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Thursday, January 17
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7:30-7:55
Breakfast Presentation :
High-Content Screening:
Multiplexing Cell Cycle with Apoptosis
Lisa B. Smith, Scientist II, Discovery Technologies,
Schering-Plough Research Institute
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Sponsored
by
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HCA in Primary and Live
Cells |
Cell Cycle Assays |
8:00-8:25
Development of Cell-Based Assays for Study of Cardiac Disease and
Drug Efficacy/Toxicity Using Primary Cardiac Myocytes and
Cardiac-Derived Cell Lines in a High-Content Format
Anthony Davies, Ph.D., High-Content Research Facility Director,
Clinical Medicine, Trinity College Dublin
We are currently engaged in the development of a range of
cell-based assays that utilize both primary adult cardiac myocytes
and immortalized cell lines. To facilitate the development of
these new assay tools, a detailed examination of both biochemical
and structural changes in primary adult cardiac muscle cells has
been conducted. These studies have yielded valuable information
regarding the behavior of primary cardiac muscle cells in their
quiescent and active states. Currently our work is focused on the
use of muscle cells derived from a cardiac myoblast cell line as a
basis for primary and secondary cardiac screens. Our ultimate goal
is to develop a stable and biologically relevant assay that can be
deployed and utilized in an automatable HCS format.
8:25-8:50 Primary
HCS in the San Diego Center for Chemical Genomics: Development of
Calcium HCS in Live Cardiomyocytes
Jeffrey
Price, M.D., Ph.D., Associate Professor, Burnham Institute for
Medical Research
Primary high-content (image-based) screens in various stages
of implementation at Burnham’s San Diego Center for Chemical
Genomics (SDCCG), a member of the NIH Molecular Libraries
Screening Center Network (MLSCN, http://mli.nih.gov/mlscn/index.php)
include screening for: 1) agonists and antagonists of VCAM-1 in
HUVECs (VCAM-1 is essential for lymphocyte adherence), submitted
by Thomas Mayer at Columbia U.; 2) inhibitors of phagocytosis in
mouse J774 macrophages, submitted by Fabienne Paumet at Columbia
U.; 3) agonists of cardiomyocyte cell cycle entry performed on
neonatal rat cells, submitted by Mark Mercola at Burnham; and 4)
compounds that alter b-catenin
distribution in HeLa cells, submitted by Pat McDonough at Vala
Sciences. Progress on implementing these screens will be
presented. As part of our screening center efforts at Burnham we
are developing an HCS system for utilizing calcium measurements
via Fluo3 and Fluo4 for detecting differentiation of cardiac
myocytes. The development of this system and results from it will
also be presented.
8:50-9:15 High-Resolution
Imaging of Living Cells in Flow Suspension Using Axial-Tomography:
3D Imaging Flow Cytometry
Spencer L. Shorte, Ph.D., Director Imagopole, Group Leader
Plateforme d’Imagerie Dynamique (PFID), Institut Pasteur
Conventional flow cytometry methods report integrated averaged
cellular fluorescence signal intensities per cell analyzed, albeit
with throughput rates of thousands of cells per second. Further,
this is combined with the powerful utility to subsequently sort
and/or retain the cells of interest. However, these methods mainly
lack the fundamental ability to reveal spatial information on the
sub-cellular compartmentalization of signals. This limitation has
prompted efforts by some manufacturers to produce state-of-the-art
commercial flow cytometry systems allowing fluorescence images to
be recorded by an imaging detector (high-content imaging cytometry).
