August 1, 2016 (Vol. 36, No. 14)

Whether They Are Used In Drug Screens or Precision Medicine, Cell-Based Assays Are All About Context

Cells are miniature laboratories that allow scientists to study key biological processes. As a result, they may provide a more natural way to assess the in vivo effects of drugs. According to MarketsandMarkets, the global cell-based assays market is expected to reach over $18 billion by 2020, from almost $11 billion in 2015.

GEN wanted to know about some of the challenges associated with the use of cell-based assays in high-throughput drug discovery and why these types of assays might be especially appropriate for precision medicine. So we decided to talk to some experts on the topic.

GEN: What are some of the challenges of using cell-based assays in high-throughput drug discovery, and how can these challenges be addressed?

Dr. Ferrick: Cell-based assays are considered a critical component of the golden era of drug discovery. With a return to the relevancy of living cells, also comes the fear of “black box” science, potentially leading to drugs that may not be fully adjudicated for efficacy and safety. Fortunately, today’s biological toolbox enables the better measurement and replication of human disease characteristics in the laboratory.

Cell-based assays are now able to measure and monitor multiple cellular pathways in real time, incorporate the preferred biological context for screening, and report phenotypic changes more reliably and with improved insight. Additionally, 3D cell cultures, microtissues, embryonic stem cell-derived cultures, and induced pluripotent stem cell-derived cultures are now able to better mimic the cell phenotype and microenvironment at the appropriate throughput levels.

Dr. Held: In cell-based assays, it is frequently necessary to control for environmental factors such as temperature, humidity, and gas composition (CO2 and O2) in order to maintain relevant physiological conditions. Also, assays can be lengthy, lasting days rather than hours, necessitating media exchanges or reagent additions at timed intervals. During these operations, assays must be kept free of bacterial contamination.

Such challenges have been addressed primarily with instrumentation and automation. Incubators, which maintain environmental conditions, can have built-in robotics to move microplates to and from liquid handlers and reader/imager devices to automate lengthy assay processes. Reagent additions or media exchanges are performed with liquid handlers, whereas detection (image based or quantitative) is carried out with a microplate reader. These systems are small enough to fit inside HEPA-filtered biosafety cabinets.

Dr. Stump: Cell-based assays enable one or more target-specific measurements amidst thousands of interdependent chemical reactions occurring inside each cell. The end goal is to identify a lead candidate based on a physiologically relevant response. To accomplish this goal, assay design (including the choice of a model system, endpoints specific to the drug target, protocols, and reagents) must be robust. Thus, methods beyond 2D cell culture may be the answer, but they drastically increase the complexity of the assay.

Adequate dynamic range of the assay is also a key challenge since nanomolar inhibitors require at least femtomolar detection for accurate curve generation. Highly specific antibodies can address this issue by substantially increasing the signal-to-noise raio. These and other elements combined must yield a reproducible assay that can achieve the throughputs desired.

Dr. Charrier-Savournin: Immortalized cell lines have been extensively used since they are cost effective and easy to manipulate genetically. However, they are not representative of normal cells, so their use for screening assays becomes outdated. Closer to native pathophysiology are fully differentiated primary cells, cancer cells, embryonic stem cells, or induced pluripotent stem cells, but they are less available, more difficult to maintain and differentiate, and more costly.

Thus, cell-based assays need to adapt. They can become more sensitive, use less sample, or even work on endogenous expression while keeping the characteristic advantages of high-throughput screening, which include robustness, ease of use, and miniaturization. Highly sensitive detection technologies, such as homogeneous time-resolved fluorescence energy transfer (TR-FRET) assays, have evolved to combine both requirements and are now fit to measure endogenous events in a screening environment.

Ms. Gitschier: Cell-based assays provide a biologically relevant environment, are amenable to high-throughput screening, and do not require the complexity and risk of whole-organism testing. A major challenge of cell-based assays involves choosing an ideal assay to represent the system of interest, while maintaining consistency and reproducibility. Cell culture and plating techniques, the use of automation, and microplate selection can be optimized to reduce variability.

Specifically for high-throughput screening, evaporation and small-volume use are concerning. For these assays, microplate seals, low-evaporation lids, and specialty microplates are useful. Finally, having vendors of instruments and microplates work closely together as technologies advance to ensure compatibility can help alleviate challenges customers face when adopting a new assay to meet their high-throughput drug discovery needs.

Dr. Collins: To more closely mimic disease, researchers are increasingly faced with the need to develop cell-based assays that accurately reflect the environment of the human body. Scientists are studying 3D spheroids as a biologically relevant model that accurately replicates the in vivo environment. For example, variable aperture confocal technology on the IN Cell Analyzer 6000 is instrumental in optimally imaging these challenging samples.

