Symposium Chairperson
Dr. Dhaval Kumar Patel, Molecular Staging Inc.
9:00 FDA-NCI Clinical
Proteomics Program: Applications at the Bedside
Dr. Cloud P. Paweletz, Co-Director FDA-NCI Clinical Proteomics Program,
Division of Therapeutic Products, Center for
Biologics Evaluation and Research, FDA
The field of molecular medicine is moving beyond genomics to proteomics. While
DNA is the information archive, proteins do all the work of the cell - and
ultimately dictate all biological processes and the cellular fate. The
challenge and opportunity within proteomics is much more than just developing
a list of all the proteins. The true scientific goal of proteomics is to
characterize the information flow within the cell and the organism. This
information flow is mediated through and by, protein pathways and networks.
The cause of most human disease lies in the
functional disregulation of protein-protein interactions. Understanding the
role that protein networks play in disease will create enormous clinical
opportunities, since these pathways represent the drug targets of the next
decade. In the future, entire cellular networks, not just one disregulated
protein, will be the targets of therapeutics. The next technologic leap will
be the application of proteomic technologies to the bedside. It will soon be
possible to analyze the state of protein signal pathways in the
disease-altered cells, before, during, and after therapy. This can herald the
advent of true patient-tailored therapy.
Our FDA-NCI Clinical Proteomics Program is
focused on the understanding of mechanisms of carcinogenesis, identification
of new drug targets, and discovery of new biomarkers for early detection in
actual human tissue tumor specimens and clinical serum specimens. Tissue-based
proteomics requires technology that can overcome the complex cellular
heterogeneity one encounters when studying disease in tissue specimens. To
that end, we employ the use of Laser Capture Micro-dissection (LCM), invented
at the NCI, for the proteomic analysis of microdissected subpopulations of
human solid tumors (prostate, breast, ovary, and esophageal) as a model for
the study of disease progression. These studies encompass and employ
high-throughput proteomic pattern profiling using surface-enhanced laser
desorption and ionization (SELDI) protein chips to identify disease-related
proteins and protein patterns directly in human sera using
artificial-intelligence based bioinformatics, and focused proteomic approaches
through the use of multiplexed phospho-specific antibody arrays, novel
reverse-phase lysate for signal transduction pathway profiling. For the first
time ever, proteomic technologies are being used in clinical trials at the NCI
through our program to monitor patients before, during and after targeted
treatment, heralding the beginning of true patient tailored molecular
medicine.
9:20 Interaction Analyses Using Whole
Proteome Microarrays
Dr. Barry Schweitzer, Protometrix, Inc.
The recent abundance of genomic data has created a need for a systematic
proteomics approach to decipher the protein networks that dictate cellular
function. To date, the generation of large-scale protein-protein interaction
maps has principally relied on either yeast 2-hybrid or mass spectrometric
techniques. In a landmark study by Snyder and coworkers at Yale University,
the feasibility of using protein microarrays to investigate the function of a
whole proteome was recently demonstrated. This technology, called ProtoP5™,
has been licensed to Protometrix Inc. and industrialized so that over 500,000
potential protein-protein interactions can now be screened per day. As a pilot
demonstration, a complete yeast protein interaction map is being generated
that will contain over 25,000,000 data points. This map contains information
that complements and significantly extends existing protein interaction
databases. Examples will also be given of how these protein arrays have been
used for screening and expression profiling applications. Finally, work in
progress on other proteomes will be described.
9:40 Development of a Pharmaceutically
Relevant Ion Channel Assay for Supported Membrane Array Chip Technology
Dr. Gerald Wiegand, Zyomyx, Inc.
We present a new approach for a platform technology aiming the functional
screening of ion channel proteins with high-throughput (HTS). It utilizes
supported lipid bilayer membranes (SMs) on microfabricated electronic chip
devices as core technology. Combined with the functional incorporation of ion
channel proteins into the SMs and an efficient electronic read-out of the chip
response by fast impedance spectroscopy, a prototype comprising all key
elements for an ion channel HTS system was built and deployed. Experiments
applying this technology with fully integrated microfluidics (six separate
assay channels) and microelectronics (36 individual electrodes) on-chip are
shown.
SMs as assay matrix have the advantage that the entire ion channel protein
/ membrane assay can be formed from solutions in a fully self-organizational
manner. The resulting protein-membrane composites are extremely stable.
On-chip membrane resistance values of the order of Gohm are reached. Two model
assays, Gramicidin and alpha-Haemolysin, were characterized successfully. The
development of the first pharmaceutically relevant ion channel assay in
supported membranes is reported. The key challenge addressed is the functional
reconstitution of ion channel proteins in supported membranes.