Nonetheless, there remains an immediate and growing need for
technologies facilitating three-dimensional (3D) fluorescence
analysis of cells in flow and/or suspension
("ultra"-high-content imaging cytometry). Here, we
report a novel methodological approach to this problem that
combines micro-fluidic-flow, and microelectrode-dielectric-field
control to manipulate, image and finally sort individual cells in
suspension. The method providing for 3D optical imaging of living
intact cells in suspension also offers important advantages for
imaging studies on cells in suspension. In particular, we report
the system’s utility for confocal axial tomography using
microrotation imaging that we demonstrate greatly enhances 3D
optical resolution sampled from single intact living cells in
suspension. |
8:00-8:25
Incorporating Protein-Protein Interaction Biosensors into Cellular
Models of Disease
Kate
A. Johnston, Ph.D., VP, Discovery Programs, Cellumen Inc.
Certain targets are not compatible with cell-based screening
either because their expression is toxic, or because methods to
measure their activity are not available. Protein-protein
interaction biosensors (PPIBs) and inducible protein systems
enable drug discovery for many such previously intractable
targets. PPIBs report on reversible protein-protein interactions
in living cells through dynamic changes in their intracellular
localization. The modular design of PPIBs enables integration in
to cellular models of disease in which expression of key targets
is regulated with inducible promoter systems. For example, a
series of biosensors to detect the interaction of several
functional domains of p53 and HDM2 were generated, and a p53-HDM2
PPIB was incorporated in to a profiling assay for disruptors of
the interaction, along with biomarkers for the effects of
regulating the expression of p53 and HDM2. Thus, incorporating
PPIBs into cellular models of disease enables not only
measurements of protein-protein interactions, but builds
additional knowledge on the relationships between these
interactions and cellular (dys)functions.
8:25-8:50 Cell Cycle Plus:
Extracting Mechanistic and Selectivity Profiles from Cell Cycle
Kinase Inhibitor Dose-Response Curves
John Moffat, Ph.D., Scientist, Biochemical Pharmacology &
Small Molecule Drug Discovery, Genentech, Inc.
Cell cycle kinases have been extensively studied cancer drug
targets, and cell cycle phenotypes are a classic HCA readout.
However, most ATP-competitive kinase inhibitor screening hits and
leads are challenged by poor selectivity. Off-target kinase
inhibition can affect the utility and veracity of the assay
readout as well as impacting decision-making in compound
progression. We have used multiparametric analysis of cell cycle
HCA data to profile a variety of cell cycle kinase inhibitors for
mechanistic fidelity, relative selectivity, and characterization
of off-target activities arising at different treatment
concentrations. Challenges and approaches to data management,
reduction, and reporting will also be discussed.
8:50-9:15 High-Content Imaging
Characterization of the Cell Cycle
Jonathan Low, Ph.D., Department of Cancer Growth and Translational
Genetics,
Eli Lilly and Co.
Understanding the regulation and progression of the cell cycle is
critical to the comprehension and treatment of cancer. Our current
understanding of the cell cycle is based upon observations that,
due to technological limitations, examined mixed populations of
cells and were unable to analyze cellular subpopulations or
determine how variability between cells affected the total
population. High-content imaging (HCI) quantifies treatment
effects on each cell and on the population of cells as a whole
using a variety of parameters. Although one parameter alone cannot
define every phase of the cell cycle, the quantification of
additional parameters further delineates cell cycle phases. The
key to understanding and defining cellular subpopulations is
multiparametric analysis using several relevant attributes that
refine the definition of each population. As more attributes are
added to the analysis additional subpopulations are often detected
and previous subpopulations become more clearly defined. We have
characterized the cell cycle of transformed mammalian cells using
HCI in conjunction with multiparametric data analysis. The
combination of these tools generates a phenotype for each phase of
the cell cycle and shows that this phenotype is reproducible
across mammalian cell lines in vitro. We illustrate that
phase-specific arrest caused by common cell cycle inhibitors
results in phenotypes identical to those observed for each phase
of the cell cycle. Multiparametric HCI cell cycle analysis quickly
characterizes where in the cell cycle experimental molecules
affect the target cells, and more importantly distinguishes the
differential effects of these molecules on individual cells and
cellular subpopulations across the total studied population. The
flexibility inherent to this system also means that
multiparametric analysis can be used in a variety of screening
settings in which one or more phenotypic markers are affected by
an experimental treatment. |
| 9:15-9:50
Networking Coffee Break |
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HCA in Model Organisms
9:50-10:15 High-Content
Analysis of Zebrafish Angiogenesis by Cognition Network Technology
Andreas Vogt, Ph.D., Assistant Professor, Pharmacology, University
of Pittsburgh
Advances in high-throughput screening and laboratory automation
have substantially improved the speed of target-based drug
discovery but it is unclear whether new drugs affecting single
targets have improved therapeutic efficacy. An increasingly
popular sentiment is that better models are needed to improve the
quality of new drug candidates. Whole organisms could provide such
models but technical challenges limit their utility as drug
screening tools. The zebrafish embryo may be amenable to
high-throughput screening methodology. We developed a fully
automated analysis of angiogenesis in a transgenic zebrafish line.