Another difficulty in developing better biological models is preparing samples in a miniaturized format. A common solution is round-bottom plates, which require a robust autofocus algorithm for automated imaging.

Finally, studies of living biology are critical to understanding dynamic cellular events. Recent speed improvements on the IN Cell Analyzer 6000 enable acquisition of beating cardiomyocytes at 42 frames per second, accurately representing cardiac physiology.

Dr. Mohan: Most high-throughput screening (HTS) in drug development is based on cell-based and biochemical assays. Although cell-based assays are useful for target validation and ADMET, they are not free from challenges. One of the greatest challenges is to develop stable cell lines that express reasonable quantities of target proteins. Another challenge is the decline in the expression level with each passage, which adds difficulty in data interpretation.

Immortalized cell lines, such as HEK293 and cancer cell lines, are widely used, but they can harbor mutations. They may not give true results, that is, results consistent with those obtained with primary native cells. Some of the challenges can be overcome by the use of 3D cell cultures, which emulate in vivo morphology as well as cell-cell and cell-extracellular matrix interactions.

Dr. Chandy: Some of the challenges in using cell-based assays are choosing a cell model that allows throughput and standardizing image analysis and interpretation for complex assays, such as time-lapse or 3D objects. In particular, imaging of 3D models may better predict reactions of or more closely mimic the in vivo environment. Previously, using live cell assays where 3D acquisition was required forced researchers to compromise between resolution and phototoxicity during z-sectioning.

Modern systems, such as the ImageXpress® Micro Confocal system, enable high-resolution screening at the speed of widefield imaging through advanced illumination techniques and optics. Integrated software packages, such as MetaXpress software, allow for multiple cell types, substrates, or cell behaviors to be automatically characterized at once, reducing variability across experiments.

Dr. Khimani: Although cell-based assays offer physiologically relevant systems for drug discovery, they traditionally provide output in just one dimension. Such output may miss effects such as molecular, morphological, and other phenotypic changes that could challenge further development of a drug candidate.

Instead, cell-based strategies including multiparametric and multianalyte readouts at the subpopulation- or the single-cell level should be considered. Multimode and multianalyte analysis tools that detect changes in a spatial context are critical in determining both the efficacy and toxicity of a drug candidate as well as better downstream outcomes. Furthermore, current advances and continued development and integration of 3D cellular models, simultaneous detection, imaging, and data analysis capabilities will further enable high-content drug discovery.

Dr. Riss: In general, the biology of the high-throughput assay system contributes more to variability than the chemistry of the detection reagents. Perhaps the biggest challenge for cell-based high-throughput screening is having uniform samples of cells in assay wells at the time the measurements are made. Variability in dispensing and culturing small numbers of cells in low-volume plates often leads to edge effects that result in variability among replicate samples.

One way to address variability is to multiplex assays to include controls to normalize data to the total or viable number of cells in the sample.

Dr. Horton: As our understanding of complex cellular systems evolves, there is a driving force to move to more predictive cellular models for drug discovery. What was incomprehensible from a bioinformatics and screening perspective just five years ago is quickly becoming the new screening norm. Traditional workhorse cell models (such as HEK293 and CHO-K1) are taking a back seat to more complex 3D cellular systems and patient-derived disease models.

The challenge to reagent suppliers is to identify and develop robust assays readouts that will function in a range of cellular backgrounds and environments while still providing physiologically relevant information. Vast improvements to high-content systems and data analysis are enabling scientists to extract a higher density of information about the cellular response to ultimately drive better decisions during drug discovery.

GEN: Why are cell-based assays especially appropriate for precision medicine?

Dr. Ferrick: Precision medicine will work most effectively if researchers can “bucket” individuals into treatable groups, as opposed to determining the unique molecular inventory of each individual. With credible evidence suggesting that there may be significant numbers of molecular pathways that lead to the same disease phenotype, researchers can now potentially identify a common intervention intersect, allowing the same drug to be effective across a range of patients with different molecular starting points.

By segregating diseases phenotypically, such as by various proliferative, apoptotic, survival, and immune escape programs, these buckets can be determined, enabling improved use of the growing array of drugs that could selectively alter these phenotypic intersects.

Dr. Held: Cell-based assays are used because their predictive value is greater than that obtained with biochemically based assays for drug discovery and development. Likewise, the use of a cell-based assay approach in conjunction with precision personalized medicine will have even greater predictive value for treatments.

For example, a number of cancer drug treatments have been shown to have a genetic component to them, where individuals with specific genetic variants will have successful outcomes whereas others with a different gene variant will not. The use of patient-specific cell-based assays would be expected to identify more of these treatment anomalies.

