2013 Archived Content

High Content Analysis - Day 2


Advanced CourseUser Group MeetingsMain Program Wednesday | Main Program Thursday
Live-Cell Imaging Workshop | Download PDF 

 

Thursday, January 10, 2013

Perkin Elmer7:30-8:15 am Breakfast Presentation

Spots to Spheres: Parasites to 3-D Cancer Models for Drug Discovery

Todd Shelper, Research Assistant, Discovery Biology (Avery Lab), Eskitis Institute for Cell & Molecular Therapies, Griffith University

The focus of our laboratory is drug discovery and how we can improve the process, or obtain more information from the data we generate. This presentation will provide exemplars of the image-based assays utilized to identify new small molecule inhibitors and modulators of parasitic diseases and cancer, as well as those used to investigate the mechanisms involved.

8:15-8:25 Award Presentations 

ThermoScientificCellome Award 

Poster Competition 

GE Healthcare LogoGE Image Competition 

 


3-D Cell-Based Assays 

8:25-8:30 Chairperson's Opening Remarks

Anthony M. Davies, Ph.D., Director, Irish National Center for High-Content Screening and Analysis (INCHA)

8:30-8:55 Overcoming the Challenges of Implementing 3-D Cell-Based Assays in Automated High-Content Screening Workflows

Anthony M. Davies, Ph.D., Director, Irish National Center for High-Content Screening and Analysis (INCHA)

To meet the needs of our in-house drug discovery and target identification programs, we have developed a novel 3-D cell culture system that has been specifically designed for use with HCS/A imaging and automated liquid handling. Unlike the 3-D assay systems currently used, our technology does not rely upon solid gel matrices, scaffolds, micro-patterned surfaces or hanging drop assay systems to achieve reproducible cancer spheroid growth, hence offering advantages in flexibility and ease of use in an HCS/HTS environment.  In this presentation we examine (i) Some of the most commonly used 3-D assay systems and their advantages and disadvantages when used in conjunction with HCA; (ii) In the same context we will also present our own 3-D cell suspension technology and the latest data derived from its use.    

8:55-9:20 3-D Hepatocyte Culture to Profile Compounds

Marc Bickle, Ph.D., Head, HT Technology Development Studio, Max Planck Institute of Molecular Cell Biology and Genetics

3-D cell culture increases the physiological relevance of cellular systems. In this context we have established a 3-D hepatocyte cell culture system, where polarized hepatocytes form bile canaliculi. In this system we have established assays to probe secretion, endocytosis, mitochondria, peroxisomes, autophagy and lipid droplets in order to profile compound action on these various cellular pathways. These assays will help predict the toxicity of compounds and establish their mode of action.

9:20-9:50 High-Content Screening of Multicellular Systems

Bahram Parvin, Ph.D., Director, Imaging and Bioinformatics Laboratory, Lawrence Berkely National Laboratory

Thermo Scientific large logo9:50-10:45 Coffee Break in the Exhibit Hall with Poster Viewing



Studying the Tumor Microenvironment:
HCA of 3-D Tumor Spheroids
 

10:45-11:10 3-D High-Content Screening for the Identification of Compounds that Target Chemo-Resistant Tumor Cells

Carsten Wenzel, High-Content Analysis, Bayer Healthcare

Limited predictability of cellular assays based on 2-D cell culture for the efficacy of drug candidates in vivo has fostered the search for novel culture techniques for drug discovery in oncology. Based on significant differences in gene expression between cells cultured in 2-D and 3-D, as well as more physiological growth conditions that resemble the situation in tumor tissue, spheroid-based 3-D cell culture is being recognized as a potential test platform to better predict drug efficacies in vivo. Here, we present our current work on evaluating and implementing different methods for the automated generation of 3-D spheroids in microtiterplates, visualization of selected markers, automated microscopy and image analysis. By this approach we identified several compounds that specifically target cells in quiescent tumor spheroid core regions. Additionally we could show that these hits, combined with cytostatics, show a significant increase in cell death in our spheroid model compared to single treatment conditions.