Images of fluorescent zebrafish embryos in 384 well plates were
acquired on a high-content reader and analyzed by object-based
image processing using Cognition Network Technology (CNT). The CNT
algorithm detected embryos regardless of orientation, partitioned
them into regions of biological relevance, and quantified the
growth of intersegmental blood vessels (ISV). The assay delivered
graded responses and documented antiangiogenic activity of a novel
small molecule microtubule perturbing agent. The results
demonstrate that it is feasible to adapt image-based high-content
screening methodology to measure complex phenotypes in whole
organisms.
Technology Showcase: Novel Biological Models for
HCS
10:15-10:30 Live-Cell Assays
and Cell Tracking
Robert
Graves, Ph.D., Senior Scientist, GE Healthcare Life Sciences
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10:30-11:00 Technology Short
Talks
Multiplex
Analysis of Nuclear Receptor Function at the Single Cell
Level
Michael A. Mancini, Ph.D., Baylor College of Medicine,
Associate Professor, Department of Molecular & Cellular
Biology
Here,
we report the development of a high throughput (HT)
image-based assay that quantifies androgen receptor (AR)
subcellular distribution and transcriptional activity at the
single cell level. Overall assay quality was superior to
previous cell based assays, achieving EC50 coefficients of
variation from 5-24%, and Z' values ranging between 0.46 and
0.91. This is achieved by the selective analysis of cells
stably expressing physiological levels of AFP tagged AR,
important because minor over-expression resulted in elevated
nuclear speckling and decreased transcriptional activity. A
panel of AR-binding ligands, including known agonists,
antagonists, and endocrine disruptors, were used in 10-point
dose response experiments. Nuclear translocation and nuclear
"speckling" were linked with transcriptional
output, cell cycle position, and specific ligands were noted
to differentially affect measurements for wild type versus
mutant AR, suggesting differing mechanisms of action.
Integration of Pipeline Pilot image analysis tools allowed
for more accurate cell identification at higher cell
densities, increasing assay robustness in heterogenous
samples, and improved throughput due to the need for fewer
images being collected. HT-based multiplex screening
provides a rapid, systems-level analysis of compounds that
may differentially affect wildtype AR or clinically-relevant
AR mutations.
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Sponsored
by
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(Sponsorship
Available. Contact Carol Dinerstein at dinerstein@healthtech.com
or 781-972-5471) |
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END-USER FORUM*
*The End-User Forum is
complimentary to paid conference attendees. Participation is
limited to end-users only and subject to approval by conference
organizers. Companies with HCA-related products or services on the market or
in development (including sponsoring and exhibiting companies)
will not receive access to the End- User Forum. Please indicate on
the registration form if you plan to attend (pre-registration is
required for attendance).
11:00-11:05 Chairperson’s
Opening Remarks
Ann F. Hoffman, Senior Principal Scientist, Roche Discovery
Technologies
11:05-11:20 Participant
Introductions
11:20-12:20 Luncheon Focus
Group Discussions
Topics Include:
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HCA Reagents and Assay
Development
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HCA Instrumentation and
Hardware
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HCA Image Analysis and Data
Management
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Novel Biological Models for
HCA
12:20-1:00 Reports from Focus
Groups and Discussion. Preparation for End-User/Vendor Panel
Discussion. |
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1:00-2:15
End-User/Vendor Panel Discussion
An updated list of panelists and discussion topics will be
posted a few weeks prior to the meeting.
2:15-2:30 Break
HCA in Tissues
2:30-2:55 Challenges and
Successes of Compound Screening in Brain Tissue
O. Joseph Trask, Jr., Ph.D., Head of Cellular Imaging
Technologies, Center for Drug Discovery, Department of
Neurobiology, Duke University
Neurodegenerative diseases are extremely difficult targets to
investigate due to the complexity and heterogeneity of the brain.
