2009 Progress Report – Executive Summary
The CCC strategy to achieve the vision of nanomedicine is
to precisely control cell function with combinatorial drugs
aimed at a specific outcome, or cell phenotype, ultimately
to advance therapeutic goals. Our platform integrates real-time
molecular detection with cell control. This approach enables
quantification of the responses of signaling nanomachineries,
such as protein-protein interactions and signal transduction
network activity, that guides cell fate decisions under multiple
types of stimulation. Using this integrative approach, synergistic
or antagonistic interactions among identified molecular complexes
are deciphered by quantifying multiple phosphorylation nodes
in the signaling network. This strategy has already been
successfully demonstrated and is beginning to link specific
signaling nanomachines to translational medicine. Successful
examples include in-vitro experiments to inhibit DNA virus
infections, to eradicate cancer cells, and to maintain human
embryonic stem (hES) cell pluripotency and self-renewal under
defined culturing conditions.
Control Cellular Function toward a Specific Phenotype
for Therapeutic Purpose
We have demonstrated a technology platform that can rapidly
search for the optimal drug cocktail to combat broad classes
of disease. Inhibition of VSV and HSV-1 viral infection,
killing non-small-cell lung cancer cells, and determining
a chemically defined culture medium for hES cells have provided
important successes. We discovered that optimized combinatorial
therapies typically reduce the amount of each drug in a cocktail
to very low doses with high efficacy and low off-target toxicity.
At the same time, we are developing a rich information data
base which allows us to explore and understand the interactions
among signaling complexes at rest and altered homeostasis
with a variety of activating stimuli.
Measurement of Responses of Signaling Complexes under
Stimulations
In signaling biology, it has become clear that individual
signaling molecules are not reflective of the state of a
system. Multiple feedback mechanisms, redundancies, and cyclic
events make modeling of cell reactivity to environmental
influences, such as drugs, particularly daunting. Moreover,
the state of the signaling machinery at any given point in
time is both a reflection of its history as well as a current
position to respond depending upon the environmental stimulus.
Systems biologists and network specialists in biomedicine
have begun to define signaling complexes, or at least begin
identifying the elements of pathways that act as surrogates
for the state of a particular pathway. This has led to approaches
that both enhance our representations of signaling networks,
as well to developing tools that use network states as 'targets'
for drug action. Clearly, since viruses and cancer work at
the level of modulating the network state, we must understand
the means by which networks regulate themselves.
Given the nature of such complex systems, the evolution of
eukaryotes in particular has chosen a path of supramolecular
complexes with transient interchangeable parts as the basic
units of decision-making within cells. Some protein elements
of such machine complexes remain as core features of these
nanomachines, while others are transiently associated with
the nanomachines as they alter the basic function of the
nanomachines, or are altered themselves. To faithfully reflect
the biology and mechanism of such supramolecular machines
and the networks in which they operate, we have chosen to
quantify the functional states of many of the sub-elements
of these systems via PhosphoFlow techniques.
Interrogate phosphorylation activities of signalosomes
by smart Petri dish
With the PhosphoFlow technique, we are measuring the phosphorylation
activities of tens of targets in different cellular supramolecular
machinery to unravel functional interactions and pathway
interconnections. We have successfully used nano-imprinting
techniques to mass fabricate TNPRs that are ideal for cellular-based
Surface Enhanced Raman Spectroscopy (SERS). We are now able
to product TNPRs in quantities that are appropriate for intracellular
delivery and investigations of signalosome activities. Our
current work is to continue improving the yield and quality
of TNPR solubilization and investigate intracellular delivery.
We are currently investigating two techniques that combines
the optoelectronic tweezer (OET) and poration technology.
The first technique is electroporation. We were successful
in delivery fluorescent molecules into the cell which maintaining
cell viability. This technique is capable of parallel processing
with single cell resolution. The second technique we are
investigating is to use femtosecond laser to create highly
localized holes in the cellular membrane. This method when
combined with OET can create large concentrations of TNPRs
at the laser-induced membrane pore allowing for highly efficient
and localized delivery.
Pathway to Medicine
Combinatorial drug technology places the CCC at a strategic
position for examining nanoscale activities, such as signaling
complex phosphorylation, to rapidly move toward translational
medicine. CCC received a clinical collaboration award. This
project is applying a rapid search scheme for lung cancer
drug discovery that will move to small animal testing in
the second year of effort.
Lung cancer is the leading cause of death in the United States
and worldwide. It is estimated that lung cancer results in
more than 1 million deaths per year worldwide and in the
United States, causes approximately one death every 4 minutes.
These devastating statistics suggest that an entirely new
approach is necessary for the treatment and prevention of
lung cancer. Although smoking cessation has made an impact
in lung cancer incidence, former smokers remain at an elevated
risk of lung cancer for many years. Our current research
is to develop a novel system for the optimization of drug
combinations for lung cancer therapy by utilizing an artificial
neural network modeling algorithm. Our approach will determine
optimal combinations of multiple drugs against panels of
well characterized drug resistant lung cancer cell lines.
These cell lines are being characterized both in terms of
their gene array expression profiles and their responses
to drugs so that we can ultimately define novel combination
therapies targeted to specific genetic abnormalities, working
through identified signalosomes.
Project Evolution
In the past two and half years, we have focused our efforts
to developing approaches and techniques that investigate
cellular responses on a network and systems level and providing
rapid pathways to translational medicine. Recently, we were
able to further our work with the combinational drug studies
by adding more clinically relevant parameters such as drug
cocktail efficacy, drug dosages, and looked at the effects
of removing dominant drugs from the overall drug mixture.
We’ve included the use of PhosphoFlow techniques to the combination
drug studies to investigate activities of signalosome during
drug cocktail stimulation. We’ve began studies of combinatorial
drugs in lung cancer and leukemia looking at drug cocktail
therapies tailored eradication tumor cells and sparing non-cancer.
We have also extended the use of the combinatory drug search
technique to the study of maintaining human embryonic stem
cells in culture. During the past year, we have also made
progress in the development of real-time monitoring techniques
of cellular signaling pathways with the success of mass fabrication
of tunable nano plasmonic resonators (TNPRs) through nano-imprinting
lithography (NIL). With mass fabrication, we are now able
to make TNPRs in quantities suitable for cellular delivery
and intracellular detection. We have also investigated different
methods incorporating optoelectronic tweezers (OET) and poration
techniques to introduce particles such as TNPRs into the
cell in a concentrated and precise method.
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