Track I: Compound Screening

TRACK I: COMPOUND SCREENING
HCA FOR COMPOUND SCREENING AND PROFILING

10:10-10:15 Chairperson’s Opening Remarks
Dr. Jeffrey R. Haskins, VP, Technology & Product Development, Cellomics, Inc.

10:15-10:45 Primary, Secondary Screening and Genotoxicity Profiling of Compounds Using High-Content Imaging Platform
Dr. Anthony Nichols, Head of Screening Technology Development, Molecular Screening & Cellular Pharmacology, Serono Pharmaceutical Research Institute
The biological applications of HCS have been implemented in our research in signaling, cell morphology and toxicology. HCS platforms have been placed within our core high-throughput screening facility. Our protein and small molecule strategies converge on diseases in therapeutic areas such neurological disorders and autoimmune diseases. Our scientists use HCS for assay development, primary, secondary screening and toxicology testing.

10:45-11:15 Use of High-Content Imaging to Profile Anti-Tumor and Anti-Angiogenic Compounds 
Dr. Mark Uhlik, Research Scientist, Angiogenesis and Tumor Microenvironment DHT, Eli Lilly & Co.
As drug discovery paradigms in many companies shift from high-throughput environments to more focused screening ones, there is an increasing need to develop tools that enable quick, efficient, and powerful means of deconvoluting small molecule activities at the cellular level. Imaging-based high-content assays provide refined methods of profiling cell-based compound activities before in vivo testing. Using high-content approaches, intricate biological processes such as apoptosis and angiogenesis may be studied in complex biological systems, such as tumor cell/endothelial cell co-cultures. To this end, the development of high-content assays for apoptosis, endothelial cell cord formation, and cell migration (wound healing) will be discussed. These assays are particularly useful within oncology drug discovery and provide data-rich information to facilitate crisper decision-making and rank-ordering of compounds prior to expensive and labor-intensive in vivo tumor models.

11:15-11:45 A Snapshot of Cellular Imaging at Eli Lilly
Dr. Jonathan Lee, Research Advisor, Lead Generation and Optimization Biology, Eli Lilly and Company
Eli Lilly has witnessed a proliferation of high-content instrument platforms and cell based imaging assays within the core biology groups and drug hunting teams. Cell imaging has been successfully applied to applications in Lead Generation, Lead Optimization, tertiary assays, Biomarker Discovery, and Toxicology. Select examples of high-content cell imaging and their impact to the drug discovery process will be discussed.

11:45-12:15 Applying High-Content Automated Imaging Assays to Compound Profiling for Kinase Inhibitor Lead Optimization
Dr. Paul Johnston, Research Associate Professor, Pharmacology, University of Pittsburgh School of Medicine
Although biochemical assays remain the predominant strategy to generate and optimize kinase inhibitor leads, only leads that demonstrate activity in an appropriate cell based model will progress. Inhibitors that interact with an ATP binding site that is highly conserved among kinases raises concerns about kinase selectivity and potential off-target effects. High-content cell based imaging assays are ideally suited to kinase inhibitor compound profiling because in addition to a direct target readout, the multi-parameter data can provide information on cell morphology and cytotoxicity, or may be designed to provide readouts on kinase targets in the same or different signaling pathways.

TRack II: Pathway Analysis

TRACK II: TARGET VALIDATION
GENOME-WIDE siRNA HIGH-CONTENT SCREENING

10:10-10:15 Chairperson’s Opening Remarks

10:15-10:45 Statistical and Functional Analysis of a Genome-Wide siRNA Cell Cycle Screen
Dr. Yan Feng, Lab Head, Genome and Proteome Sciences, Novartis Institute for BioMedical Research
We used a multi-parameter imaging cytometry based assay and genome-wide siRNA knockdown to characterize genes and pathways that regulate cell cycle progression. Cells from various cell cycle stages were categorized by a combination of clustering and machine learning algorithms. A multi-parameter cytometry fingerprint was then generated for each gene. System level analysis was performed by putting the cell cycle fingerprint into a function genomic context.

