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R21 CA126664-01A2 2008 BARBAS, CARLOS F SCRIPPS RESEARCH INSTITUTE Zinc Finger Recombinases for Endogenous Genome Tailoring
This proposal seeks to develop a strategy of genomic recombination suitable for applications in both cancer research and therapy, and is a revised resubmission of RA-CA-126664 responsive to RFA-CA-07-002. At present, there is no method for targeted and site-specific recombination of the endogenous human genome. Such `genome surgery' would enable the genetic dissection of cancer biology, as well as, empower a new approach to gene therapy. The rationale for the proposed work is that RecZFs, with their capacity for efficiently excising, inverting, or integrating large segments of DNA in many sequence contexts, are a unique and powerful tool for both introducing and repairing genetic defects related to cancer. This strategy contrasts with current gene therapies, which provide only transient gene expression or risk non-specific genomic integration of exogenous DNA. The proposed research focuses on the TP53 locus, a crucial tumor-suppressor, which is functionally inactivated in many different kinds of human cancer. The overall objective of this research proposal is to develop tools for efficient and selective integration into any genomic site, using the TP53 locus as a model target. The central hypothesis is that RecZFs, constructed from novel zinc finger proteins and evolved catalytic domains, will be able to perform this task. As powerful tools for genetic manipulation, we anticipate that RecZFs could facilitate the creation of novel animal models of cancer, the application of gene therapy, and the elucidation of cancer genetics.
 
R21 CA125277-01A2 2008 CAMP, ROBERT L [Completed by Agarwal, Seema] YALE UNIVERSITY Novel Methods to localize protein-protein interactions in fixed cells and tissues
Although analyses of DNA, RNA, and protein expression can elucidate the phenotype of a cell, ultimately it is the interactions between proteins that determine metabolic function. Understanding protein-protein interactions is vital to the study of cancer, and the aim of most new chemotherapeutic drugs is to disrupt aberrant interactions. Recent advances in the quantification of tumor biomarkers in patient samples show great promise in predicting patient outcome and response to treatment. However, there is no good way to assess the co-localization of proteins in tissue samples. The successful evolution of bio-specific therapies and associated pharmaco-diagnostics may ultimately require quantitative measures of protein-protein interactions within each patient's tumor. From the basic science perspective, new methods in co-localizing proteins in tumor samples will advance the understanding of carcinogenesis and the subsequent development of targeted therapies. We hypothesize that engineering robust methods for assessing protein-protein interactions in human tumor samples will significantly improve the analysis of patient specimens and ultimately speed the development of targeted patient-specific therapies. Protein interactions are the gears which drive cells. In tumor cells, many of these processes are corrupted. Today, we can study the expression of individual proteins in patient tumor samples; however, we cannot look at the interactions between these proteins in patient tumors. Our goal is to create new methods for recognizing and studying such interactions. We hope that these methods will improve our understanding of cancer and provide means to assess which tumors will respond to specific therapies.
 
R21 CA126622-01A1 2008 CHAPUT, JOHN CHARLES ARIZONA STATE UNIVERSITY-TEMPE CAMPUS A High-throughput Route to Synthetic Antibodies for Array-based Cancer Detection
The ability to monitor the levels of large numbers of proteins for indicators of disease (biomarkers) holds great promise for the detection and treatment of many diseases, including cancer. Although most analytical techniques for detecting protein levels in human serum currently rely on ELISA-based assays or mass spectroscopic analysis, protein capture arrays have gained considerable interest as a future technology for proteome-wide analysis. The studies described in this application seek to develop a comprehensive array of high affinity synthetic antibodies (synbodies) that can be used to detect and profile cancer and cancer-related proteins in human blood and saliva. In contrast to immunoglobins, for which high costs and slow production rates limit the availability of suitable quality affinity reagents, the rapid synthesis of artificial antibodies from low cost chemical reagents offers a possible solution to the protein ligand problem. The goal of this application is to develop synthetic antibodies to five known cancer biomarkers using a technology that we call systematic recombination of ligands and linkers (SRLL). This technology combines the multiplex capability of surface plasmon resonance (SPR) with the nanometer-scale precision of DNA self-assembly to identify optimal peptide pairs and peptide pair separation distances needed to produce multivalent binding agents from monovalent ligands previously identified by screening or selection methods. We have termed the ligands that emerge from this process ""synbodies"". The Specific Aims of this proposal are to: 1. Determine the minimum number of selection steps needed to produce modest affinity peptide ligands to five well-established cancer biomarkers. 2. Generate an array of high affinity synthetic antibodies to the five cancer biomarkers targeted in Specific Aim 1.The relevance of the proposed research resides in the potential for early cancer detection based on routine proteome-wide analysis using synthetic antibody arrays to detect cancer biomarkers presymptomatically.
 
R21 CA134250-01 2008 DOW, THOMAS A; EDWARDS, JACK RAY; MUDDIMAN, DAVID C. (contact) NORTH CAROLINA STATE UNIVERSITY RALEIGH Improved Cancer Biomarker Detection Using Novel Air Amplifier Designs in ESI-MS
The identification and quantification of candidate biomarkers specific for a given cancer (e.g., ovarian, prostate) using tools such as liquid chromatography-mass spectrometry (LC-MS) is necessary for the diagnosis and treatment of the disease. Improved limits of detection for LC-MS and LC-tandem MS (LC-MS/MS) by implementation of devices such as the air amplifier proposed here will further these efforts enabling better patient care. Design using computer modeling and simulation, and fabrication using precision engineering methodologies will result in a stable, low tolerance air amplifier. The improved limits of detection by a well-designed and implemented air amplifier device will result in detection and quantification of low abundance candidate biomarkers in biofluids for both discovery-based and targeted proteomic applications. Systematic evaluation of the new air amplifier designs will utilize electrospray ionization mass spectrometry (ESI-MS). This will compare the original air amplifier design to the newly designed air amplifier based on the metrics of ionization time (MS analysis), limits of detection, and number of protein identifications from LC-MS/MS. After establishing a reproducible protocol for the air amplifier, it will be directly applicable for biomarker discovery and target based analysis. Multi-site evaluation of the improved air amplifier is proposed to determine inter-laboratory reproducibility. As a final element of this proposed research, the air amplifier design and application instructions will be disseminated on the internet for open access.
 
R21 CA125397-01A2 2008 EDWARDS, JEREMY S. UNIVERSITY OF NEW MEXICO Sequencing Chr. 6 in Melanoma Patients
Herein we propose to further develop and utilize an ultra-high throughput DNA sequencing technology to generate large amounts of genome sequence to identify and characterize genomic variation in a population of people diagnosed with invasive melanoma. Specifically, we will ultimately sequence chromosome 6 from ten carefully chosen individuals. As a quantitative milestone, we will report the sequence for chromosome 6 to at least 85% completion for these ten people at better than 99.999% assembled accuracy. In a future R33 proposal, we will collect sequence for 290 additional people. In the end, we will have a robust platform for generating high-throughput nucleic acid sequence and converting these data to genetic markers that identify people who are predisposed to progressive malignant melanoma. Current progress towards our goals is at an advanced stage. We are able to routinely sequence bacterial genomes and we have the potential to generate enough data to rapidly sequence a single human chromosome >20x coverage in less than a month. Therefore, we feel that much of the technical risk has been reduced by our previous work, and there are substantial rewards to be gained by pursuing the goals described here.
 
