HCA for RNAi Screening and Pathway Analysis

8:30-8:55     Application of High-Content Fingerprinting to Oncology Drug Discovery: Focus on in vitro and in vivo Phenocopying and Cancer Stem Cell Analysis
Louis Stancato, Ph.D., Research Advisor, Cancer Growth and Translational Genetics, Eli Lilly and Company
We have previously reported on the analysis of HCI data using a subpopulation-based informatics method to develop high-content fingerprints of the eukaryotic cell cycle.  As a result we are now able to better separate the effects of cellular perturbations on the cell cycle (small and large molecules, RNAi, etc.) than was possible using total population readouts. The cell-level resolution afforded by our informatics tools is ideally suited to both target-based and phenotypic drug discovery.  This presentation will highlight the continued development of HCI subpopulation analysis tools and their application to oncology drug discovery, with a particular emphasis on bridging the gap from in vitro to in vivo discovery. In addition, high-content characterization of glioblastoma stem cell populations using these tools will also be presented.

8:55-9:20     Effective Support of Oncology Driven Drug Research by Functional High-Content Analysis
Stefan Prechtl, Ph.D., LDB Screening, High-Content Analysis,
Bayer Schering Pharma AG
A reasonable selection of High-Content Analysis applications is indispensable to effectively support preclinical drug research projects. This applies for target identification and validation, for lead identification and generation as well as for in vivo pharmacology studies. Critical decisions based on profound knowledge are crucial for successful project progression. Reasonable HCA applications can help to build such a profound knowledge data base. Here, we present a number of HCA assays that we have deployed to support oncology driven projects. They encompass a panel of assays based on fixed cell analysis as well as assays utilizing living cell analysis. Reliability of results coming from established GFP-technologies is compared with novel technology approaches such as self-labeling techniques. Complex image analysis routines for the analysis of histological preparations were established and are in place to facilitate the work of in vivo pharmacologists.

9:20-9:35     Sponsored Presentation (Opportunity Available.) Contact Katelin Fitzgerald, Manager, Business Development, at 781-972-5458 or kfitzgerald@healthtech.com.

9:35-10:00  Profiling p53 Networks by Cell-Based Multiplex  Imaging
Rami Hannoush, Ph.D., Scientist, Protein Engineering, Genentech, Inc.
A multiplex cell-based imaging assay was developed to investigate activation of the p53 pathway, a key signaling mechanism that is mutated in several types of cancer. The assay utilizes cellular markers of activation of p53 in a 384-well plate format, and quantitatively reports on protein translocation and cellular levels. Using this assay, RNA interference and small molecule screens were conducted in parallel to identify regulators of the p53 pathway. Systems relationships were derived about the p53 cellular interaction network and its pathway regulation.

HCA in Primary Cells

8:30-8:55              High-Content Cell-Based Assays Used for Study of the Toxic Effects of Antiviral Drugs in Cardiac Cells
Anthony Davies, Ph.D., Director, High-Content Research Facility, Clinical Medicine, Trinity College, Ireland
Over the last decade there has been a huge increase in the production and testing of new biologically active compounds towards an ever-increasing range of therapeutic targets. Despite the increase in drug research and development, and the technological advances, which accompany this, the drug discovery process is still costly with regard to both time and financial resources (typical development costs are $1 billion over 12 years/new drug). Cardiotoxicity of both cardiac and non-cardiac drugs is well-recognized and costly problem in drug development programs. For this reason we have developed several functional based multi-parametic assays to measure cell stress and metabolic damage markers as indicators of cytotoxicity. To demonstrate their utility, we include new and comprehensive data sets from a study, conducted to investigate the potential toxic effects of antiviral drugs on cardiac cells.

8:55-9:20              Primary HCS for Immunomodulation
Carmel B. Nanthakumar, Ph.D., R & II Discovery Technology Group, GlaxoSmithKline
Conventional HCS assays utilize recombinant cell lines, however, with increasing interest in the use of primary cells in early drug discovery, HCS also provides an ideal technology platform to study proteins in their native state.  We have combined HCS and primary cells to develop robust assays measuring endogenous receptor internalization, wound repair and migration, proliferation and differentiation for immune-modulators.  Front-loading physiologically relevant cellular systems in early drug screening may increase the potential to identify compounds with greater probability of demonstrating in vivo efficacy.  However, developing HCS assays in immune cells remains a challenging area.  

9:20-9:35              Sponsored Presentation
Title to be Announced

9:35-10:00            High-Throughput High-Content Screening in Primary and Stem Cells
Francesca Santini, Ph.D., Research Fellow, Automated Biotechnology, Merck Research Laboratories
Implementation of physiologically relevant cell systems and integration of high-content screening data with in-house databases and data mining tools are key drivers to the successful drug discovery at Merck & Co.