Structure-function considerations as well as lipid-protein interactions and
reconstitution biochemistry play an important role for the incorporation
procedure and the assessment of the activity of ion channel protein in this
assay format.
10:00 RCA Immunoassay Microarrays
Dr. Dhaval Kumar Patel
10:20-11:00 Refreshment Break with Exhibit
and Poster Viewing
Saturday, September 7
8:30 am Coffee Break with Exhibit
and Poster Viewing
|
TRACK 2
|
NANOTECHNOLOGY:
Tumor Biology and Its Exploitation in Therapy |
|
T1 |
T3 |
Home |
Symposium Chairperson
Dr. Stephen Lee, The Ohio State University
9:00 Tumor Physics and Biology: Impact of
Aberrant Vascular Architecture on Therapy
Dr. Lance L. Munn, Harvard Medical School and Massachusetts General
Hospital
It is known that the vasculature of tumors is unusual in many respects—flow
is sluggish and intermittent, permeability is focally high and the network
architecture is not well-organized. Recent studies have shown that the
structure and integrity of the vascular endothelium are also abnormal, a fact
that potentially influences drug delivery and metastatic cell dissemination.
Defects in the endothelial wall include regions of shed endothelium, extremely
thin endothelial leaflets, gaps between endothelial cells and deficient
basement membrane. Interestingly, the appearance of these defects appears to
be influenced by the growth characteristics of the tumor. In other studies, we
have also found that tumor cell intravasation into the vascular compartment is
a relatively inefficient process, with few viable cells shed into the
bloodstream. Even so, some cells are able to successfully leave the primary
tumor and colonize distant sites. In the process, these cells vary the
expression of various genes, including matrix-degrading enzymes, that may
influence the structure of the vessel wall, resulting in the observed defects.
Therapies aimed at "normalizing" the vasculature may have multiple
benefits, restoring the barrier to metastasis and establishing drug delivery
to previously inaccessible regions of the tumor.
9:20 Affinity-Based Methods for Tumor Targeting
Dr. Erkki Ruoslahti, Burnham Institute
We use libraries of phage-displayed peptides to identify specific changes
in tumor vasculature (Ruoslahti, 2002). By combining ex vivo screening of the
libraries on cell suspensions prepared from tumors and in vivo screening for
tumor homing, we have identified new peptides that home to MDA-MB-435 breast
cancer or HL-60 leukemia xenografts grown in nude mice. One of our new
peptides (F3) recognizes blood vessels in various tumors and another (LyP-1)
recognizes lymphatic vessels in the MDA-MB-435 tumors (Porkka et al., 2002;
Laakkonen et al., submitted). Both peptides also bind to the tumor cells and
show little or no binding to normal tissues. The presence and importance of
blood vessels in tumors is well established, but it has only recently been
found that lymphatic vessels can also be present within tumors. These vessels
were previously thought to occur only in the normal tissue surrounding tumors.
The phage that displays the LyP-1 peptide homes to MDA-MB-435 tumors when
injected either into the bloodstream or subcutaneously. The phage and
fluorescein-labeled LyP-1 peptide accumulate in vessel-like structures in the
MDA-MB-435 tumors that stain for lymphatic vessel markers, but not for markers
of blood vessels. The phage and the peptide do not accumulate in normal
tissues, indicating that the PL-1 peptide distinguishes lymphatic vessels in
MDA-MB-435 xenografts from normal lymphatic vessels. Targeting drugs to
lymphatic vessels in tumors with this peptide may be effective in preventing
tumor metastasis.
The F3 and LyP-1 peptides have the remarkable
property of accumulating in the nucleus of the target cells. Thus, these
peptides may be particularly suitable for targeting anti-cancer drugs that act
in the nucleus. Our current main priority is to establish the specificity of
these peptides for other breast cancers and other types of tumors, and to
identify their cell surface receptors.
We have previously shown that vascular homing
peptides can be used to target drugs into tumors, and that the targeting
enhances the efficacy of a drug and reduces its side effects. Recently, we
used this approach to direct a proapoptotic peptide to the vasculature of the
normal prostate. The treatment caused partial destruction of the prostate and
delayed the development of prostate cancers in transgenic prostate cancer
(TRAMP) mice (Arap et al., 2002). We have also recently shown that it is
possible to target biomolecule-coated inorganic nanostructures to specific
tissues in vivo (Akerman et al., submitted). We used quantum dots for the
targeting. Qdots are inorganic nanocrystals that possess unique optical
properties and can be readily derivatized with peptides (and other
biomolecules). We coated red- and green-luminescent ZnS-capped CdSe qdots with
vascular targeting peptides and showed that the peptides specifically direct
qdots to the appropriate targets after intravenous injection into mice. In the
future, this targeting approach may find application to drug nanocrystals as
well as more complex nanosystems.