R21 CA132806-01 2008 GODFREY, TONY E; MORRISON, TOM B (contact); WILLEY, JAMES C. BIOTROVE, INC. Standardized NanoArray PCR for Gene Expression Profiling of Lung Cancer
Molecular characterization of cancer, in particular by gene transcription profiling, has great potential to improve prognosis, therapeutic selection and clinical outcomes. However, the potential for using expression signatures for cancer prognosis and treatment selection is hampered by lack of readily deployable test kits with the accuracy, low RNA requirement and inter-site concordance required for routine clinical use. We propose an innovative solution based on two well-validated PCR technologies whose combination uniquely addresses the problem of diagnostic assay reproducibility. Our plan is to implement Standardized RT (StaRT)-PCR, a proven competitive PCR method developed at the UNIVERSITY of Toledo, in a novel nanofluidic PCR platform developed by BioTrove Inc. in order to streamline the fluidic workflow, improve measurement throughput, and at the same time reduce test cost and maintain low RNA input. As compared to existing hybridization or real-time qPCR approaches, Standardized NanoArray PCR (SNAP) will provide the same dynamic range and quality as RT-PCR, yet require less RNA input and be more readily clinically deployable. The development will entail step-wise integration of proven technologies. First, real-time qPCR TaqMan assays will be developed for 16 lung tumor prognostic genes. These assays will be converted to StaRT-PCR by creation of competitive template and a competitor specific dye-labeled probe. Adding a pre-amplification step to StaRT-PCR will reduce the RNA input requirements to enable thousands of tests per sample. Finally, moving the assays into the OpenArray nano-PCR plate will streamline fluid handling. Using RNA isolated from lung clinical tumor resections, dynamic range and precision equivalent to real-time qPCR will be demonstrated for the integrated platform. After the initial development phase is complete, we will compare SNAP and real time qPCR in two critical gene expression profiling experiments. First we will compare the minimum amount of RNA required for each method by monitoring loss of precision as a function of decreasing RNA sample input. Second we will demonstrate lower inter-site variability, a critical factor for deploy-ability, by measuring gene expression profiles of seven lung tumor resection samples in three laboratories. Meeting these Specific Aims will lead to seeking further funding for multi-site prognostic validation studies involving formalin-fixed, paraffin-embedded (FFPE) lung specimens with extensive clinical history.
 
R21 CA126701-01A2 2008 JIN, SONG UNIVERSITY OF WISCONSIN MADISON Ultrasensitive Nanoscale Magnetic Sensors for Label-free Analysis of Cancer
In this resubmitted application responding to Innovative Technologies for Molecular Analysis of Cancer (FOA CA-07-033), we seek to develop a novel type of sensor technology that exploits the emerging nanoscale dilute magnetic semiconductor (DMS) materials to enable ultrasensitive label-free electrical detection of DNA molecules for genetic analysis of cancer. The sensing mechanism is the following: the semiconducting properties of the properly biofunctionalized DMS nanomaterials are modulated by surface charges upon specific binding of target molecules to surface probe molecules, which leads to significant magnetic property changes that can be detected using highly sensitive giant magnetoresistance (GMR) magnetic field sensors found in computer hard disks. Outperforming any current technologies, the proposed sensors promise to be simple, specific, high throughput, robust, potentially compatible with high density arrays and multiplexing, and potentially boast low cost and good manufacturability on a large scale. In this application, we will demonstrate the proof-of-principle operation and validate the extremely high sensitivity of the proposed sensors using two families of DMS nanomaterials and model DNA samples from the p53 gene. The specific steps are: 1) synthesize and characterize nanomaterials of transition metal doped ZnO and Cr-doped ZnTe, and demonstrate robust ferromagnetism near room temperature; 2) develop and optimize strategies to attach suitable oligonucleotides onto the surface of these nanomaterials for use as the sensing elements of proposed sensors; 3) demonstrate and understand the magnetic property changes upon applying complementary target oligonucleotides from p53 genes to surface modified nanomaterials using a SQUID magnetometer; 4) construct integrated sensor devices using commercial GMR devices, and demonstrate proof-of-principle real-time electrical sensing measurements with model analytes of target oligonucleotides; and 5) evaluate and quantify the sensor response versus DNA concentrations and demonstrate the detection of complementary target p53 oligonucleotide down to concentration of 0.1 femtomolar (fM), and the differentiation of wild type and single nucleotide mismatched mutant p53 at the concentration of 10 fM through the optimization of sensors. The success of this project will enable inexpensive table-top or even handheld turn-key electrical devices that will allow heathcare professionals to perform robust high throughput but yet highly sensitive analysis of urine, serum, and blood samples for the detection and diagnosis of cancer, the monitoring of cancer progression, and the determination of response to therapy with little training and sample preparation in point-of-care settings.
 
R21 CA126635-01A1 2008 KEELY, PATRICIA J UNIVERSITY OF WISCONSIN MADISON Using New Optical Techniques to Study Cell Signaling in 3D Matrices
Mammary epithelial cells undergo ductal morphogenesis only in compliant 3D extracellular matrices (floating collagen gels or Matrigel), but not in otherwise identical matrices that are stiff (attached 3D collagen gels) or have increased collagen density. An understanding of how cancer cells interact with local tissue environments requires the ability to observe the relationship between signaling molecules, subcellular structures, and components of the ECM within a 3D environment. Few studies have investigated signaling events in 3D, and to our knowledge none have done so using live cell imaging or FRET approaches. 3D imaging adds complexity to these fluorescence studies, including the inherent challenge of a 3D volume as opposed to a 2D image, problems of overlapping spectra, low signals, and light scatter. The goal of this project is to develop imaging techniques in the context of 3D culture systems to directly visualize signaling pathways relevant to breast tumor cell behavior. Our underlying hypothesis is that the Rho signaling pathway is the sensing mechanism by which the physical properties of the microenvironment are conveyed to signaling effectors within the cytoskeleton. We propose the following Aims: Aim 1: Characterize endogenous signals of the 3D matrix and tissue environment within which we wish to investigate signaling events A) Characterize the multidimensional signals of endogenous fluorophores within cells within a 3D collagen matrix of various densities and compositions The ""fingerprint"" of endogenous fluorophores (NADH, FAD, and collagen) within collagen matrices will be mapped spatially using multiphoton laser-scanning microscopy (MPLSM) and Spectral and Fluorescent Lifetime Microscopy (SLIM and FLIM). B) Characterize the endogenous signals in the mouse mammary gland during tumorigenesis Endogenous signals (NADH, FAD, and collagen) will be characterized by MPLSM, SLIM, and FLIM in models of mouse mammary tumorigenesis, generating novel data regarding metabolic changes during tumorigenesis. Aim 2: Develop strategies for directly visualizing Rho/ROCK signaling pathways within 3D matrices using Multiphoton Laser-Scanning microscopy (MPLSM) and Fluorescent Lifetime Microscopy (FLIM) A) Investigate the matrix-dependent activation of Rho GTPase. Fluorescent probes coupled to Rho and a Rho binding domain will be used as FRET pairs to determine Rho activation spatially and temporally in a 3D context within collagen gels and definable microfluidic channels. B) Apply FRET/FLIM techniques to characterize the coupling of Rho to its effector ROCK in response to collagen density. Coupling of activated Rho to ROCK, and regulation of myosin-mediated contractility will be determined spatially and temporally within 3D collagen matrices.
 
