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High Content Analysis - Day 3


Friday, January 15

7:30-8:15 Breakfast Presentation or Morning Coffee

Sponsorship available. Contact Katelin Fitzgerald, Manager, Business Development, at 781-972-5458 or kfitzgerald@healthtech.com 

HCA for siRNA Screening

8:30-8:35 Chairperson’s Opening Remarks

8:35-9:00 The Pros and Cons of siRNA Use in HCS

Eugenio Fava, Ph.D., Director, Services and Facilities, German Centre for Neurodegenerative Diseases

The use of siRNA in high-content screens (HCS) in mammalian cells has become a valuable method to identify and describe genetic relationships in both basic biology and disease mechanisms. However, the use of siRNAs in HCS has been hampered by an inherent problem of siRNA, namely off target. Off target is the phenomenon that takes place when genes with incomplete complementarity are downregulated by the siRNA. This leads to problems in data interpretation and overall difficulties in the determination of hits. Although there are different strategies to reduce siRNA target, ranging from design to chemical modification, avoiding it is very difficult if not impossible. Here we analyze and discuss a complementary method approach that allows us to evaluate the siRNA off target from the HCS data. We used this method to compare and contrast different siRNA technologies and the results will be discussed.

9:00-9:30 Phenotypic Drug Discovery to Identify Novel Oncolytics using High-Content Fingerprinting

Louis Stancato, Ph.D., Senior Research Advisor, Cancer Growth & Translational Genetics, Eli Lilly & Co.
The development of high-content imaging subpopulation analysis tools has enabled a high resolution look into cancer cell function, and as a result is now an integral technology in Lilly Oncology. One area in particular that has experienced a renaissance is phenotypic drug discovery, a discipline ideally suited to the application of HCI. By incorporating custom in-house informatics tools we are able to advance molecules with novel mechanisms of action through the lead generation process, in particular chemical series previously discarded due to perceived failure in conventional “targeted” approaches. This presentation will focus on how the HCI informatics tools are used to identify, phenotypically fingerprint and subsequently advance in vivo, novel anti-cancer molecules using both cultured cell and cancer stem cell models.

9:30-9:55 A High-Content Assay to Screen for Modulators of the miRNA Machinery

David Shum, Ph.D., Assay Development Specialist, HTS Core Facility, Memorial Sloan Kettering

MicroRNAs are small non-coding regulatory RNAs reducing stability and/or translation of fully or partially sequence-complementary target mRNAs in plants and animals. Their upregulation has been reported in many malignancies and proposed to regulate several tumor suppressor pathways; the understanding of their biogenesis may offer novel therapeutic approaches and intervention. There are no well documented modulators to help us study the pathway and unravel its biological significance; for this purpose, we set out to develop a high-content assay using a stably transfected HeLa S3 cells expressing EGFP under miR-21 regulation as the reporter gene carries a sequence with perfect complementarity to miR-21 in its 3‘ UTR region, thus decrease in miR-21 levels would induce increase in the expression and maturation of the EGFP reporter which can easily be imaged using automated microscopy. We will present and discuss our assay development and validation against both a pilot chemical library and a custom siRNA library targeting known genes of the miRNA pathway together with few essential housekeeping genes to assess transfection efficiencies. Our high-content assay and its dual adaptation would allow screening and discovery of novel modulators of the miRNA pathway and a better understanding of the regulatory components of its maturation machinery.

HCA for Toxicity Profiling

8:30-8:35 Chairperson’s Opening Remarks

8:35-9:00 Refinement of Predictive Cytotoxicity Assays Using High-Content Analysis and Extension into Different Cell Lines

Peter O’Brien, D.V.M., Ph.D., Veterinary Clinical Pathologist, Pathology, University College Dublin, Ireland

Our HepG2 predictive cytotoxicity assay using HCA was refined, along with the associated process and data analysis and applied in new capacities, including screening in academic “drug discovery” activities. It was streamlined and made more user-friendly, reproducible, and cost-effective at the biological reagents, assaying, and data reduction and analysis ends. Its effective application was extended into several other cell lines, including lymphoid, monocytic, and intestinal, and into other programs and disciplines, including in cancer drug evaluation, food health, and environmental monitoring.

Sponsored by
9:00-9:15 Use More Cells! High-Content Alternatives for in vitro Toxicity Assessment

Mark A. Collins Ph.D., Director, Marketing, Thermo Fisher Scientific
Finding toxicity late in the drug discovery process is not only frustrating but costly and current methods are often insufficient to find the problems in time. There is a clear need for innovative strategies to address this challenge. An automated imaging approach to detecting physiological changes in individual cells can generate a variety of phenotypic measurements, which can then be used to assess each cell’s reaction to a toxic insult. This cellular profile provides more information to better determine the toxicity risk of a potential drug candidate, enhancing decision making. We will discuss the advantages of cell imaging assays over traditional plate reader assays and review recent literature that has demonstrated substantial impact of such approaches on candidate attrition. We will show that using the Thermo Scientific ToxInsight IVT Platform, which combines new instrumentation, software, reagents in a simple intuitive workflow, you can benefit from these new tools for assessing toxicity risk in liver, kidney and other organ models.


