January 15, 2007 (Vol. 27, No. 2)

Obtaining Relevant Contextual and Physiological Data from Individual Cells

The pressure during drug discovery to detect failure compounds earlier and at decreased cost has increased the relevance of high-content screening (HCS), according to scientists who will be presenting papers at the upcoming Cambridge Healthtech Conference, “High-Content Screening and Analysis,” in San Francisco.

More and more, HCS is being used to assess the impact of phenotypic and cellular changes that are brought about by gene modification or drug/compound treatment. HCS, when combined with ADMETox studies, can provide a lot of useful and detailed information in a cellular and physiologically relevant context.

The development and availability of sophisticated instruments, automated data capture, and analysis software and multiplexed assay chemistry in the past few years have aided the explosion in HCS. Some of these advancements will be discussed at the San Francisco conference.

“The genomics era was focused on genome sequencing and functional genomic studies with the attendant proteomic analysis. Tools and technologies were scaled to accommodate this functional and high-throughput approach. Now there is a definite shift to a systems biology era where the focus is on biology and cellular systems. With systems biology gaining importance, tools are needed to obtain relevant contextual and physiological information from individual cells. Thus, high-content imaging tools are becoming increasingly relevant,” says Philip G. Vanek, Ph.D, director of marketing at BD Biosciences(www.bd.com).

Dr. Vanek will present a seminar at the conference on how BD is enabling discovery through its live-cell confocal automated bio-imaging (BD Pathway™ 435 and 855) platform offering.

“The BD Pathway systems were designed to explore the context of living cells and address unmet needs such as automation and advanced image-analysis software. The BD Pathway 435 allows confocal imaging of cells and tissues with high optical precision and exploration of cells accurately on an XYZ plane. The 435 system targets the end-point assay-market segment.

One can run multiplexed experiments with fluorescent antibodies and/or dyes followed by rapid and sequential analysis. A unique feature is that the optics move and hence can be used for study of suspension as well as adherent cells,” says Dr. Vanek.

The BD Pathway 855 is a fully automated live-cell confocal imaging workstation. It includes all the features of the BD Pathway 435 and offers complete environmental control and fluidics capability, allowing the researcher to study the dynamics of living cells. A binocular eyepiece and 16 excitation filters provide added flexibility to the instrument platform.

“These systems are being utilized by researchers to do live-cell kinetic imaging, study cell invasion, and run novel angiogenesis and neurite outgrowth assays, among other applications,” adds Dr. Vanek.

Dr. Vanek will also discuss the company’s bioimaging certified reagents that have been qualified for use in high-content and microscopy applications. These reagents are built upon BD Biosciences Pharmingen’s antibodies, a number of which have been conjugated to Alexa dyes for multiplexing experiments and can be used directly for cell-imaging applications.

GPCR Analysis

Intracellular calcium assays have become the industry standard for screening compound libraries in GPCR studies. The Fluorescent Imaging Plate Reader (FLIPR) to monitor calcium mobilization from Molecular Devices (www.moleculardevices.com) is widely used in primary screens.

The Molecular Devices Transfluor assay is used to assess b-arrestin recruitment during the GPCR receptor desensitization. Transfluor directly visualizes the intracellular localization of b-arrestin, making it a universal assay for GPCRs, and thus complements HTS calcium-mobilization assays.

Molecular Devices will discuss the advantages of running the FLIPR and Transfluor assays as a combination assay using the same cells in the same well. A key feature of combining the two assays is that it is homogenous. Cells are labeled with the calcium and Transfluor reagent prior to the experiment. The calcium flux is measured first on live cells using FLIPR. The cells are fixed, following the calcium-flux measurement, and the Transfluor response can be analyzed at a later time.

“Our combination assay allows us to study the activation and desensitization biology of the same receptor in one single assay,” explains Pierre Turpin, Ph.D., product application scientist at Molecular Devices. This combination assay approach was validated with a study using the Library of Pharmaceutically Active Compounds (LOPAC1280) to screen for agonists or antagonists of the Angiotensin Receptor.

“The overlap between the compounds identified in the FLIPR assay and the Transfluor assay corresponds to the real hits to follow up on. This assay can greatly help decrease the cost and increase the speed of the compound characterization process,” says Dr. Turpin.

The FLIPR response was measured on the Molecular Devices’ FLIPRtetra, Transfluor response was imaged with the ImageXpressMICRO system, image analysis was performed with MetaXpress data-analysis software, and multiparameter analysis was performed using Molecular Devices’ AcuityXpress cellular informatics software.