R21 CA122878-01A2 2008 KELLEY, SHANA O UNIVERSITY OF TORONTO Development of DNA-templated IR quantum dots
Functionalized semiconductor quantum dots have previously been demonstrated to bind to markers on cells. Through their spectrally-narrow optical emissions, they illuminate tumors and other harbingers of disease. They are enabling the highly specific detection of a range of diseases at the earliest stages. We propose to pursue a new and improved class of semiconductor quantum dots. We have shown already that, by seeding the growth of nanoparticles using a DNA template, we are able to produce quantum dots that are efficient, stable emitters in the infrared wavelengths (so-called biological window) in which living organisms are much more transparent than in the visible wavelengths, and in which living organisms' autofluorescence is orders of magnitude lower than in the visible. These materials retain their luminescence properties over time even in biological media such as plasma at 37o C. The collaborating team of Dr. Shana Kelley, a nucleic acids chemist, and Dr. Edward Sargent, an optoelectronics engineer, bring the expertise required to optimize the DNA-grown nanoparticles for applications in medical diagnosis. The strategy of directing the growth of luminescent nanoparticles using DNA provides a one-pot route towards the strong coupling of light-emitting tags with DNA-based aptamers for programmable specific-binding. The project is divided into the following Specific Aims: 1) Direct the growth, and thereby maximize the performance, of DNA-templated quantum dots using designer DNA sequences; 2) Discover optimized DNA sequences for specific binding assays using in vitro selection. We will thereby develop new means of creating highly luminescent nanomaterials for medicine and biology. The team, with its complementary expertise in biomolecular chemistry and optical materials, is equipped with the infrastructure and resources to make a significant contribution to the realization of improved visible and infrared fluorophores for diagnosis.
 
R21 CA134299-01 2008 KEMP, MELISSA LAMBETH; LU, HANG (contact) GEORGIA INSTITUTE OF TECHNOLOGY MICROFLUIDIC SYSTEM FOR HIGH-THROUGHPUT EVALUATION OF T CELL FUNCTIONALITY WITH H
Adoptive transfer of T cells is a promising clinical cancer therapy that relies on enhancing the adaptive immune response to target tumor cells in vivo. Widespread application of this therapy, however, has been hindered by the necessary expansion of large populations of T cells for each patient (often selected for tumor antigen specificity) and loss of functionality of the T cells post-transfer. Our long-term objective is to understand how T cell activation is dampened in vivo by the tumor milieu and to be able to evaluate the responsiveness ex vivo-expanded T cells accurately for cancer therapy. Microfluidic chips are ideal for high-throughput parallel experimentation and automation. In addition, microfluidics also provides the relevant length scales (~microns) and unique physical phenomena (e.g. laminar flow) to handle cells. The type of multiplex data that we can obtain from this technology will enable quantitative modeling of T cell activation and better understanding and characterization of anergy. The objective of this R21 project is to engineer a multiplex microfluidic assay to quantify T cell activation on a small population of cells with high temporal resolution. The hypothesis is that capturing the early dynamics of T cell activation of ex vivo expanded clones would improve upon current measures of T cell functionality. The first component of this project is to develop the high-throughput microfluidic system for multiple time-point stimulation and lysis of cells; in parallel, we are to develop biochemical assays to characterize the performance of the system and the cell state. The second component is to perform in vitro characterization of ex vivo expanded T cells for distinguishing anergic versus responsive behavior. The approach is innovative because the technology developed here dramatically increases the capabilities and throughput of existing assays in evaluating T cells for adoptive transfer. Furthermore, this work proposes and tests a new paradigm in T cell evaluation using multiplex quantitative means. The proposed research is significant because it is expected to expand the toolbox of cancer therapy and possibly other related quantitative biosciences and medical technologies.
 
R21 CA118592-01A2 2008 KNUDSEN, BEATRICE S FRED HUTCHINSON CANCER RESEARCH CENTER Tissue Lysates for Studies of Protein Phosphorylation
The long-term objective of this project is to measure phosphoproteins in human cancer tissues. As the first step towards this goal, the objective of this R21 grant is to identify optimal conditions for preparation of tissue samples by testing multiple combinations of tissue fixation and protein extraction buffers. Xenograft tissues are frozen or fixed in a non-crosslinking fixative or in formalin. Proteins are extracted with 4 buffers and analyzed for protein phosphorylation using a dot-blot assay. In Aim 1, we measure global phosphorylation on tyrosine and threonine. We also use proQ Diamond to detect all phosphorylated proteins. In Aim 2 we evaluate specific phosphorylation sites in proteins that are of clinical significance as drug targets or predictive biomarkers. We use statistical methods to identify sample preparation protocols that are (1) reproducible, (2) provide a high yield of extracted proteins, and (3) preserve protein phosphorylation during the extraction process. We rank the results and compare the top ranked protocols to a reference sample preparation procedure. We anticipate that by identifying sample preparation protocols for measurement of global and specific protein phosphorylation in cancer tissues, we will improve drug selection and response rates for many patients with solid tumors. Public Health Relevance Statement: Phosphoproteins represent effective predictive and prognostic biomarkers in human cancers; however, their expression is unstable and their measurement difficult. The objective of this project is to test novel sample preparation protocols that may better preserve protein phosphorylation in human cancer tissues for precise and reproducible measurement.
 
R21 CA132700-01 2008 KOIDE, SHOHEI UNIVERSITY OF CHICAGO High-performance affinity reagents for peptide epitopes
Cancer results from the disregulation of signal transduction networks. Thus, quantitative detection, functional assessment and isolation of proteins involved in cancer biology are critically important for molecular analysis of cancer. Short peptide motifs within many of this class of proteins and posttranslational modification thereof play central roles in signal transduction regulation through controlling protein function and interactions. However, there are a very small number of affinity reagents (reagents that bind to a target with high affinity and specificity) to this class of high-value epitopes in cancer analysis. The long-term goal of this project is to develop a powerful technology platform for generating high-performance affinity reagents for short peptide epitopes. The primary products will be novel affinity reagents termed ""Epitope Clamps"". Our strategy is distinct from many others in that we aim to develop distinct types of affinity reagents, with each type specific to a particular class of short peptide epitopes. This contrasts with the conventional antibody-based approaches where a single, general- purpose platform is used for engineering distinct types of targets. Our innovative protein engineering strategy harnesses the inherent specificity present in the so-called interaction domains and dramatically enhances their affinity and specificity by attaching an ""enhancer domain"". This makes it possible to generate protein libraries predisposed to binding to a particular motif, and thus it dramatically increases the chance of successfully engineering high- performance affinity reagents. Our proof-of-concept experiments have successfully demonstrated this new paradigm in affinity reagent engineering. These proposed studies will establish the full potential of the Epitope Clamp technology, and will generate high-performance affinity reagents to phosphopeptide epitopes in proteins that are critically involved in cancer biology. The technology and tools developed in this project will make a major impact on molecular analysis of cancer, and more broadly on proteomics and biotechnology. Accurately measuring the amounts of proteins of interest in cells and tissues and assessing their functional state are major technological challenges in molecular analysis of cancer. This application aims to establish a powerful technology platform for facile generation of high- performance reagents that tightly bind to a predefined segment within a protein of interest. We aim to develop such ""affinity reagents"" to diverse protein segments that carry a chemical signature of activated proteins. This technology will fill a major void in the current technology portfolio for molecular analysis of cancer.
 