HCA for RNAi Screening and Pathway Analysis
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10:30-10:55 From Assay to Molecular Target and from Diabetes to Hepatitis
Fred Levine, Ph.D., Professor and Director, Sanford Children’s Health Research Center, Burnham Institute for Medical Research
We have performed a number of high-content cell-based screens based upon an assay in which insulin promoter activity can be measured. A number of positive small molecule and siRNAs have been identified. One of these, a compound that inhibits insulin promoter activity, is a potent inhibitor of HNF4alpha. This is one of the first examples of a cell-based assay that has been carried through to the identification of a specific molecular target.

10:55-11:10  Sponsored Presentation
Title to be Announced                   

11:10-11:35  Studying  GPCR Trafficking Using Automated HCS Tools
Ying-Jie Zhu, Ph.D., Research Scientist II, Lead Discovery, Bristol-Myers Squibb Co.
The endocytic membrane trafficking tightly controls the activity of G-protein-coupled receptors (GPCRs).  Following endocytosis from the plasma membrane, GPCRs may be either recycled to the plasma membrane or sorted for lysosomal degradation. GPCR endocytosis from the plasma membrane most commonly occurs in a GRK- and b-arrestin-dependent manner. The subsequent recycling or sorting for lysosomal degradation is also regulated by (associated with) b-arrestin.  We have used two systems to examine trafficking of a GPCR upon stimulation with various agonist ligands: the CHO line expressing the GPCR-GFP and the U2OS line expressing the GPCR and GFP-beta-arrestin.  Different receptor recycling kinetics was observed with different compounds.  The in vivo biological relevance of these data will be discussed.

HCA in Stem Cells

10:30-10:55         Multiresolution Texture Analysis of Human Embryonic Stem Cells for Monitoring Pluripotency
Paul Sammak, Ph.D., Associate Professor, ObGyn Reproductive Sciences, University of Pittsburgh
Pluripotency of human embryonic stem cells can be monitored non-invasively at the level of colonies, at low resolution using algorithms for texture analysis of phase contrast images. We evaluate a variety of growth media and passaging conditions and identify characteristics of pluripotent colonies that will be useful for real-time monitoring of cell culture without requiring destruction of cells. In addition, pluripotency can be evaluated at the subcellular level by the degree and pattern of chromatin condensation, which show measurable changes during differentiation. High-resolution confocal imaging shows that quantitative measures of chromatin condensation are associated with DNA and histone methylation. The analysis of nuclear texture also provides a genome-wide marker for the state of pluripotency.

10:55-11:20  Multi-Color Live Cell Immunofluorescence Timecourse Analysis of Human-Induced Pluripotent Stem Cell Formation
Thorsten M. Schlaeger, Ph.D., Head, hESC Core Facility, Harvard Stem Cell Institute, Children’s Hospital Boston Stem Cell Program
Human embryonic stem cells (hESCs) promise to revolutionize biology, drug discovery, and regenerative medicine. However, ethical concerns related to the source of these cells and the inability to efficiently produce patient- or disease-specific hESC lines have impeded progress. It was recently shown that forced expression of transcription factors can ‘reprogram’ patient-derived somatic cells into so-called human induced pluripotent stem cells (hiPSCs) that are virtually indistinguishable from hESCs. However, proper reprogramming is a slow and rare event, making it difficult to study. We used the BD Pathway platform to study the reprogramming process by 4-color live cell imaging of fibroblasts as they undergo reprogramming. A large area was monitored every 2-3 days for >2 weeks, and over 24k images were studied per experiment. This analysis revealed important novel insights into key molecular events of reprogramming that could not have been revealed by other methods such as FACS or gene expression analyses.

11:20-11:45         Implementation of HCA for Chemical Screening: Applications to Anti-Viral and Stem Cell Modulators Discovery
Hakim Djaballah, Ph.D., Director, HTS Core Facility, Memorial Sloan Kettering Cancer Center
I will be presenting examples of successful implementations of high-content assays for chemical screening for anti-viral agents blocking entry, replication or spread and reporting on the first proof of concept HCA for human embryonic stem cells yielding to the discovery of their first small molecule modulators. These hits have categorized them into two main groups, either they induce differentiation and in some cases followed by cell death or simply help maintain these cells in a self- renewal state. I will discuss some of the challenges we faced as we progressed through assay development and validation.

HCA for RNAi Screening and Pathway Analysis
(continued)

12:45-1:10  Combining HT-RNAi with Multi--Parametric Microscopy-Based Readouts for Discovery of Novel Oncology Targets
Christophe Echeverri, Ph.D., Chief Executive Officer and Chief Science Officer, Cenix BioScience GmbH
Abstract unavailable at time of printing.