10:45-11:15 Genome-Wide RNAi Screen Identifying Novel Cell Cycle and Cancer Targets
Dr. Daniel R. Rines, Institute Fellow, Lead Discovery, Genomics Institute of the Novartis Research Foundation (GNF)
The application of highly parallelized methods to examine cellular phenotypes can enable the study of gene activities at the level of the genome. Towards this end, we have assembled genome-wide collections of siRNAs and cDNAs. Combining these libraries with high-throughput methodologies for parallel transduction of various cell-types and high speed microscopy platforms, we have executed cell-based assays to examine a diverse range of activities. In particular, we have recently used a RNA library of 49,000 double-stranded (ds)RNAs, targeting approximately 24,000 genes, in a genome-wide loss-of-function screen for essential mitotic chromosome segregation genes. Multi-parametric quantitative analysis of the image-based data has allowed us to isolate over 200 known and novel genes. The application of this functional profiling technology has led to the elucidation of several novel gene activities, and will likely play an integral role in the understanding of gene function on a global scale.

11:15-11:45 Genome-Wide High-Content RNAi Screening to Identify Regulators of Endocytotic Pathways
Dr. Eberhard Krausz, HT-Technology Development Studio, Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)
We are applying siRNA libraries to multi-parametric high-content assays using an automated high-throughput microscope. In primary viral infection screens, kinases were identified to regulate two independent endocytotic pathways each specifically hijacked by a virus. Primary 'hits' were further characterized in a set of six secondary assays that allow pathway dissection. Complex regulative networks were discovered. A subsequent genome-wide screen has been implemented.

11:45-12:15 Rapid, High-Content Genome-Wide Assays Using Cell Microarrays 
Dr. Anne E. Carpenter, Novartis Fellow of the Life Sciences Research Foundation, Sabatini Laboratory, Whitehead Institute for Biomedical Research
Cell-based microarrays allow the preparation of thousands of individual cell samples on a single microscope slide using conventional microarrays. This allows testing an entire genome for high-content phenotypes on 4-8 slides. Each spot on the slide is a cluster of several hundred cells that are perturbed by a single gene expression plasmid, small molecule, or RNA interference reagent (RNAi), and can be imaged using high resolution microscopy. Because existing commercial software had limitations, we developed CellProfiler cell image analysis software to measure a variety of interesting phenotypes of cells on these arrays. By knocking down each gene in Drosophila by RNAi and analyzing cells with CellProfiler, we are obtaining a high-quality, high-content readout of the effects of knocking down each gene on a variety of cellular phenotypes.

LUNCHEON TECHNOLOGY SHOWCASE

1:00-1:15
Turnkey High-Content Screening with an Integrated Imaging Platform Dr. Michael Sjaastad, Director of Marketing, Imaging, Molecular Devices Corp. High-content screening with fixed and live cell-based assays provides extensive multi-parametric cellular data. However, successful implementation or HCS requires a complete, turnkey environment for image acquisition, analysis, and data management. Most importantly, to leverage the extensive data generated, an informatics platform must be fully integrated into a solution. This presentation will provide a clear overview of the components and new options for the MDC HCS platform. Using novel data, we will demonstrate turnkey cellular informatics and review developments in AcuityXpress, our cellular informatics software for improved analysis of HCS data.

1:30-2:00 Technology Short Talks
Additional Sponsorship Available. Contact: Carol Dinerstein at 781-972-5471 or dinerstein@healthtech.com.

HCA FOR IN VITRO TOXICOLOGY

2:15-2:45 Applications of High-Content Screening in Discovery Toxicology
Dr. Peter O’Brien, Head of Discovery Toxicology Biomarkers, Safety Sciences, Europe, Pfizer Limited
High-content screening (HCS) was evaluated for its capability to assess the potential of ~250 compounds to produce human toxicity. Changes in cell proliferation, mitochondrial activity, nuclear features, calcium concentration, membrane permeability, and oxidative stress were simultaneously monitored as HCS biomarkers in a human cell line. The HSC assessment was compared with that of seven conventional cytotoxicity assays. The latter were found to have unacceptable sensitivity, although reasonable specificity. However, there was high correlation (>80%) between sub lethal changes in HCS biomarkers and the occurrence of human toxicity.