R21 CA126764-01A1 2008 KRON, STEPHEN J. UNIVERSITY OF CHICAGO MALDI Imaging of Cancer Signaling Signatures
Detection of the activation of cellular kinases associated with oncogenic signaling can serve as a valuable molecular marker for cancer diagnosis and as a predictive tool for selection of therapy. In human squamous cell carcinoma of the head and neck (SCCHN), EGFR activation is associated with therapeutic resistance, increased metastasis and poor outcomes. Histology and tumor architecture provide complementary and critical information about cancer stage and grade. By adapting and extending emerging technologies for kinase sensor biochips, we propose to develop the capability to image the distribution of cancer signaling in tumor tissue obtained by biopsy or surgical excision. We and others have developed robust, sensitive and specific biochip-based assays for kinase activity in cellular lysates, using immunodetection, radionuclide incorporation or MALDI-TOF MS analysis as a read-out. We now intend to adapt these methods to create multiplexed assays whereby multiple peptides, serving as specific substrates for kinase involved in oncogenic signaling, will be linked reversibly to the surface of a biochip. This multiplexed biosensor will be exposed to thick sections of tumor biopsies by ""tissue print"" to allow phosphorylation of the peptides. The cellular material will be washed away and the biochip will be interrogated by MALDI-TOF MS imaging to detect relative peptide phosphorylation, creating an image of the multiple kinase activities across the tumor section. These kinase activity images can be correlated with conventional histology and immunocytochemistry to enhance diagnosis and prognosis. To establish proof-of-principle in the R21 phase, methods will be developed using human SCCHN tissue culture cells and xenograft tumors of grown in athymic nude mice. We will take advantage of the activated EGFR kinase characteristic of this tumor and the availability of the specific EGFR inhibitors gefitinib and erlotinib to develop and validate our biochip sensor and imaging capabilities. We anticipate achieving sufficient sensitivity and resolution to detect SCCHN tumor islands embedded in extensive stroma.
 
R21 CA126727-01A1 2008 LANDEGREN, ULF DAG UPPSALA UNIVERSITY Diagnostic analyses of endogenous protein interactions
Detection of patterns of interactions among proteins can prove of great conceptual and diagnostic value in malignancy and other diseases, but efficient methods have been lacking to characterize interactions both in research and diagnostics. We will take advantage of a unique set of tools developed in our lab to now establish methods to: 1) Characterize interactions among large sets of proteins directly in patient samples using the proximity ligation technique and a recently established paired-tag array read-out, for interaction biomarker discovery. 2) Develop and validate smaller-scale diagnostic assays of interacting protein molecules using a combination of array read-out and a novel in situ interaction analysis that we have also recently established. The procedure we will use in the characterization phase involves immobilization of in situ crosslinked interacting proteins to a solid support. A series of antibodies specific for proteins of interest, conjugated with oligonucleotides including antigen-specific tag sequences, are allowed to bind to proteins in the biological samples in so-called proximity ligation reactions. The presence of interacting proteins results in co-localization of specific pairs of antibodies, which in turn brings their attached oligonucleotides in close proximity, allowing these to be joined by enzymatic ligation. The resulting DNA molecules are combinations of tags that reflect the identity of the detected pairs of interacting proteins. The ligated molecules are amplified, restriction digested, and sorted on an array containing all possible pairs of tag sequences for the proteins under investigation. Paired-tag sequences properly hybridized to the arrayed molecules can be circularized, and the oligonucleotides immobilized on the array next serve as primers for rolling circle reaction where only correctly ligated, circular molecules are amplified. The resulting rolling circle products contain tandem repeats of generic detection sequence, permitting hybridization of a fluorescent probe for array detection. The method will be established and applied to study interactions among members of the Smad protein family, intracellular effectors of the transforming growth factor (TGF)-ยจ signaling pathway that play well-documented roles in both tumor suppression and tumor progression. In the second phase, which will temporally overlap with the first, assays adapted for diagnostic use will be developed, and interacting pairs of proteins will be validated as markers using this assay, and also by studies in tissue sections from patient samples, with the help of so-called proximity ligation in situ assays. In these assays, proximal binding of pairs of antibodies with attached oligonucleotides result in the formation of circularized DNA strands for localized rolling-circle replication reactions that permit even single interacting pairs of proteins to give rise to clearly detectable signals. The proposed procedures allow endogenous protein complexes to be observed without the requirement for cloning or transfecting exogenous components, permitting dynamic interaction networks to be investigated in normal, precancerous, and cancerous cells. Furthermore, any pair-wise interactions among a given set of proteins can be monitored, and the paired-tag system is scalable and transferable to study different pathways. The procedures and results of this project will establish protein interaction events as markers for tumor diagnostics, and for evaluation of drug treatment.
 
R21 CA126669-01A1 2008 LAZAR, MARIA IULIANA VIRGINIA POLYTECHNIC INSTITUTE AND STATE UNIVERSITY Microfluidic MALDI-MS Device for High-Throughput Proteomics & Biomarker Discovery
Cancer is a leading cause of death in the United States with over 1,000,000 new cases being diagnosed every year. As a result of the high sensitivities and specificities that are required to justify large-scale population screening, only very few single protein biomarkers are routinely used today in the clinical setting. It is of critical priority to develop novel technologies that will enable the rapid detection of a plethora of biomarkers relevant to early diagnosis, prognosis, staging and treatment response. The long-term objective of this research is to combine the emerging technology of microfluidics with state-of-the-art mass spectrometry (MS) detection to enhance our capacity for analyzing molecular structure and function in biological systems. This application capitalizes on the distinguishing capabilities of microfluidic architectures that enable process integration, multiplexing, fast and high-throughput processing of minute amounts of sample, and the power of MS detection that provides the sensitivity, specificity and resolving power necessary for unambiguous detection of trace level components. Specific Aim 1: Develop a compact, low-cost and disposable microfluidic analysis platform with matrix assisted laser desorption ionization (MALDI)-MS/MS detection for high-throughput proteomics that will enable the study of protein co-expression patterns and biomarker discovery. The microfluidic device will carry out parallel liquid chromatography (LC) separations and will integrate a novel microchip-MS interface to enable sensitive MALDI-MS/MS detection directly from the chip. Specific Aim 2: Demonstrate the effectiveness of the microfluidic MALDI-MS/MS platform for the detection of multiple cancer biomarkers in cellular extracts. Cellular fractions from the MCF7 breast cancer cell line will be analyzed for target proteins that are involved in essential cellular processes driving cancer on-set and development (cell proliferation, cell cycle regulation, DNA repair, apoptosis and invasion/metastasis).
 