Ger Brophy, Ph.D., general manager of discovery sciences at GE Healthcare (www.gehealthcare.com), discussed the use of high-content analysis to study radiation-induced changes in non-small-cell-lung carcinoma (NSCLC). “Radiotherapy treatment, in the form of ionizing radiation to kill tumors, is often administered on drug-resistant tumors. Often times, NSCLCs with the same histology exhibit differential radioresponses to ionizing radiation. Recent discoveries, performed by our collaborators at University of Texas, indicate that a mutation in a tyrosine kinase domain (TKD) of an epidermal growth factor receptor (EGFR) gene, in certain subsets of NSCLCs, increase tumor responsiveness to the EGFR tyrosine kinase inhibitor gefitinib.

“However, the effects of ionizing radiation on EGFR-expressing tumors were not known. We performed a study to look at the effects of ionizing radiation on EGFR-expressing (mutant and nonmutant EGFR) NSCLC cell lines. This study correlated the presence of such mutations to NSCLC ionizing radiation sensitivity,” explains Dr. Brophy.

A typical approach for such a study would have involved multiple steps and assays such as irradiation of a wide range of cell lines, several end-point assays to assess effect of ionizing stimuli, tedious individual microscopic analysis, and flow-cytometry experiments.

“We conducted the study with GE’s high-content IN Cell Analyzer 1000, which allowed us to integrate all these different assays onto one platform. Nineteen different EGFR-expressing cell lines were treated with multiple configurations of ionizing radiation on a microtiter plate and analyzed with the IN Cell Analyzer 1000. Several endpoints such as surviving cell numbers, morphological changes, BrdU incorporation (to assess proliferation), and mitotic index were assayed,” explains Dr. Brophy.

The IN Cell Analyzer 1000 has a high-resolution automated microsope and offers multiple objectives, filter sets, multiplex analysis capability, and environmental liquid-handling control on deck, so cells can be studied live (in-cell). Effects of stimuli can be directly monitored on the instrument deck. The analyzer is also coupled to a high-powered IN Cell Investigator image analysis software and Spotfire® data-visualization software so data can be analyzed in a rapid manner, according to the company.

“The IN Cell Analyzer is ideal for conducting complex multiparametric pharmacogenomics study. It is critical to have the pharmacogenomic profile of a drug mapped out at times of market launch. Such studies can also be translated into modifying radiotherapy in sub populations of patients who may have resistant or non-resistant strain of NSCLC,” says Dr. Brophy.

Cellular Technologies

PerkinElmer (www.perkinelmer.com) plans to enter the HCS-provider arena with a suite of offerings. Their entry will be enabled by their acquisition of Evotec Technologies (www.evotec-technologies.com). Evotec currently provides systems for confocal imaging, cell handling, ultra-high throughput screening (uHTS), as well as image capture and cellular analysis software.

“PerkinElmer already offers a variety of cellular science platforms such as the CellLux (an automated cellular fluorescence imaging platform), LumiLux™ (automated cellular luminescence imaging platform), and assay chemistries. These plate readers are complementary with Opera™ and Acapella™, a confocal imaging system and sophisticated imaging software, respectively, both of which will be acquired through Evotec Technologies. The combination of these systems significantly boosts the number of applications that PerkinElmer can serve in cellular screening,” says Mary Duseau, business unit leader for detection and analysis systems at PerkinElmer.

Other technologies of interest are the microchannel flowthrough device, which is essentially a cell processor with dielectric elements. It allows individual cells to flow through the system and be imaged. This device also enables localization studies in live cells.

“Customers are looking to add cellular capability to their existing HTS setup. We will be able to offer the EVOscreen® uHTS system, which has the ability to integrate multiple platforms and is amenable to HCS,” adds Duseau.

HCS can provide a lot of useful information if one can effectively sift through and analyze the enormous amount of data generated from such screens. Charles Y. Tao, Ph.D., laboratory head of genome and proteome sciences at Novartis Institutes for BioMedical Research (www.novartis.com), describes data analysis strategies for high-content screening. “High-content screens result in a huge amount of data. For example, a whole- genome (comprised of about 25,000 genes) screen with a simple reporter gene assay would typically result in 50,000 data points per screen. These data points contain only the intensity information.

“However, a whole-genome screen with the HCS approach could potentially result in billions of data points per screen after image quantification. The data overload is often a bottleneck, and it is critical to have effective data-analysis strategies in place,” comments Dr. Tao.