Sponsored by
GE Healthcare
9:15-9:30 High Content Analysis for Characterization and use of Cell Lines Developed to Model Tumor Cell Drug Resistance

Robert Graves, Ph.D., Senior Applications Scientist, GE Healthcare

Monitoring mitochondrial status and targeting mitochondrial function and are important strategies for the assessment of toxicity and development of potential therapeutic drugs.  A persistent challenge in the development of effective therapies targeted at solid tumors is the acquisition of drug resistance, and hence continued survival of a population of constituent cells within the tumor.  A number of studies have indicated that tumor cells become resistant to mitochondrial toxicants by bypassing oxidative phosphorylation under high glucose conditions.  As models of drug-resistance, we have developed cell lines differentially conditioned into high and low glucose conditions.  High content analysis with a flexible automated imaging system has been an essential technology throughout the process of cell line characterization, assay optimization and screening with test compounds and siRNAs. Automated analysis approaches are employed to improve assay statistics and extract from each individual cell a wide range of measurements that can be used to assess cell phenotype. Features such as manual microscopy mode, on-line cell counting, and image restoration have improved both the workflow and data quality, facilitating the capture of phenotypic data that may be informative in developing better cancer therapeutics.

9:30-9:55 In vitro Toxicity Profiling in Non-Adherent Cells by Automated High-Content Microscopy

Michael Prummer, Ph.D., Scientist, High-Content Screening, Discovery Technologies, Pharma Research, F. Hoffmann-La Roche Ltd.

Most High-Content Screening assays are run with adherent cells and not with suspension cells due to practical limitations. However, there are established cellular models for conventional microscopy in which suspension cells are used. We are presenting here a novel and universal approach to immobilize non-adherent cells in multi-well format for automated microscopy that fills this gap. As a first application we report on a high-content toxicity assay at the single-cell level using an innovative new preparation tool. For image segmentation and object classification we have developed an adaptive and iterative algorithm in collaboration with Definiens AG. To extract a toxicity score from the derived data, non-parametric statistical methods were employed. Taken together, these developments open up new routes for high-content screening with suspension cells.

9:55-10:20 Networking Coffee Break

HCA for Pathway Analysis

10:20-10:45 High-Content Screening of Combinatorial Library for Identifying Biomarkers

Hang Chang, Ph.D., Project Scientist,
Life Sciences Division, Lawrence Berkeley National Laboratory

A combinatorial fluorescence library of small molecules is used for exploring (i) differential labeling of different cell lines (e.g., do they localize differentially for a panel of cell lines) as a result of different types of treatment (e.g., radiation, drug treatment), (ii) flux analysis (e.g., what is uptake and retention of each ligand), and (iii) the relationships between structure and activity of each ligand and their corresponding subtypes. All data
and results are then registered against a database of response for bioinformatics analysis.

10:45-11:10 Targeting Cytokine Receptor-JAK-STAT Signaling Pathways - Development of an HCS Assay to Identify Selective Inhibitors of STAT-3 Phosphorylation and Nuclear Translocation

Paul A. Johnston, Ph.D., Research Associate Professor, Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine

The Signal Transducers and Activators of Transcription (STATs) are transcription factors that mediate the effects of growth factors and cytokines to regulate the expression of genes involved in cell proliferation, differentiation, inflammation, migration, and apoptosis. Activated STAT3 is an oncogene that directs tumor cells toward proliferation and survival, induces angiogenesis and alters the tumor microenvironment, and promotes tumor metastases through its effects on cell migration and invasion. In sharp contrast activated STAT1 is considered a tumor suppressor because it is a potent inhibitor of tumor growth, promotes tumor cell apoptosis, and enhances tumor immunity. Consequently, the discovery of a STAT3 selective inhibitor would be a highly desirable goal for the development of an anti-cancer drug. The development of an HCS assay to identify selective inhibitors of STAT-3 phosphorylation and nuclear translocation will be described.