2:45-3:15 Comparison of Imaging and Conventional Assays as Readouts for Toxicity
Mrs. Marjo Simonen, Discovery Technologies, Novartis Pharma AG 
We compare a small set of frequent hitters from reporter gene assays plus some known toxic compounds in four different commercially available toxicity assays and in imaging-based assays using IN Cell Analyzer 3000. The toxicity assays measure either reducing units or ATP levels of the cells, or the amounts of dead cells in the culture. With IN Cell Analyzer 3000 several markers of toxicity and apoptosis are measured, like cell number, nuclear intensity and size, annexin V staining, caspase activity and leakage of cytochrome c from the mitochondria.

3:15-3:45 High-Content Screening in Support of Discovery- Applications from an Open Access Cytometry Core
Mr. David F. Gebhard, Principal Scientist, Lead Assessment Team, Pfizer, Inc.
High-Content Screening is increasingly utilized in many aspects of contemporary drug discovery and development. This presentation will discuss the implementation of a HCS platform in a “classic” flow cytometry core laboratory, and the subsequent integration of a site based HCS informatics initiative. Application of HCS to early compound characterization-secondary assays, insight into process control for discovery lab procedures, and support for early in vitro safety assessment will be discussed.

LUNCHEON TECHNOLOGY SHOWCASE

12:30-12:45
Adenoviral Vectors in Pathway Analysis and Target Validation Dr. Stephen Game, Department Director, Molecular and Cell Biology, GE Healthcare The new Adenoviral Vector Gene Delivery System from GE Healthcare expands the possibilities for drug target validation by enabling the development of cellular assays that can greatly aid secondary screening and early-stage drug discovery. The system includes a range of ready-to-use recombinant adenoviral preparations and data will be presented of a range of assay targets and applications.
12:45-1:00 Recent Developments in siRNA-Based RNA Interference

1:00-1:30 Application of Ingenuity Pathways Analysis and RNAi technology in drug discovery programs
Dr. Keith Joho, Senior Vice President Research and Product Management, Ingenuity Systems, Inc Ingenuity Pathways Analysis (IPA) enables biologists and bioinformaticians to model, analyze and understand the complex biological systems at the core of life science research. IPA 3.1 allows scientists to elucidate and customize biological pathways for particular targets, biomarkers, disease areas, and processes, leveraging Ingenuity's broad knowledge base of biological relationships between genes and proteins, cells, tissues, and diseases. This web-delivered application enables effective interpretation of critical pathways that are activated or disrupted upon RNAi knockdown. Target identification studies in which Ingenuity Pathways Analysis has been used in conjunction with RNAi to elucidate pathways for potential drug targets and to assess genome-wide effects of a knockdown of that target will be presented.
1:30-1:45 RNAi Goes Genomic: Elucidating Gene Function with siRNA Libraries
Dr. David Dorris, Vice President RNAi Technologies, Ambion, Inc Four keys to successful RNAi screens in human cells are: using highly effective siRNAs, reproducibly and efficiently delivering those siRNAs, ascertaining RNAi effects with a robust assay, and carefully controlling experiments. We will discuss the latest developments in siRNA design and delivery as they relate to RNAi screening, and present data from experiments using siRNA libraries to identify genes involved in apoptosis, cell proliferation and cell cycle progression. We will also present data on use of siRNA pools versus multiple individual siRNAs, as well as data illustrating the need for multiple carefully chosen siRNA controls for siRNA screening experiments.
1:45-2:00
Technology Short Talks Additional Sponsorship Available. Contact: Carol Dinerstein at 781-972-5471 or dinerstein@healthtech.com

2:15-2:45 Phenotypic Fingerprinting of shRNA Effects on Mitotic Progression Using High-Content Imaging
To Be Announced
Successful application of RNAi at scale combined with high-content imaging has the potential to greatly increase our understanding of a wide range of cellular processes. Lentiviral-based shRNA delivery is a particularly effective method that allows for RNAi evaluation in a wide range of cell types, including non-dividing primary cells. This presentation focuses on high-content analysis of both on and off targets effects of lentiviral delivered shRNAs on mitotic progression in human cells with the aim of identifying phenotypic fingerprints derived from protein knockdown. Analysis of high-content data captured using Cellomics VTI technology, with an emphasis on the mitotic markers cyclinB1 and phospho-histoneH3, will be presented. RNAi technology comparison data (shRNA vs. siRNA) will also be included.