11:10-11:35 Elucidation of Cell Signaling Using High-Content Imaging of Patient-Derived Breast Tumor Spheroids with Cancer Stem Cell Characteristics

Fredika M. Robertson, Ph.D., Professor, Experimental Therapeutics, Center for Targeted Therapy, Translational Therapeutics Laboratory, University of Texas MD Anderson Cancer Center

Using tumor spheroids, we have identified specific gene signatures and genetic abnormalities that are involved in cancer metastasis which we find to be associated with changes in cell signaling pathways that can be targeted using small molecule inhibitors. This presentation will discuss the use of the combination of high-content live-cell imaging, confocal microscopy, flow cytometry, and histological approaches that allow imaging of these surrogates of metastasis and the evaluation of a patient's tumor cells for drug responses in real time.

11:35-12:00 pm 4-D MAME Models for Live-Cell Imaging of Interactions between Breast Tumor Cells and Their Microenvironment:  Adapting for Compound Screening

Bonnie Sloane, Ph.D., Distinguished Professor and Chair, Pharmacology and Karmanos Cancer Institute, Wayne State University

To define druggable pathways involved in breast cancer progression, our laboratory has pioneered the development of techniques for functional imaging of protease activity associated with live human breast cells and of 3- and 4-D co-culture models that recapitulate breast tumor architecture as well as the cellular and non-cellular tumor microenvironment, i.e., MAME (mammary architecture and microenvironment engineering) models. Use of these techniques and models in concert with various types of imaging probes has allowed us to image, quantify and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways.

12:00-12:25 The Multicellular Tumor Spheroid: A Systems Biology Approach for High-Content Imaging and Cancer Drug Discovery

Daniel V. LaBarbera, Ph.D., Assistant Professor, Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado

In vitro 3-D models, particularly multicellular tumor spheroids (MCTS), offer a systems biology approach to bridge information from in silico, molecular target, and 2- D cell-based screening, with in vivo models to increase the predictability of cancer drug discovery. Here we will discuss our current research targeting the metastatic phenotype in cancer using MCTS models suitable for
high-content imaging.

12:25-1:55 Enjoy Lunch on Your Own



HCA of Stem Cells 

1:55-2:00 Chairperson's Opening Remarks

Hakim Djaballah, Ph.D., Director, HTS Core Facility, Molecular Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center

2:00-2:25 Development of Screening Platforms for Modifiers of Cell Fate in Human Pluripotent Stem Cells

April Pyle, Ph.D., Assistant Professor, Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles

Differentiated cells from human pluripotent stem cells (hPSCs) provide an unlimited source of cells for use in regenerative medicine. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hPSCs. One caveat is that hPSCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hPSC, and hampers production of large-scale culture of hPSCs. Improving our understanding of pathways that are important for hPSC growth could improve our ability to culture, differentiate and derive new hPSCs. Our large scale screening work provides a comprehensive evaluation of chemical space for modifiers of cell fate in hPSCs.

2:25-2:50 Patient-Specific Cell-Based Disease Models for Drug Discovery

Anne Bang, Ph.D., Director, Cell-Based Disease Modeling and Screening, Sanford-Burnham Medical Research Institute

Patient-specific primary cells and induced pluripotent stem cells (iPSCs) complement traditional cell-based drug discovery assays, allowing testing on differentiated features not reflected by immortalized lines. We used patient cells to develop a phenotypic assay for muscular dystrophy that distinguishes between affected and unaffected siblings, faithfully recapitulating key molecular features of the disease. A high-content screen of patient cells was conducted with the goals of identifying early treatment candidates, and probes to gain a greater understanding of underlying cellular defects. We will discuss screening results and development of iPSC-based models for testing of drugs on disease relevant cell types.

2:50-3:15 HCS to Discover Drugs for Heart Failure

Mark Mercola, Ph.D., Professor and Director, Muscle Development and Regeneration Program, Sanford-Burnham Medical Research Institute

There is an urgent need for therapies that reverse the course of ventricular dysfunction in heart failure, which remains a leading cause of morbidity and mortality in Western countries. Current therapies do not treat the underlying cause—death or damage of heart muscle cells combined with increased scarring. Our research is focused on developing high-content screening assays and instrumentation to discover targets and screen for molecules active in cardiac regeneration and cardiomyocyte contractility. The presentation will discuss modeling regeneration using stem cells in high-throughput analysis of cardiomyocyte physiology, and demonstration of small molecules that promote differentiation and RNA molecules that sustain contractility in the failing heart.