R21 CA131920-01A1 2008 LEVCHENKO, ANDRE JOHNS HOPKINS UNIVERSITY Microfluidic Devices for Studying Cancer Signal Transduction
The emergence and progression of various cancers is heavily influenced by extracellular cues such as growth factors and inflammatory cytokines. The intracellular signaling pathways activated by these cues often have altered behavior and new dynamic properties, leading to neoplastic behavior. Furthermore, in a given cell, multiple signaling pathways may be activated leading to complex interactions known as 'cross-talk' and making it difficult to identify key carcinogenic pathways, or to propose targeted treatment. Therefore, a comprehensive, systems-wide view of cancer signaling is required. An important barrier to achieving a truly systemic analysis of dynamic signaling behavior in cancer cells is the lack of a tool to systematically, efficiently, and reproducibly measure the immediate signaling outputs. Here we propose to develop and enhance novel microfluidic devices that can overcome this barrier. The devices contain dozens of miniature chambers which can be filled with cells. Cells in each chamber can be independently stimulated and monitored for a distinct signaling event using conventional immunocytochemistry methods. In addition to the large number of parallel measurements that can be made in a single device, microfluidics offers precise control over experimental conditions, enhancing reproducibility and making quantification more accurate. Our existing prototypes have been tested for all major required functions and provide proof-of-principle of our technological approach. We propose to further develop the existing prototypes to enable very high-throughput experiments cataloging cell signaling signatures in diverse cancer cells. The main focus of this application is to increase the size and capacity of the device and supporting equipment, and demonstrate its functionality through a pilot screen of signaling in cancer cell lines and cells from malignant melanoma biopsies. These pilot screens may reveal new signal transduction-based markers for cancer diagnosis and suggest optimal chemotherapeutic targets. We anticipate that the device will become a powerful research tool to study signal transduction. Furthermore, the use of the device could potentially translate to clinical laboratories, specifically in its ability to perform multiple experiments directly on a patient's cells. We envision clinicians will eventually be able to use the device in personalized therapy, for example, by using a small biopsy to screen for various signaling features that mark drug susceptibility or by directly testing the efficacy of chemotherapeutic agents.
 
R21 CA125698-01A1 2008 LIOTTA, LANCE ALLEN GEORGE MASON UNIVERSITY Surrogate and Sentinal Technologies to Monitor Stability of Cancer Phosphoprotein
The phosphorylation, or activation state of kinase-driven signal networks embodies information concerning disease pathogenesis and the ongoing state of kinase associated therapeutic targets. Profiling the tumor phosphoproteome using human tumor biopsy specimens is a crucial component of the perceived upcoming revolution of individualized cancer therapy. A critical unmet need addressed by this application is tissue phosphoprotein stability data, standardized protocols, and novel technologies which can be used in the real world clinical setting (e.g. operating room, outpatient clinic biopsy, radiological suite needle aspiration) for seamless collection, immediate preservation, and real time stability monitoring of phosphoproteins. Our multidisciplinary team has previously developed Laser Capture Microdissection (LCM) and reverse phase protein microarray (RPA) technologies to conduct phosphoproteomic analysis of the tissue microenvironment. We will standardize the RPA technology to simultaneously measure at least 100 known phosphoprotein endpoints with high precision and sensitivity in a human core needle biopsy or fine needle aspirate. This technology will be used to collect previously unknown quantitative information about the tissue half-life of phosphoproteins representing a wide range of signaling pathways, cellular compartments and phosphorylated residues over time zero to 48 hours. These data will become the basis to identify a subset of highly representative and most-labile endogenous phosphoproteins which can be employed as novel surrogate quantitative markers of tissue preservation and phosphoprotein stability. We will employ this stability data and novel surrogate endpoints technology to propose definitive quantitative guidelines for tissue perishability limits. We will create exogenous phosphoprotein sentinel nanoparticles to record the processing history of the specimen for ongoing Quality Assurance. The RPA, surrogate markers, and sentinel technology will be used to rank candidate preservative solutions that stabilize kinases, phosphatases, and phosphoproteins for 24-48 hr at room temperature. The desired outcome will be a complete standardized technology kit which can be employed for routine clinical collection, shipping, early warning of perished tissue, and real time monitoring of tissue phosphoproteins for molecular profiling.
 
R21 CA126720-01A1 2008 MACBEATH, GAVIN HARVARD UNIVERSITY Quantitative, multiplexed and high-throughput: macroarrays of lysate microarrays
Mammalian signaling networks comprise biochemical pathways with shared components, common inputs, and overlapping outputs. Understanding how information flows through these pathways requires information on signaling networks as a whole, rather than on one or two components. To study signaling at a systems level, we need ways to measure the abundance and post-translational modification of many proteins in a parallel, quantitative, and reliable manner. In addition, since an understanding of signaling requires the frequent temporal sampling of many proteins under multiple conditions, these methods must be high-throughput. Here, we describe technology that mimics an immunoblot, but in a multiplexed and extremely miniaturized format. Cells are cultured in 96-well plates and subjected to a variety of perturbations (stimulation with epidermal growth factor in the presence of selected shRNA's or cDNA's). The cells are then lysed and the lysates arrayed at high spatial density onto glass-supported nitrocellulose pads, also arranged in a 96-well format. By probing each pad with a different antibody, the `state' of the signaling network is assessed. Currently, high- throughput multidimensional readouts can be obtained either by automated fluorescence microscopy or by multiplexed flow cytometry. Although both techniques provide the ability to track more than one protein simultaneously, they rely on the use of different colored fluorophores and hence can only follow about a dozen proteins. In contrast, the technology described here enables a single sample to be replicated thousands of times on separate microarrays and is thus easily scaled. This application details efforts to make lysate microarray technology rigorously quantitative and outlines automation strategies that render it high-throughput and reproducible. In addition, since one of the biggest challenges in analyzing cancer at a systems level is to go beyond a mere description of the data, a strategy is also presented to build predictive models of cell signaling using Bayesian methods. As proof-of-concept, we will focus on epidermal growth factor signaling in A431 cells. Although this system is relatively well-understood, our approach should capture higher-order interdependencies between proteins that are not evident from traditional studies. More importantly, our strategy should provide a general way to uncover causal relationships in less well-studied networks using data derived from our high-throughput microarrays.
 