11:10-11:35 Development of a Multiparametric Assay to Measure Cell Phenotypic and Pathway Parameters Indicative of ROCK Activity

Danli L. Towne, Scientist II, High-Content Screening, Lead Discovery, Abbott Laboratories

Rho kinase (ROCK) is a serine/threonine kinase which is activated by Rho GTPase. Activated ROCK phosphorylates many substrate proteins, and consequently regulates various downstream signaling pathways that control multiple cell type-specific cellular functions including smooth muscle contraction, cell migration, proliferation and neurite outgrowth. All these effects require reorganization of the actin cytoskeleton. ROCK can also increase Myosin Light Chain (MLC) phosphorylation through inhibition of Myosin Light Chain phosphatase, which may contribute to actin reorganization as well. We have developed a cell-based high-content assay using HUVEC cells to measure a combination of phenotypic (actin reorganization) and pathway (MLC phosphorylation) parameters associated with ROCK inhibition. Our results show that MLC phosphorylation and actin reorganization can be decoupled. The assay is also valuable for detecting off-target compound effects. In addition, our data suggests that the HCS assay provides a valid approach to measure ROCK inhibition in intact cells. 

11:35-12:00 High-Content Cellomics Cell Cycle Analysis of the Effects of HSP90 Inhibition

Susan Lyman, Ph.D., Research Scientist, Exelixis, Inc.

Cell cycle analysis has traditionally been carried out by FACS – or more recently, by combining a measurement of DNA content with imaging of one or more phase-specific readouts, such as phospho-histone H3. We have used the latter approach to develop a novel and robust high-throughput Cellomics-based cell cycle assay that accurately reports the phase status of a cell (G1, S, G2, or M) as well as its DNA content (2n, 4n, >4n). We have applied this technique to examine the cell cycle perturbations caused by inhibition of HSP90, a molecular chaperone that enhances the stability of a wide spectrum of client proteins. HSP90 clients include cell cycle proteins such as PLK1 and CDC2 as well as cancer-associated proteins such as EGFR, ERBB2, and MET, making it an attractive cancer target. This presentation will illustrate the Cellomics cell cycle technique and will highlight the strikingly similar complex cell cycle perturbations induced by 3 different small-molecule HSP90 inhibitors in a large panel of immortalized cancer cell lines.

Neuronal Screening

10:20-10:45 Using HCA to Identify a Transcription Factor Family that Regulates the Intrinsic Ability of Neurons to Extend Axons

Murray Blackmore, Ph.D., Research Assistant Professor, The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine

As neurons age, they lose the ability to extend axons, resulting in failure of regeneration after injury to the central nervous system. We used HCA to screen genes that were differentially expressed during development in retinal neurons and cortical neurons. We identified a family of transcription factors that regulate axon growth in vitro and in vivo. In the course of these studies we found that some types of primary neurons are more suitable for detecting increases in neurite growth than other types of neurons.

10:45-11:10 High-Content Analysis of Synapse Formation in Primary Neuronal Cultures for Developmental Neurotoxicity Screening

William R. Mundy, Ph.D., Integrated Systems Toxicology Division, U.S. Environmental Protection Agency

Cell-based assays can model neurodevelopmental processes including neurite growth and synaptogenesis, and may be useful for screening and evaluation of large numbers of chemicals for developmental neurotoxicity. This work describes the use of HCA to detect chemical effects on synaptogenesis in vitro. Pre-synaptic puncta were associated with the cell body and dendrites of primary cortical neurons, and increased over several weeks in culture. Several chemicals were identified that decreased synapse number during development in vitro.

11:10-11:35 Screening Neuronal Pattern Formation in vivo

Sebastian Munck, Ph.D., Coordinator, Light Microscopy and Imaging Network LiMoNe, Department of Molecular and Developmental Genetics, VIB, K.U. Leuven

We would like to describe an assay for imaging-based high-content analysis of the network formation in vivo. The neurons in the optic lobe of Drosophila melanogaster form a stereotypical pattern. In the optical system neuronal /electrical activity is believed to be a crucial part in the pattern formation process. We want to dissect the genetic and activity depended processes. The aim of the project is to identify the key players of the pattern formation and to modulate them by combining the power of a genetic screen in drosophila with the imaging based read out of the formed pattern.

11:35-12:00 Development of a Composite Primary Neuronal High-Content Screening Assay for Huntington's Disease Incorporating Non-Cell Autonomous Interactions

Linda S. Kaltenbach, Ph.D., Research Scientist, Neurobiology, Duke University

Huntington’s Disease, a fatal neurodegenerative disease caused by expansion of a polyglutamine repeat in the Huntingtin protein, incorporates obligatory non-cell autonomous pathways involving both the cortex and the striatum.  We developed a fully automated high-content screen in high-density composite primary cultures including both cortical and striatal neurons as well as their supporting glial cells.  Cortical and striatal neurons are transfected separately with different fluorescent protein markers such that image-based high-content analysis can be used to assay these neuronal populations separately, but still supporting their intercellular interactions within the same culture wells.  The results of a screen of ~500 selected small molecule compounds and preliminary data on a neurite-based endpoint will be described.

12:00 Close of Conference