Thermo Scientific large logo3:15-4:15 Refreshment Break in the Exhibit Hall with Poster Viewing



RNAi Screens 

Chairperson's Remarks

Hakim Djaballah, Ph.D., Director, HTS Core Facility, Molecular Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center

4:15-4:40 SeedSeq—Off-Target Transcriptome Database

Karol Kozak, Ph.D., Head, Data Handling Unit and High-Content Screening, ETH Zurich

Owing to a tolerance for mismatches and gaps in base-pairing with target transcripts, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs (off-targets) besides their intended target transcripts (on-targets). The Reference Sequence (RefSeq) collection aims to provide a comprehensive, integrated, non-redundant, well-annotated set of sequences, including mRNA transcripts. We performed a detailed analysis of off-targeted transcripts based on latest RefSeq version. We developed SeedSeq, an extended version of the RefSeq database including information about off-target transcripts.

4:40-5:05 A Genome-Wide RNAi Screen Using C. elegans Identifies a Drug that Protects against Toxicity Associated with Protein Aggregation

Stephen C. Pak, Ph.D., Assistant Professor, Pediatrics, University of Pittsburgh School of Medicine

The accumulation of misfolded protein aggregates within cells is a common cause of tissue injury and degenerative disease (e.g., Alzheimer's, Huntington's, ALS and alpha-1-antitrypsin (AT) deficiency). To better understand the genetic factors that influence pathogenesis of protein aggregation disorders, we modeled AT-deficiency in a microscopic nematode, C. elegans. Transgenic animals expressing the mutant AT protein accumulated large intracellular aggregates, similar to those detected in human hepatocytes. Using the ArrayScanVTi imaging platform, we developed a high-content assay for the quantification of misfolded protein accumulation. Using whole, live animals, we performed a genome-wide RNAi screen to identify genetic modifiers of AT-deficiency. Moreover, using computational methods, we found compounds with known inhibitory activity against the genetic modifiers. When tested in human cell models of AT-deficiency, one compound significantly reduced accumulation of mutant AT protein aggregates.

5:05-5:30 High-Content Assays to Assess Cellular Stress Responses

Hakim Djaballah, Ph.D., Director, HTS Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center

High-content assays provide the ultimate measure of cellular stress responses. We explored this feasibility by investigating responses upon exposure to chemicals, or during DNA/RNA transfection using an in-house developed panel of indicators/reporters. This has allowed for the first time the ability to classify both performance and cytotoxicity of these various agents. I will describe our established platform and discuss our results, especially regarding the use of transient transfection in chemical and RNAi screening.

Phenotypic Drug Discovery 

8:25-8:30 Chairperson's Opening Remarks

Jonathan A. Lee, Ph.D., Senior Research Advisor, Quantitative Biology, Eli Lilly & Co.

8:30-8:55 The Value of Phenotypic Assays to Drug Discovery

David C. Swinney, Ph.D., CEO, Institute for Rare and Neglected Diseases Drug Discovery (iRND3)

Drug discovery strategies include target-based molecular approaches and phenotypic-based empirical approaches. Our recent analysis revealed the phenotypic approach as the more successful strategy for first-in-class medicines. We rationalized that this success was influenced by the unbiased identification of a molecular mechanism of action (MMOA) that contributed to a useful therapeutic index. The value and success of phenotypic approaches will be further increased through efforts to bridge the gap between molecular approaches and phenotypic approaches such that once an effective MMOA has been confirmed with phenotypic assays, molecular approaches can be efficiently utilized to optimize leads to medicines. 

8:55-9:20 Modern Phenotypic Drug Discovery Is a Viable Pharma Strategy

Jonathan A. Lee, Ph.D., Senior Research Advisor, Quantitative Biology, Eli Lilly & Co.

The majority of first-in-class NMEs originate from phenotypic screening (PS); however, this approach is not commonly used by the pharmaceutical industry. We addressed perceived issues with PS by conducting an
anti-angiogenesis MTS. Identification of novel chemical scaffolds that are differentiated, structurally and mechanistically, from anti-angiogenic standards of care and which modulate molecular targets not previously associated with angiogenesis demonstrates that PS interrogates relevant biology regardless of target validation status. Modern PS combines the biological complexity of physiology-based systems with the high-throughput compound testing capacity and operational robustness of biochemical methodologies.

