope binding bins. This method generates competitive binding profiles of each antibody against a defined set of known or unknown reference antibodies for binding to epitopes of a target antigen. Because there is no requirement for purification or direct labeling of the antibodies, this method is well suited for characterizing antibody candidates during the early discovery phase.

Another therapeutic area of great promise involves the engineering of autologous patient T cells to express chimeric antigen receptors (CAR-T) for use in adoptive cellular therapy of malignancies. Martinez et al. ²⁴ describe a novel HTFC approach that allows the concurrent measurement of T-cell-dependent cellular cytotoxicity and the functional characterization of engineered T cells expressing chimeric antigen receptors, T-cell phenotype, and activation status in a single assay. Because of a focus on targeting solid tumor malignancies, optimized protocols are also presented for using adherent cells in suspension assays of cell–cell interactions.

An important issue in developing screening campaigns is understanding the relative merits of different platforms for assessing biological responses of interest. In this context, Ding et al. ²⁵ perform head-to-head comparisons of cytokine quantification profiles generated using a commercial HTFC immunoassay kit and several other commonly used immunoassay methods. Results correlate well across all methods when evaluating 12 cytokines in four different formats, although disparities are observed for some cytokines with respect to absolute quantities detected.

Response Profiling of Primary Cells and Cell Lines

Flow cytometry is an important tool for single-cell analysis in a broad range of clinical and basic research applications. The advent of HTFC has significantly expanded the efficiency with which large collections of samples can be rapidly analyzed, producing high-content data sets for diagnostic, prognostic, and research purposes. An additional benefit of many HTFC platforms is the ability to utilize very small sample volumes and to analyze each sample in its entirety with little measurable loss of cells as dead volume. This enables wringing of more high-quality information from the limited quantities of primary tissues typically accessible from patients. An excellent example is presented in the paper by Fan et al. ²⁶ in which a 384-well HTFC assay measures multiple parameters of T-cell activation and dendritic cell maturation induced by monocyte-derived dendritic cells in a mixed-lymphocyte reaction setting. The assay is miniaturized and enabled using 10 times fewer primary cells than the number of cells required in more traditional flow cytometry methods. Another example is the paper by Perez et al., ²⁷ which presents an HTFC approach to screen a collection of FDA-approved tyrosine kinase inhibitors in a panel of T-cell-lineage acute lymphoblastic leukemia (T-ALL) cell lines, T-ALL patient samples, and patient-derived xenografts. The assay evaluates cytotoxic effects of the inhibitors under normal and hypoxic conditions associated with chemotherapy resistance. The authors identify several clinically approved tyrosine kinase inhibitors targeting T-ALL in a hypoxic environment similar to that in bone marrow. Javarappa et al. ²⁸ describe a multiplexed moderate-throughput flow cytometry assay for evaluating myelosuppressive effects induced by chemotherapeutic drugs. A nine-color antibody panel is used to evaluate the effects of a panel of 15 chemotherapeutic drugs on the differentiation and maturation of megakaryocytes and myeloid cells differentiated from CD34-positive hematopoietic progenitor cells, purified from peripheral blood and bone marrow samples from healthy donors. The assay allows for temporal monitoring of multiple rare cell populations and can serve as a valuable tool in preclinical studies screening for potential adverse effects of novel anticancer compounds.

An additional paper relevant to high-content cell response profiling ²⁹ describes the development of a novel SynScreen software that allows rapid visual and mathematical determinations of synergy (or antagonism) with respect to effects of drug combinations on single or multiplexed cellular responses. The utility of the software is demonstrated in four replicated HTFC leukemia cell line cytotoxicity screening experiments, each involving 25 pairings of repurposed and chemotherapeutic drugs in orthogonal 8-point dose–response assessments (64 dose combinations per drug pair). The software is especially useful for analyzing multiplex HTFC drug combinations and provides three-dimensional data visualization to allow for simultaneous assessment of the combination responses in comparison with the individual drug responses.

Overall, the collection of papers in this special issue presents a wide range of recent HTFC screening applications as well as describing several HTFC sample preparation and sample acquisition automation systems and data analysis solutions for facilitating HTFC applications in drug discovery. Flow cytometry has evolved from a low-throughput technology requiring operation by specialists to a medium- to high-throughput drug discovery technology amenable for nonspecialists to operate. Advances in the HTFC field have transformed flow cytometry into a very attractive platform for drug discovery and high-content, single-cell profiling.