9:40 Tumor Immunology and Particulate
Vaccine Approaches
Dr. David Persing, Corixa Corporation
The concept of cancer vaccination borrows heavily on immunological themes
learned from parasite immunology, including the coexistence of humoral immune
responses with chronic low-level parasitemia, antigenic variation, and active
immunosuppression of the host. In keeping with this concept is the requirement
for identification of cancer-specific vaccine targets, along with the use of
vaccine adjuvants and delivery approaches to generate strong cytotoxic T and T
helper cell responses to tumor cells. Corixa has used a variety of genome-wide
scanning techniques for discovery of cancer-specific targets, and is now
engaged in preclinical and clinical development of vaccines employing
additional novel technologies: 1) the use of synthetic lipid A mimetics to
arouse innate and adaptive immune responses, and 2) the use of biodegradable
microspheres to encapsulate and deliver antigens in particulate form. The
synthetic lipid A mimetics, by virtue of activation of TLR-4, induce
maturation of and antigen presentation by dendritic cells, and microspheres
provide an easily assimilated phagocytic target containing a high
concentration of the tumor-specific antigen. The combination of these
technologies is likely to lead to new therapeutic approaches for prevention of
cancer recurrence in the next few years.
10:00 External Control of Biomolecular
Function via Covalently Attached Nanocrystal Antennas
Dr. Kimberly Hamad-Schifferli, Massachusetts Institute of Technology
Metal nanocrystals can be used as antennas for biological systems which
control their activity. The authors present results in which DNA and
proteins are linked to 1.4nm diameter gold nanocrystals. The
nanocrystals are inductively heated, which is achieved by placing the sample
in an external magnetic field (frequency ~1GHz) to induce alternating eddy
currents in the nanocrystals. As a result, the nanocrystals transfer
heat to surrounding molecules. Induction heating of nanocrystals linked
to DNA oligonucleotides in solution has been shown to dehybridize the DNA in a
manner that is localized and reversible. In addition, nanocrystals have
also been attached to the enzyme Ribonuclease S, allowing reversible and
specific control of hydrolysis of RNA. Applications in biology and
nanotechnology will be discussed.
10:20-11:00 Refreshment Break with Exhibit
and Poster Viewing
Saturday, September 7
8:30 am Coffee Break with Exhibit
and Poster Viewing
|
TRACK 3
|
HYBRID BIO/ARTIFICAL
MICRODEVICES |
|
T1 |
T2 |
Home |
Symposium Chairperson
Dr. Jeffery D. Carbeck, Princeton University
9:00 Microfabricated Devices for
Biomolecular Detection
Dr. Scott Manalis, Massachusetts Institute of Technology
9:20 Title and Speaker to
be
Announced
9:40 Soft Plumbing for Integrated
Nanofluidic Devices
Dr. Carl Hansen, California Institute of Technology
We have been using soft lithography to make microfabricated chips for
ultrasensitive analysis of single DNA molecules and cells. There are numerous
advantages to fabricating chips out of polymeric materials, and as a result we
have been able to rapidly and inexpensively fabricate active devices with
moving parts, such as pinch valves and peristaltic pumps. This technology
allows fabrication of highly integrated devices with thousands of valves. We
have developed a series of microfluidic devices for cellular and molecular
analysis, ranging from protein crystallization to a microfabricated
fluorescence activated cell sorter. The novel valve and pump components for
on-chip fluidic manipulation that we developed in the course of this research
will be useful for fabricating more complex chip designs for a variety of
biotechnological applications. Our ultimate aim is to create a set of tools
giving the ability to perform biology on nanoliter volumes, rather than the
current microliter standard.
10:00 Patterning of Cell Adhesion Proteins
via Colloidal Assembly: Protein Organization Directs Cell Behavior
Dr. Jeffrey D. Carbeck
This talk describes patterning of proteins at surfaces via colloidal
assembly, and effects of specific protein patterns on cell organization and
behavior. Protein coated colloidal particles are used to pattern proteins on
two length scales: the size of individual particles (500 nm – 2 microns),
and of micropatterns of particle arrays produced via self-assembly and soft
lithography (10 – 100 microns). Interfaces produced in this way show that
the organization of cell adhesion proteins on sub-cellular length scales can
directly affect cell adhesion, shape and spreading. In particular, by varying
the density of particles coated with fibronectin we switched fibroblast cells
from a morphology consistent with a static, adhesive state to a morphology
consistent with a dynamic, migratory state. In the past, such changes had been
seen only in response to changes in protein composition on surfaces. We show
that these changes can be directed through protein organization on surfaces.
10:20-11:00 Refreshment Break with Exhibit
and Poster Viewing