9:20-9:45 High-Content Phenotypic Screening for Target Identification at Pfizer: High-Throughput Flow Cytometry and Live-Cell Imaging of Human Primary Cells

Regis Doyonnas, Ph.D., Senior Principal Scientist, Stem Cell Engineering and High-Content Screening Lab, Hit Discovery and Lead Profiling, PDM-NCE, Worldwide Research & Development, Pfizer

Thermo Scientific large logo9:45-10:45 Coffee Break in the Exhibit Hall with Poster Viewing



Phenotypic Drug Discovery (continued) 

10:45-11:10 A Chemical Approach to Controlling Cell Fate

Sheng Ding, Ph.D., William K. Bowes, Jr. Distinguished Investigator and Professor, Pharmaceutical Chemistry, Gladstone Institute of Cardiovascular Disease, University of California San Francisco

Recent advances in stem cell biology may make possible new approaches for the treatment of a number of diseases. A better understanding of molecular mechanisms that control stem cell fate as well as an improved ability to manipulate them are required. Toward these goals, we have developed and implemented high-throughput cell-based phenotypic screenings of arrayed chemical libraries to identify and further characterize small molecules that can control stem cell fate in various systems. This talk will provide the latest examples of discovery efforts in my lab that have advanced our ability and understanding toward controlling stem cell fate, including self-renewal, survival, differentiation and reprogramming of pluripotent stem cells.

11:10-11:35 Screening of Protein Kinase Inhibitors to Identify Target and Liability Kinases in Axon Growth

John L. Bixby, Ph.D., Professor, Pharmacology and Neurological Surgery; Vice Provost for Research, University of Miami

We screened 995 protein kinase inhibitors (PKIs) in primary neurons to identify critical kinase targets and "hit" PKIs that promote growth of neuronal processes. We analyzed the activity profiles of hit and non-hit PKIs against a kinase panel, narrowing the set of informative kinases. A machine learning model was trained using these kinases and will be used to perform in silico searches for novel hits using experimentally acquired or predicted activity profiles. Our novel approach allows us to search for hits based on biochemical profiles rather than SAR, and addresses the problem of PKI polypharmacology.

11:35-12:00 pm Zebrafish as a Tool for Rapid, in vivo Identification of Complex Behavioral Chemotypes

Andrew J. Rennekamp, Ph.D., Postdoctoral Researcher, Cardiovascular Research Center and Division of Cardiology, Massachusetts General Hospital, Harvard Medical School

Zebrafish larvae are well suited for in vivo, high-content screens and yet they are neurocompetent organisms exhibiting complex behavioral phenotypes with neurophysiology that is remarkably similar to humans. Using zebrafish, we have identified several reproducible behaviors amenable to high-content screening. By probing these behavioral phenotypes with compounds having known pharmacological targets in humans, we are able to identify neurological pathways relevant to human diseases. Subsequently, we are able use the zebrafish as a tool for the identification of novel drugs which perturb those important pathways via previously unknown mechanisms.

12:00-12:25 Image-Based High-Dimensional Profiling of Cell Response to Perturbation

Auguste Genovesio, Ph.D., Group Leader, Image Analysis, Broad Institute of MIT and Harvard

From each image in a high-content experiment, hundreds of cells can be detected and analyzed leading to millions of high-dimensional cell profiles for an experiment. These profiles may be a rich source of information about the corresponding RNAi or small molecule treatments, revealing complex and subtle phenotypes and identifying families of treatment conditions. How to maximize the insights gained from these huge datasets is currently a challenging question for the community. In this talk, we discuss the data analysis of high dimensional datasets of this kind and show applications in drug discovery and basic research.