1:10-1:35     Identification and Characterization of Host Cell Factors Involved in Infection Processes
André Paul Mäurer, Ph.D., Molecular Biology, Max Planck Institute for Infection Biology
Pathogens such as Helicobacter pylori (Hp), Chlamydia trachomatis (Ctr) and Influenza represent a major threat for public health as they can cause a broad range of severe diseases, including gastric inflammation, infection-associated cancers or respiratory tract infections, respectively. The department of Molecular Biology at the MPI for Infection Biology (MPIIB) focuses on molecular interactions of these pathogens with the host cell and underlying host cell responses essential for maintaining the infection. For this purpose an RNA interference screening platform was established. Using cell-based assays, such asa stable p65-GFP fusion protein for Hp or an infection and infectivity count assay for Ctr and Influenza the role of host cell factors important in these processes were investigated. This includes robotics, sophisticated high-content automated microscopy, image analysis as well as data handling, data mining and analysis leading to the identification of primary targets and providing data for answering questions on a more global range such as pathway or network analysis. Screens are finished with a pilot kinase siRNA library for these pathogens as well as on a genome wide basis. Here we present the main strategies for performing siRNA-based screens for infection processes as well as core results from these screens.

HCA in Model Organisms

12:45-1:10            Automated Phenotyping of Zebrafish Embryos by High-Content Analysis
Andreas Vogt, Ph.D., Research Assistant Professor, Pharmacology, University of Pittsburgh
Currently there is no vertebrate animal model that is compatible with contemporary paradigms of drug discovery encompassing rapid screening of large compound collections. The zebrafish has the potential to become a viable model for high-throughput chemical screens, but tools required for automatic image analysis are lacking. We have developed an automated method to analyze transgene expression in specific subdomains of zebrafish larvae arrayed in multi-well plates. The application is independent of embryo orientation and imaging platform, applicable to a variety of phenotypes and deals effectively with morphological, toxicity, and imaging artifacts.

1:10-1:25              Sponsored Presentation
Fish Are Friends Not Food: High-Content Screening
of Zebra Fish

1:25-1:50              Whole Organism-Based Assays for the Identification of Chemical and Genetic Modifiers of Human Misfolded Protein Disorders Using C. elegans
Stephen C. Pak, Ph.D., Research Assistant Professor, Pediatrics, University of Pittsburgh School of Medicine
C. elegans has proven to be a useful model system for investigating numerous human diseases and physiological processes. Recently, there has been growing interest in using C. elegans as a tool for drug discovery. Their small body size and ability to grow in liquid culture makes them ideal for whole organism-based, high-content screening. We have developed a C. elegans model of misfolded protein accumulation similar to that seen in human alpha-1-antitrypsin deficiency. In addition, we have optimized an automated assay that allows rapid detection and quantification of the disease phenotypes. The described model and assay should have powerful implications for alpha-1-antitrypsin deficiency and also for other human diseases caused by misfolded protein accumulation.

LIVE CELL IMAGING

3-D Models for HCA

2:15-2:40              Ultra High-Throughput Production of Consistent Three-Dimensional Cellular Aggregates
Mark Ungrin, Ph.D., Post-Doctoral Fellow, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Canada
From embryonic stem cell differentiation to tumor spheroid susceptibility to chemotherapeutic agents, cells in 3-dimensional aggregates exhibit differences in behavior from the same cells cultured as monolayers. We present a novel microwell-based approach for production of uniform cellular aggregates, suitable for both scale-out (parallel assessment of multiple conditions) and scale-up (increased quantity under a given condition) applications. We have produced > 900,000 aggregates in a single experiment without automated liquid handling or excessive manual processing, and further scaling is limited only by the availability of input cells. Several hundred aggregates can be produced within a single well in standard 96-well microtiter plate format, and aggregates can be cultured with refeeding in situ once formed.

2:40-3:05              High-Content Screening of Multicellular Systems
Bahram Parvin, Ph.D., Adjunct Professor, Electrical Engineering, Lawrence Berkeley National Laboratory
Several novel technologies are introduced for high-content screening of tissue sections and cultured multicellular systems that share a common informatics framework for associating quantified cell-based assays with the molecular data. While tissue sections are stained through immunohistochemisty and imaged in bright field, cultured multicellular systems are imaged in full 3D through fluorescence microscopy. A novel method for scoring samples imaged in bright field is introduced, followed by new advances in quantifying of multicellular systems imaged through fluorescence microscopy. A unified representation captures quantified information from both assays, which can be subsequently explored through a web-based imaging bioinformatics with heatmaps, plots, and scatter diagrams. Next, HCS assays are associated with the molecular data for indentifying those genes that are predictive of a specific phenotype. As an example, genes associated with a metastatic phenotype are identified through correlative analysis. In summary, the framework provides HCS for cell culture models, cultured mulicellcular systems, and tissue sections, which are associated with molecular data through an imaging bioinformatics system.

3:05-3:30              Designer Self-Assembling Peptide Nanofiber Scaffold for 3D Cell Cultures: Implications for High-Content Drug Discovery
Shuguang Zhang, Ph.D, Center for Biomedical Engineering, Massachusetts Institute of Technology
Biomedical researchers have become increasingly aware of the limitations of conventional 2-D tissue cell culture systems including the coated Petri dishes, multi-well plates and slides to fully address many critical issues in cell biology, cancer biology, neurobiology and high-content drug screen, such as the 3-D microenvironment, 3-D gradient diffusion, the 3-D cell migration and 3-D cell-cell contact interactions.  In order to fully understand how cells behave in the 3-D system, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known for effective high-content analysis.