2:45-3:15 RNAi-Based Pathway Analysis Using HCA of Non-Adherent Primary Cells
Dr. Orian Shirihai, Professor, Pharmacology, Tufts University Medical School
To study cellular functions in heterogeneous populations of cells, such as tissue-derived cells, there is a need to continuously monitor functional parameters at a single cell resolution in a large number of cells. This is becoming a unique challenge when the cells of interest are non-adherent, as in the case of blood or bone marrow samples. To overcome this limitation, we used Molecular Cytomics' Optical LiveCell™ Array, a device containing a densely packed array of transparent micron-sized wells. This technology enables real-time, ongoing observation of cellular events from thousands of individual adherent or non-adherent living cells, followed by structural and post-fixation studies on the same cells, using bright field and fluorescent microscopy. We employed this technology for investigating mitochondrial proteins involved in the heme biosynthetic pathway during differentiation of hematopoietic cells. We suggest this approach as a robust tool for HCA studies in heterogeneous populations of cells and as a revolutionary platform for imaging non-adherent cells.

3:15-3:45 Cytoskeletal and Cell Viability Assays for RNAi-Based Validation of Oncology Targets Associated with Rho/Rac Signaling
Dr. Robert Blake, Senior Scientist, Target Discovery, Exelixis Inc.
Conserved members of the Ras super-family Rho, Rac, and CDC42 are key regulators of the cytoskeleton that have roles in promoting cellular proliferation and are implicated in tumorigenesis. We have combined RNAi knockdown techniques and automated fluorescence imaging of cytoskeletal organization, cellular apoptosis and proliferation to investigate the role of candidate genes in these pathways. Knockdown analysis of Rho/Rac/CDC42 proteins or known pathway regulators / effectors, supports the view that Rho and Rac signaling are necessary for cell viability and proliferation and that knockdown of specific pathway components leads to well-defined cytoskeletal alterations. For example RhoA siRNA is associated with reduced myosin light chain phosphorylation. siRNAi of Rac1 or -3, or CDC42 is generally associate with increased actin content and in some cases with reduced cell/matrix adhesion. We discuss assay development for siRNA screens and target validation, and efforts to distinguish primary cytoskeletal alterations from those arising more indirectly from apoptosis or perturbations in other cellular processes.

HCA STANDARDS

5:00-5:30 Controlling the Extracellular Matrix for Standardizing Cell Environment
Dr. John Elliott, Research Scientist, Biotechnology, National Institute of Standards and Technology (NIST)
Extracellular matrix (ECM) proteins provide adhesion sites and signaling cues to cells in culture. Even cells cultured on polystyrene are responding to ECM proteins adsorbed to the substrate surface. Since the molecular features of the adsorbed extracellular matrix proteins can have a dramatic effect on the adhesion and phenotype of adherent cells, it is important to control the tissue culture substrate for optimal intra-experimental and intra-laboratory comparison of high content assay data. Self assembly of ECM protein, such as collagen, into films on alkanethiol monolayers is a controllable process that provides a highly reproducible, highly homogeneous matrix environment for cells, which can be independently validated by surface chemistry analytical techniques. Using this approach, we have studied how subtle variations in ECM preparations can alter cell phenotypic response in dramatic ways. Thus, control of the ECM environment is also critical for assuring the appropriateness of cell response in pharmacological screening, and for allowing correct interpretation of the assay results in terms of signaling pathways. This approach to fabricating ECM substrates can be used to benchmark cell response and may aid in the standardization of data that is available from high content analysis.

5:30-6:00 Panel Discussion 

6:00-7:00 ThinkTank Roundtable Discussions
The concurrent roundtable discussions (open to all delegates) provide a small-circle forum for discussing key issues and meeting potential partners. The discussion facilitators will present an update the following morning. Please visit www.highcontentanalysis.com for an updated list of topics and discussion facilitators. (You must be a registered attendee to participate.)

Discussion Topics Include:
• Compound Screening
• In Vitro Toxicology
• Target Validation
• Neuronal Screening
• Assay Design for HCA
• Imaging Biosensors
• Live-Cell Imaging
• HCS Data Analysis
• HCA Standards
• ROI Analysis on HCA Technology