Dr. Stump: Precision medicine focuses the spotlight on an individual patient’s response instead of a statistical result from a double-blinded, placebo-controlled trial. Tumors are being placed into mice with the hope that xenografts will yield actionable information on drug susceptibility. Such approaches are time-consuming, complex, and difficult to sustain and scale for precision medicine.

Cell-based assays provide the complexity required for drug-based responses, offer mechanistic insights, and are amenable to faster assay times. Simply put, timely development and testing of in vitro models has great potential for producing patient-centric data for targeted therapies. Robust reagents will be pivotal to achieving success.

Dr. Charrier-Savournin: Pathophysiologically relevant environments are a big challenge with cell-based assays, and procedures that would match the right drugs to the right patients are an option only if patient-derived samples are available. Researchers need to monitor the same readout in widely different biological backgrounds, such as synthetic lethality models in breast cancer or patient’s peripheral blood mononuclear cells.

The reliable analysis of biomarkers with cell-based assays, such as assays for cytokine quantification, enable rapid functional tests of patients’ cells that have been exposed to a panel of potential therapies. Such an approach could help identify therapies that would likely be unsuccessful in a given population.

Ms. Gitschier: As technologies emerge to make high-throughput cell-based assays more amenable for use with stem cells and patient samples, these assays are becoming increasingly important for precision medicine. These cells often elicit responses or display resistance differently during drug exposure compared to alternative cellular models and cell lines.

Further, as tools enabling 3D cell culture continue to emerge, more physiologically relevant microenvironments for high-throughput drug discovery and precision medicine are being realized.

Dr. Collins: Cell-based assays are well suited for precision medicine. They provide more pertinent in vivo results compared to biochemical assays. With recent advances in stem cell biology, induced pluripotent stem cells can now be collected from patients in high-throughput cell-based assays. These studies are ideally suited for precision medicine because genotypic variations are eliminated.

Also, image-based high-content analysis allows researchers to answer many questions using a combinatorial approach. When optimized assay design is coupled with multiparametric analysis, one can minimize genetic and environmental variations that are intrinsic to precision medicine.

Dr. Mohan: Precision medicine is based on differences in genetic and environmental makeup of individuals that can affect the outcome of therapy. Cell-based assays could provide important information on changes in expression of “biomarker” proteins as prognostic factors.

Cell-based assays can also be used to assess safety of novel experimental drugs. They can be useful in quality control testing of stem cells for cell-based therapies in regenerative medicine, as well as to test immunogenicity of therapeutic antibodies to avoid the development of antidrug antibodies, which can reduce efficacy through rapid clearance or neutralization.

Dr. Chandy: Primary cancer cells can be used in cell-based assays to identify drugs that target specific genome types or traits. Compared with adherent cells grown in 2D monolayers, 3D growth conditions are believed to more accurately model the in vivo environment. Cellular transformation/tumorigenicity assays using cultures of cells in semisolid media better correlate with tumorigenicity in animal models, for example, in mouse xenografts.

These assays were once labor-intensive and inconsistent. New tools, such as the MetaXpress 3D image analysis toolkit, enable 3D cell-based assays to be used as a screening tool to reproducibly assess more relevant compound effects, proliferation, and viability of cancer cells.

Dr. Khimani: In precision and personalized medicine, the relevance of cell-based assays to the target(s) and associated disease becomes an integral part of drug discovery. Hence, clinical relevance of the assay on a sample enables evaluation of the efficacy and toxicity of a drug on that individual.

For example, in precision medicine against cancer, functional assays performed ex vivo on tumor cells become a powerful tool to evaluate targeted therapies. In other diseases, induced pluripotent stem cells may constitute cell-based assay models for phenotypic and molecular profiling.

Dr. Riss: There is variability among individuals in a population just as there is variability between primary breast cells isolated from a patient’s tumor compared to MCF7 cells. There is much promise for using extremely sensitive viability assay technologies.

Assays such as the CellTiter-Glo® Luminescent Cell Viability Assay for ATP detection can enable miniaturized screening of known chemotherapeutic drugs and drug combinations using cells derived from patient biopsies. Completion of clinical trials will likely support the hypothesis that tumor cells isolated from an individual more accurately predict the in vivo therapeutic response of that individual.

Dr. Horton: With the escalating cost of healthcare, the ability to design personalized treatment options is going to be instrumental in curbing healthcare expenses while still improving patient outcomes. The ability to leverage cell-based assays on patient-derived cellular models will allow healthcare professionals to intelligently design customized treatment options that will have the greatest chance of success.

Cellular assays are never going to be a direct replacement for the real thing. They do, however, present an attractive balance between cost and speed in their ability to provide predictive treatment options in days, as opposed to the months or years necessary for animal studies.

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