R21 CA126728-01A1 2008 MCCAWLEY, LISA JOY VANDERBILT UNIVERSITY MEDICAL CENTER Parallel Capillary Bioreactors for Leukocyte Transendothelial Migration Analysis
The tumor microenvironment, and in particular tumor associated inflammation, is a driving force of tumor progression. Immune cell (i.e. leukocyte) infiltration into sites of inflammation requires the coordinate regulation of multiple steps including arrest on endothelium, migration through the endothelial barrier and directed migration through connective tissue. A potential key regulator of leukocyte infiltration is a member of the matrix metalloproteinase family, MMP3. Mice which are null for MMP3 demonstrate reduced infiltration of leukocytes in a variety of model systems, and a number of matrix and non-matrix MMP substrates identified include those known to affect immune cell function. We hypothesize that MMP3, as well as other MMPs, directly enhances leukocyte extravasation during tumor progression Direct analysis of the roles of MMPs is possible through analysis of primary cells isolated from mice with genetic ablation of individual family members. However, current technologies to assay leukocyte extravasation either do not recapitulate key physiological parameters such as the microfluidic shear and apical-basolateral organization of endothelium, or they require extensive tissue samples that excludes their use with primary cells isolated from mice. The goal of this proposal is to exploit the expertise of our collaborative team of Cancer Biologists and Engineers in applying soft-lithography microfabrication technology to the biological challenge of the study of tumor- associated leukocyte infiltration ex vivo. We propose a multidisciplinary approach in the development of planar and multilayer Parallel Capillary Perfused Bioreactors (PCPB) that 1) better approximate the spatial constraints and architecture of blood vasculature, 2) can provide regulated shear flow and 3) are high- throughput in design requiring minimal cell samples for assay conditions. The development of the planar and multi-layer PCPBs for application in studying leukocyte transendothelial migration are detailed below. Completion of these aims will generate novel devices that will provide an ex vivo system that more closely approximates physiological vasculature facilitating novel insights into leukocyte recruitment from circulation. Specific Aim 1: To develop a planar parallel capillary perfused bioreactor (PCPB) A) Design and fabricate planar PCPB with a recirculating nutrient supply system B) Apply the planar PCPB to the assay murine derived leukocyte attachment to endothelial monolayers. Specific Aim 2. To develop a multilayer parallel capillary perfused bioreactor (PCPB) that will support endothelial polarization into apical and basolateral surfaces A) Design and fabricate a multilayer PCPB that will incorporate a filter system; B) establish culture conditions that allow for endothelial polarization across filter of multilayer PCPB and C) define parameters for leukocyte attachment, rolling and transendothelial migration in multilayer PCPB as compared to traditional TEM assay.
 
R21 CA132039-01 2008 NELSON, EDWARD L UNIVERSITY OF CALIFORNIA IRVINE Application of a Novel Nanotechnology for Molecular Profiling of Tumor Cellular E
Our increasing appreciation of 1) tumor genetic and cellular heterogeneity, 2) the recent descriptions of cancer stem, endothelial progenitor, & myoepithelial cells, among others, and 3) disparate responses to treatment even for histologically similar tumors; raise fundamental questions as to the relative contributions of various tumor cellular subsets to the biologic behavior of a tumor. However, technologies permitting the prospective characterization of discrete tumor cellular elements and recovery of selected viable cells from a tumor have yet to be developed. We have developed a novel nanotechnology consisting of an array of microfabricated SU8 polymer elements that permits the isolation and recovery of individual adherent cells. This advanced nanotechnology combined with multicolor immunofluorescence and advanced confocal microscopy enables us to propose application of this technology to the simultaneous identification, recovery, and evaluation of selected molecular profiles from viable primary adherent cell populations representing the various cellular elements within individual tumors. The HYPOTHESIS for these studies is that the pallet array nanotechnology will permit identification, enumeration, and recovery of the following individual cellular tumor elements: cancer stem, endothelial progenitor, myoepithelial, epithelial, and inflammatory cells leading to the molecular characterization of these cellular subsets within individual tumors and will be tested by pursuing the following Specific Aims: AIM #1. Refine the pallet array for maximum cell capture, detection, and recovery of tumor cellular elements. AIM #2 Apply pallet array to Fine Needle Aspirate (FNA) samples of primary breast tumors to identify and isolate individual cells from discrete tumor cellular elements. AIM #3 Establish feasibility of molecular analysis of recovered individual rare cells, e.g. single cell RT-PCR. These studies represent a convergence of biomedical engineering, advanced laser optics, cell biology, immunology, and clinical oncology and will drive future studies to address fundamental biological and clinical questions.
 
R21 CA125336-01A2 2008 NOLTE, DAVID D PURDUE UNIVERSITY WEST LAFAYETTE Multiplexed Assays on the BioCD for Acute Lymphocytic Leukemia
The BioCD is an emerging label-free assay technology with potential for high multiplexing and high throughput to screen for many analytes across many samples simultaneously. The basis of the BioCD technology is rapid optical interferometric scanning. Interferometry is the most sensitive and quantitative means of direct optical detection. It is faster than fluorescence, with better signal-to-noise, and it requires no labels, which is essential for multiple analyte detection. The broad, long-term objectives of this proposal are to apply the BioCD for the first time to the prognosis of cancer. The goal is to assay multiple biomarkers across a large cohort of 300 patients, at a level previously inaccessible to immunohistological arrays, to predict patient outcome in response to chemotherapy. The specific aims of this proposal are to establish standard response curves for a set of biomarkers relevant for predicting chemotherapy outcome for patients with acute lymphocytic leukemia (ALL), and to do a comparative study between assays performed on the BioCD and assays performed previously on a limited number of tissue microarrays. This is followed by the aim to scale up the capacity of the BioCD to screen for an expanded biomarker set across a set of samples already collected from the cohort of 300 patients with acute lymphocytic leukemia. The research design and the methods for achieving the stated goals rely on high- capacity automated protein spotters and high-speed laser interferometric readers. Standard concentration curves will be established using commercially available antigens in tissue lysates, followed by measurements of marker concentrations in healthy tissue lysate. These measurements set the gold standard against which the expanded marker set measured across the large patient cohort will be compared. The relevance of this research to public health is the expansion of the marker and sample base to establish stronger clinical confirmation of the prognostic value of inactivation of a molecular pathway in patients with standard-risk acute lymphocytic leukemia, and to establish the BioCD as a novel high-capacity resource for diagnostic and prognostic applications for cancer.
 
R21 CA128620-01A1 2008 RABINOWITZ, JOSHUA D PRINCETON UNIVERSITY Mass Spectrometry Methods for Probing Metabolic Dynamics in Normal & Cancer Cells
Many of the most clinically important chemotherapeutic agents inhibit the metabolism of tumor cells. Our overarching goal is to develop a complete and quantitative understanding of the metabolic differences between normal and cancer cells, and to use this knowledge to guide the rational design of novel anticancer regimens. To achieve this goal, we are developing methods that apply state-of-the-art liquid chromatography- electrospray ionization-triple quadrupole mass spectrometry technology to probe cellular metabolism in a dynamic, quantitative, and comprehensive manner. To date, we have succeeded in developing methods for measuring metabolite concentrations and fluxes from several microbes. Here we propose to extend these methods to enable reliable measurement of metabolite concentrations and fluxes in normal and cancer cells. Specifically, we aim to enable quantitation of the concentrations of at least 150 different known, structurally-defined intracellular metabolites. We also aim to measure, using isotopic tracers, the fluxes through central carbon, lipid, amino acid, and nucleotide metabolism. We will apply the analytical technology that we develop to map major metabolic differences between normal and cancer cells and to study the dynamic response of these cells to treatment with anti-metabolite anticancer drugs. The methods developed here will have long-term value for understanding the mechanism of action (and toxicity) of anti-metabolite anticancer drugs, for characterizing the metabolic differences between drug-responsive and resistant cancer cells, and for suggesting new approaches to inhibiting metabolism that will specifically kill cancer cells.
 