For such reasons, simple non-neuronal cell line-based screens have
proven to be limited in identifying promising new clinical
candidates. We have thus developed an acute rat brain slice-based
assay platform for Huntington disease (HD), a genetic
neurodegenerative disease for which there is currently no
effective treatment or cure. In this platform, novel imaging
techniques are used to quantitate directly the neuroprotective
properties of small molecule drug candidates against
neurodegeneration induced by biolistically introduced mutant HD
DNA constructs. In this talk I will discuss the challenges and
successes of assay development, validation, and results of
screening compounds in this model.
2:55-3:20 Application of
High-Content Screening to Measure In Vivo Pharmacodynamic
Response in Aurora Kinase Inhibitor Program
Douglas Bowman, Ph.D., Senior Manager, Imaging Sciences, Molecular
and Cellular Oncology, Millennium Pharmaceuticals, Inc.
CS and related imaging technologies are widely used for in
vitro assays throughout a drug discovery program including
target identification, lead optimization, and pre-clinical
toxicology. These image-based assays provide us with an
understanding of the biological effect of our compound, whether it
is an upstream, direct, or downstream effect of target inhibition.
We have extended HCS technologies to preclinical and clinical
studies to characterize the mechanistic effects (pharmacodynamic
response) of the drug. I will discuss these assays and the
inherent challenges of adopting HCS techniques to tissue in our
Aurora A Kinase inhibitor program.
3:20-3:40 Refreshment Break
HCA in Stem Cells
3:40-4:05 High-Content
Screening for Molecules that Modulate Pancreatic Beta-Cell Growth
and Differentiation
Fred Levine, M.D., Ph.D., Professor, UCSD Department of
Pediatrics; Adjunct Professor, The Burnham Institute for Medical
Research
Regeneration of pancreatic beta-cell by neogenesis from
stem/progenitor cells or by enhancing beta-cell replication is an
attractive approach to treating diabetes. Unfortunately, little is
understood about the mechanisms that control regeneration. We have
been using high-throughput, high-content screening of cell line
models and human primary pancreatic cells to detect molecules that
modulate beta-cell growth and differentiation.
4:05-4:30 A Chemical Approach
to Stem Cell Biology
Sheng Ding, Ph.D., Associate Professor, Departments of Chemistry
and Cell Biology, The Scripps Research Institute
Recent advances in stem cell biology may make possible new
approaches for the treatment of a number of diseases. For better
understanding of the molecular mechanisms that control stem cell
fates as well as an improved ability to manipulate these cells, we
have developed and implemented chemical and functional genomic
tools, including high-throughput cell-based phenotypic screens of
arrayed chemical, cDNA and RNAi libraries, and performed genomic
and proteomic profiling of homogenous
undifferentiated/self-renewing or selectively differentiated cell
populations under chemically defined conditions. In-depth
biochemical and functional assays in vitro and in vivo,
have been applied to identify and further characterize small
molecules and genes that can control stem cell fate in various
systems.
4:30-4:55 Non-Invasive
Monitoring of Human Embryonic Stem Cell Growth, Death and
Differentiation by Texture Analysis of Images
Paul Sammak, Ph.D., Research Associate Professor, Magee Women’s
Research Institute, Department of Obstetrics, Gynecology and
Reproductive Sciences, University of Pittsburgh
Quantitative monitoring of the expansion and fate of stem cells is
important for routine culture and for drug screening. We have
found that multiscalar texture analysis can be used to classify
stem cell colonies into distinct groups based on overall
morphology of phase contrast images and be used to monitor cell
health and phenotype. This capacity will be especially important
for reproductive toxicology using primitive human stem cells of
various lineages. Varying densities of mouse embryonic fibroblasts
were used to support human stem cell self-replication and neuronal
differentiation. Statistically different categories of
differentiation were determined based on non-destructive imaging
and validated by molecular markers including Oct-4 (pluripotent
cells) and nestin (neurectoderm and neural stem cells). Texture
analysis can be used to measure amorphous biological samples where
standard geometric measurements of shape and size are insufficient
for quantitative, statistically significant measures of cell
morphology.
4:55-5:00 Closing Remarks
5:00 Close of Conference |
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