12:25-1:55 Enjoy Lunch on Your Own



Digital Pathology and Tissue Diagnostics 

1:55-2:00 Chairperson's Opening Remarks

Joe Trask, Ph.D., Head, Cellular Imaging Core, The Hamner Institutes for Health Sciences

2:00-2:25 Digital Pathology: Development, Challenges and Strategies Using HCA to Measure in vivo Responses

Joe Trask, Ph.D., Head, Cellular Imaging Core, The Hamner Institutes for Health Sciences

In this talk I will discuss the similarities between digital pathology and high-content analysis and how these disciplines when taken together are used to extract in-depth multiparameteric data to better understand the biological outcome from in vivo response from tissue sections. In a test case scenario of a small animal study, rats were treated with a synthetic steroidal antiandrogen drug cyproterone acetate (CPA) or 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD); whole liver sections were isolated, processed, and labeled with multiple probes to identify cellular damage including apoptosis using activated caspase-3, TUNEL, and nuclei with either fluorescent or chromagen indicators. I will discuss the limitation of imaging technology to capture tissue sections, the challenges and the methods employed to address autofluorescence in liver tissue and development of a multiplexed bioprobe assay using automated image analysis techniques.

2:25-2:50 High-Content Analysis of Peripheral Patient Tissues for Analyzing Biological Processes and Drug Response in Neurodegeneration

Justin D. Boyd, Ph.D, Scientist, Laboratory of Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Harvard Medical School, Brigham and Women's Hospital

Neurodegeneration is ranked among the top causes of death in the United States. The majority of neurodegenerative diseases have no effective therapy. A challenge to discover disease-modifying drugs for neurodegeneration is that the diseases manifest in the brain. But what if we could use blood to monitor a disease process? Processes that contribute to the degeneration in the brain have been reported in blood. We are developing a platform to monitor a disease process in blood cells from neurodegenerative disease patients to 1) characterize different forms of the disease as they relate to these processes, 2) to identify drugs that elicit a modification of a disease-causing dysfunction and 3) to identify patients most likely to respond to specific treatments.

2:50-3:15 High-Content Analysis and Tissue Diagnosis with Label-Free Multi-Spectral Molecular Vibrational Imaging

Stephen T.C. Wong, Ph.D., Systems Medicine and Bioengineering, The Methodist Hospital Research Institute, Weill Medical College, Cornell University

We introduce an emerging technology of label-free microscopy that provides an alternate route of optical molecular imaging without the use of contrast agent or fluorescent label. The label-free imaging approach, coherent anti-Stokes Raman scattering (CARS) microscopy, has gained much attention recently as it provides high spatial resolution images with good intrinsic contrast and high chemical specificity. CARS chemical spectra capture molecular signatures of tissues based on their intrinsic chemical compositions and can be used to form distinct spectra or spectrum-based images. We will illustrate application examples of automated CARS microscopy in drug screening. In addition, coupling with fiber optics technology, multi-spectral CARS imaging can provide real-time microscopic visualization of morphological patterns and molecular signatures of tissues at the cellular level could, ideally, replace tissue extraction biopsy and open up many new vistas for instant diagnosis and intra-operative intervention of cancer. Integrated with intra-operative or image-guided navigation, the label free microscopy technique would also allow early detection of cancer, identify cancer surgical margins, avoid nerve damage during intervention, and improve cancer surgical outcomes.

Thermo Scientific large logo3:20-4:15 Refreshment Break in the Exhibit Hall with Poster Viewing



Digital Pathology and Tissue Diagnostics (continued) 

4:15-4:40 Real-Time Fluorescence Imaging of Reactive Oxygen Species in vivo

Adam J. Shuhendler, Ph.D., Postdoctoral Fellow, Department of Radiology, Stanford School of Medicine

Reactive oxygen and nitrogen species (RONS) are important in acute (sterile or ischemic/reperfusion injury) and chronic inflammation (bacterial infection, tumor growth, cardiovascular disease or arthritis). Local RONS generation precedes inflammatory cell arrival, providing molecular imaging probes capable of detecting these pro-inflammation chemicals, the advantage of early inflammation detection. This presentation will describe a dual-color fluorescent biocompatible nanoprobe for real-time, whole-body in situ detection of RONS. This nanoprobe enables for the first in vivo visualization of the complete progression of inflammation from sub-clinical time points prior to histological changes, through to restitution. It also allows whole animal and intravital investigations of tumor therapy, providing the first real-time evidence for the RONS-mediated anti-vascular effects of dichloroacetate on tumors in vivo. Our nanoprobe demonstrably serves as a platform for systemic, in situ and real-time imaging of microenvironmental changes associated with pathological processes.

 

 



Advanced CourseUser Group MeetingsMain Program Wednesday | Main Program Thursday
Live-Cell Imaging Workshop | Download PDF