R21 CA131859-01A1 2008 REHEMTULLA, ALNAWAZ UNIVERSITY OF MICHIGAN AT ANN ARBOR HTS for FADD kinase inhibitors using molecular imaging
Using differential expression profiling, quantitative two-dimensional (2-D) gel electrophoresis and data mining we recently identified a new prognostic biomarker, Fas-associated death domain (FADD), which is overexpressed in a number of human malignancies such as lung, head and neck, brain and adult male germ cell tumors. Studies in lung cancer revealed that overexpression of FADD significantly associated with poor clinical outcome. Immunohistochemistry-based tissue microarray analysis confirmed the association between FADD over-expression and the poor outcome, and also revealed the presence of nuclear localized phosphorylated FADD (p-FADD). Tumors with increased p-FADD expression also showed elevated NF-?B activation. Taken together, published results from our lab and others suggest a causal relationship between the phosphorylation of FADD and NF-?B activation, a hallmark of an aggressive therapy resistant cancer phenotype. Thereby, we hypothesize that inhibiting FADD phosphorylation in tumor cells may sensitize cancer cells to chemotherapeutic agents. To aid in experimentation of this hypothesis we have resorted to molecular imaging tools and developed a pan FADD kinase reporter (FKR) which non-invasively senses FADD-kinase activity in real time. In Specific Aim 1, we will characterize the sensitivity and specificity of FKR. In Specific Aim 2A we will perform a high throughput screen to identify molecules from a diverse set of compound libraries that target FADD phosphorylation. Utilizing secondary screens with cells expressing either mutant FKR or luciferase, the toxic and less sensitive lead molecules will be eliminated. In Specific Aim 2B we will evaluate the relative efficacy of the candidate molecules by quantifying IC50 of the top leads. In Specific Aim 2C the specificity of candidate molecules in inhibiting FADD kinases will be investigated using western blotting and protein kinase arrays. The utility of these compounds and their derivatives in the treatment of cancers will be investigated in subsequent years.
 
R21 CA126716-01A1 2008 REVZIN, ALEXANDER UNIVERSITY OF CALIFORNIA DAVIS Microfabrication Tools for In Vitro Monitoring of Cancer Cell Metabolism
The physiological or pathophysiological state of cells and organs is reflected in their energy metabolism. For example, deviations in glucose metabolism is almost always observed in tumors. The energy metabolism of cancer cells differs strikingly from normal tissue with glycolysis and subsequent lactate production being much more prominent in cancer cells, even under aerobic conditions. Increasingly, therapeutic strategies for cancer treatment are designed to target metabolic deviations, making levels of energy metabolites such glucose or lactate potential correlates of drug efficacy. The goal of this proposal is to develop a lab-on-chip platform for in vitro monitoring of the effects of drug candidates on energy metabolism of cancer cells. The proposed platform will intimately integrate small groups of glioma (brain cancer) cells with miniature glucose biosensors in a microfabricated device, where the cellular microenvironment can be precisely defined and easily modulated. This platform will be used to: 1) establish tumor-mimicking microenvironment conditions (e.g. hypoxic, acidic, nutrient-limiting) in glioma cell cultures; 2) challenge the cells with pharmacological inhibitors of kinase signal transduction pathways central in energy metabolism; and 3) monitor local extracellular glucose levels. Thus the cell culture/biosensor platform will connect tumor microenvironment, energy metabolism and anti-cancer drug efficacy, and will help to delineate conditions making cancer cells more susceptible to therapy. The proposed technology will be a valuable tool for the development of therapeutic anti-cancer agents, and will help illuminate molecular aspects of metabolic adaptation of cancer cells.
 
R21 CA134386-01 2008 SZMACINSKI, HENRYK UNIVERSITY OF MARYLAND BALTIMORE Time-resolved fluorometric method for assay of multiple biomarkers
In this application we propose the development of technology with potential of at least 100-fold improved sensitivity over current fluorometric methods. Specifically we integrate a metal enhanced fluorescence (MEF) with time-resolved detection technique to obtain high sensitivity and ability for real time monitoring of biomolecular interactions. We propose to specifically integrate Metal-Enhanced Fluorescence (MEF) phenomena and time-resolved phase-modulation (PM) detection technique with surface-based assays to obtain high sensitivity and large analyte concentration range as well as to simplify the biochemical procedure. The new approach represents a significant advance on fluorometric analyses of biomolecule interactions, with sensitivity comparable to ELISA and a simplified sample procedure. Specifically, we will demonstrate the potential of MEF-PM technology using a panel of cytokines such as IFN?, TNFa, IL-5, IL-8, IL-16, VEGF, and RANTES. Currently, detection of multiple cytokines requires the use of the most sensitive detection technologies such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay and chemiluminescence because of their low concentration in human blood. Within projected work we will perform feasibility studies on reproducible fabrication of fluorescence enhancing substrates, functionalization and optimization of surface chemistry, performing feasibility on clinical assays that require high sensitivity and reduction in cost, and optimization of time-resolved detection modality, and integration of components into practical systems for clinical diagnostics and for research. This will be accomplished by (1) Developing a procedure for reproducible fabrication of substrates with metallic nanostructures. (2) Optimization of biomolecules immobilization on the surface of the MEF substrates. (3) Validation of MEF-PM method using a panel of cytokines with comparison with ELISA method. The proposed technology will meet requirements for high sensitivity (1-10 pg/ml), broad analytical range (4 - 5 orders of magnitude), ease of use, and versatility. MEF-PM will be of broad use in basic and cancer research applications, and will provide a tool for proteomics, bioassay developments and clinical diagnostics.
 
R21 CA128628-01A1 2008 TURCHI, JOHN J INDIANA UNIVERSITY-PURDUE UNIVERSITY AT INDIANAPOLIS Development of methodologies for the analysis of DNA repair capacity to predict t
Following a cancer diagnosis, determining the best course of treatment is of paramount importance. Along with recent advances in understanding the biology and pathways involved in the initiation and progression of certain cancers have come advances in individualizing treatment based on the molecular analyses of these pathways. The most convincing case involves analysis of breast cancer to determine which individuals will most likely require and benefit from adjuvant therapy. Expanding this type of analysis to other cancers holds the promise of similarly impacting cancer therapy. Considering numerous very effective therapies, including cisplatin, induce DNA damage one pathway that is directly related to how individuals respond to certain therapeutic treatments is DNA repair. In the context of cisplatin based cancer chemotherapy, reduced DNA repair capacity is associated with increased sensitivity, while increased repair activity is associated with resistance. The goal of the research described in this application is to develop methodologies to accurately determine DNA repair capacity in cancer tissue, focusing on the nucleotide excision repair (NER) pathway. The NER pathway is also responsible for removing DNA damage resulting from exposure to a variety of insults including cigarette smoke. Our hypothesis is that reduced DNA repair capacity increases the risk of smoking induced carcinogenesis and also contributes to the dramatic initial tumor regression often observed upon administration of cisplatin based therapies for treating lung cancers. The relatively short-lived response and subsequent resistance severely limits the utility of platinum based therapies. Our hypothesis is that the observed resistance is impacted by increased repair in the resistant tumors. To further test these hypotheses an accurate measure of DNA repair activity is required. Measuring gene expression or protein expression, while useful, does not always correlate with bone fide NER repair activity. Numerous NER proteins are regulated not only at the level of mRNA or protein expression, but also by posttranslational modification and protein-protein interactions. Therefore this application focuses on the development of novel methodologies to determine the extent of specific posttranslational modifications of key NER proteins and actual repair activity.
 