He developed a data-analysis platform at Novartis to address this problem. The goal is to mine HCS data for information relevant to drug discovery using various statistical and bioinformatic approaches and to ensure the quality of data.

“We perform data analysis at every stage of our HCS campaign. The first step is quality control and data normalization based on preselected parameters. This is an often ignored aspect of data analysis. Errors are detected and systematic errors are corrected, if possible.

“At the second step, we apply various statistical methods to analyze the data. For example, in a cell-cycle study, machine-learning approaches are taken to classify the millions of cells into the different phases of the cell cycle in order to generate a cell-cycle profile for each gene or compound, while in a nuclear translocation study, dose-response profiles are generated. Finally, bioinformatic methods, such as pathway analysis, are applied to further prioritize the hits,” explains Dr. Tao.

As a case-study example, Dr. Tao will discuss a whole-genome HCS. With the data-analysis platform, “we were able to identify both known and new cell-cycle regulators,” says Dr. Tao.

Kenny Guo, research scientist in the applied genomics department at the Bristol-Myers Squibb Pharmaceutical Research Institute (www.bms.com), will discuss strategies to effectively streamline and analyze the complex and high-volume data that is generated by HCS. Bristol-Myers Squibb conducts high-content screening for target validation, compound-library screening, and determination of compound mechanism of action.

“Our high-content screens are run using a high-resolution, fluorescent-based imaging system. Cells are labeled and assayed for multiple endpoints. The output combines numeric and image-based data. For example, if we run a four-channel assay with three primary measurements per channel and 1,000 cells per well, the resultant twelve combinations generate 12,000 data points from a single well. When you add in the image data at ten images per well (1,000 images per plate), the final output can easily reach a million data points from a single 96-well plate,” says Guo.

“To deal with this type of high-volume data, we use an integrated system that is based on a company-wide database. This database is built upon scanned data and results data. It starts with a hierarchical storage system for images and a relational database for numeric results. This data is linked to a data-management system.

“Data annotation and data QC are then conducted in an automated fashion within the data-management system. Customized tools are used to rank and sort the potential hits. This data is then published for company-wide use. The data-management system is also integrated into data-visualization and -analysis software tools to maximize efficiency and productivity,” explains Guo.

An example apoptosis study, conducted with multiple markers to delineate the molecular mechanisms of compound cytotoxicity, will be presented at the conference.

Also scheduled to talk at the Cambridge Healthtech Conference, Steven Suchyta, Ph.D., technical specialist at Ambion (www.ambion.com), an Applied Biosystems (www.appliedbiosystems.com) business, says he will describe how the R&D group used targeted siRNAs to alter survivin expression. A series of biochemical and cell-based assays were performed on the transfected cells to assess known indicators of apoptosis to better understand the role of survivin in cancer.

Elucidating Gene Function

“Survivin (BIRC5) is a 17-kDa bifunctional protein that plays critical roles in the regulation of both cell division and survival. Survivin expression is linked to a broad range of cancers,” says Dr. Suchyta.

Ambion’s Silencer® Pre-designed siRNAs targeting survivin were first evaluated for silencing using TaqMan® Gene Expression Assays from Applied Biosystems. “It is important to always include more than one siRNA per target gene in a study to rule out results caused by non-specific effects,” notes Dr. Suchyta.

Thus, two siRNAs that effectively silenced survivin expression were chosen to proceed. These validated siRNAs were then used to study the functional effects of survivin down regulation in HeLa cells. Apoptosis was assessed by monitoring cell survival/proliferation, nuclear condensation and chromosomal fragmentation, phosphatidyl serine externalization, and caspase activation. The magnitude of the induced biological effects was correlated with that of siRNA-induced silencing at the mRNA and protein levels using a timecourse study. “Together, the data indicates that reduction of survivin protein levels induced apoptosis-related events, although it failed to activate pro-caspase-3,” notes Dr. Suchyta.

Two key challenges in high-content analysis are how to analyze the data and how to effectively use the information obtained from the HCS. “For example, the apoptosis assay results in a cell death number, but also provides additional information such as caspase activation, membrane integrity, and morphological changes,” says Donald Jackson, Ph.D., senior research investigator at Bristol-Myers Squibb.

“The challenge at the end of the day is to determine which of these treatments is killing the cells and makes sense of the additional data in a contextual manner. In spite of these obvious challenges, the biggest benefit of HCS is that one can detect patterns and exclude artifacts to generate a truly valuable data set.”

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