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Center of Cell Control

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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