R21 CA120742-01A2 2008 WANG, TZA-HUEI JOHNS HOPKINS UNIVERSITY Nanobiosensing Method for Point Mutation Detection of Cancer
Detection of point mutations in tissues and body fluid DNA have wide-spread implications in studying molecular etiology of cancer as well as in developing new technologies for future clinical applications. In this application we propose to develop a clinically relevant genetic analysis technology that enables multiplex detection of point mutations in unamplified genomic DNA using limited amounts of clinical samples. This amplification-free detection technology will be developed using a combination of two innovative technologies, single-molecule detection (SMD) and quantum dot (QD)-mediated fluorescence resonance energy transfer (qFRET). Preliminary studies have yielded promising results indicating that this integrative SMD-qFRET technology is able to detect DNA targets at extremely low concentrations (~ 5 fM), obviating the need for target amplification. When incorporated with allele-specific oligonucleotide ligation, this technology can enable detection of low- abundance point mutations in unamplified genomic DNA. This project consists of three Specific Aims. First, we will develop an amplification-free point mutation detection method and evaluate it by analyzing four representative point mutations in the KRAS gene (at codon 12 and codon 13) and one commonly occurring mutation in the BRAF gene (at codon 599) in unamplified genomic DNA from ovarian serous tumors. Second, we will enhance the sensitivity and resolution of this new method to 0.5 fM and 0.5 % (mutant/wild-type ratio of 1:200) respectively by optimizing both the design of the QD-mediated fluorescence energy transfer system and the ligation reaction conditions. Third, we will increase the analysis throughput and mass detection efficiency of the assays by implementing this new detection method in a multiplex, microfluidic format. We will design and fabricate a microfluidic array device and use it to dispense and guide micro-volumes of genomic DNA samples for multiplex analysis using SMD spectroscopy for seven mutation assays simultaneously. It is expected that, as compared to conventional PCR-based mutational analysis, this new technology will provide a more rapid and reliable measure in detecting point mutation using a 5 ?l or less assay volume. If successfully established, it could provide a relatively straightforward molecular diagnostic platform for cancer detection and can potentially be performed in many laboratories and clinical settings.
 
R21 CA132815-01 2008 WEIER, HEINZ-ULRICH G. UNIVERSITY OF CALIFORNIA-LAWRENCE BERKELEY LAB Accelerating Cancer Research with Single Cell Arrays
This proposal addresses the sensitive detection of chromosomal changes such as small translocations, rearrangements or genomic imbalances in apparently normal individuals, benign neoplasia, premalignant lesions, and cancer. Current techniques for full karyotype analysis of individual cells require metaphase cells, and cells in interphase or non-viable cells cannot be analyzed. Many cells that can be obtained from human tumors are not in metaphase. The objective of the proposed research is the development of technologies to support the cytogenetic analysis of small amounts of fresh, fixed or archival tissues regardless of the cells' proliferative stage. A highly sensitive, fluorescence in situ hybridization (FISH)-based technology platform termed Single Cell Arrays (SCAs) will allow the detection of small rearrangements in interphase and metaphase cells by combining the high-resolution DNA in situ analysis with sensitivity in the kb range. This will be achieved by immobilizing cell nuclei on glass slides and controlled stretching of chromatin in specially designed micro-chambers followed by cytogenetic analysis using FISH. The Specific Aims of this R21 feasibility study are 1. Demonstrate the feasibility that interphase cell nuclei can be immobilized in a defined pattern and reproducibly extended for subsequent cytogenetic analysis. We will demonstrate the feasibility of preparing SCAs comprised of individual cell nuclei arranged in a defined pattern inside microscopic reaction chambers and elongated/stretched by a constant force. Importantly, the extent of chromatin stretching will be controlled by cell fixation and adjusting environmental parameters such as buffer, chamber temperature, and humidity, and the force applied to pull the chromatin. 2. Develop an optimized assay for the sensitive, high-resolution cytogenetic analysis of SCAs. We will develop a protocol for a FISH-based multi-locus cytogenetic analysis of SCAs. The assay is expected to provide near kilobase sensitivity for the detection of single copy nucleic acids with a resolution in the order of 10-20 kb, while minimizing the overall loss of DNA. The assay will be tested by analyzing SCAs prepared from different breast or thyroid cancer cell lines. SCAs will become powerful tools in basic and applied/clinical research, where chromosomal changes often affect a cell's phenotype and the fate of its progeny. In clinical practice, for example, such a sensitive assay may support cell classifications, thereby benefiting patients with de novo translocations or premalignant lesions as well as cancer patients. Furthermore, SCAs will allow the analysis of very small samples, regardless of their integrity or cell cycle stage. This will open new avenues for the analysis of small samples like those obtained by fine needle biopsies as well as the analysis of circulating or exfoliated tumor cells. Public health relevance statement: At present, no technology exists to prepare small samples of non-proliferating cells and screen them for karyotypic abnormalities. Highly sensitive, FISH-based assays termed Single Cell Arrays (SCAs) will provide a platform technology with which one can develop a multitude of tests tailored to specific diseases and cell or tissue samples. Due to their versatility, SCAs may become powerful tools in basic and clinical research, thereby benefiting patients with de novo translocations or premalignant lesions as well as cancer patients.
 
R21 CA126733-01A1 2008 WONG, DAVID T UNIVERSITY OF CALIFORNIA LOS ANGELES Collection, Stabilization and Storage of Saliva Samples for Cancer Research
The ability to diagnose cancer at an early stage will greatly enhance chances of treatment success and reduce the mortality and morbidity. Currently there is no molecular test that can diagnose or screen for oral cancer non-invasively. We were able to identify a 7-gene signature that can predict the presence of an oral cancer with an accuracy of 82%. Since 2004, the characterization and diagnostic use of human salivary RNA has been actively pursued in our laboratory. Our proposal, ""Collection, Stabilization and Storage of Saliva Samples for Cancer Research,"" responds to the RFA titled ""Innovations in Cancer Sample Preparation"" (RFA CA-07-037). We will develop optimal and standardized collection, RNA stabilization, storage and processing protocols to perform gene expression analysis of salivary mRNA. We propose three Specific Aims to address the multifaceted nature of this RFA. Aim 1 will establish optimal extraction methods for mRNA from saliva. In Aim 2 we will determine the intra-individual fluctuation of mRNA levels, define adequate endogenous transcripts for normalization and validate our oral cancer markers and additional new candidates in an independent cohort. In Aim 3 we will determine stability of saliva and expression patterns over periods of up to 6 months with different storage conditions and with stabilizing reagent. In all aims we will apply a new method for the multiplex reverse transcription and pre-amplification with subsequent quantitative PCR of a variety of mRNA transcripts endogenous to saliva. In addition, our set-up allows the implementation of several spike sequences that will yield important insights in the extraction and storage and are ideal controls for clinical application. We are confident that our research can be translated directly into a study for the large validation of oral cancer markers and spark the initiation of a multitude of projects and sample collections for the use of saliva as a diagnostic tool for oral and systemic diseases. Our studies will contribute to the advancement of human saliva as a clinically important body fluid for molecular diagnosis to improve cancer-directed health care.
 
R21 CA120693-01A2 2008 YANNELLI, JOHN R. UNIVERSITY OF KENTUCKY Cryopreparation of PBMC for Immunotherapy and Immune Assessment Studies.
There are many immunotherapy trials being conducted throughout the world ranging from vaccines to T cell transfer and antibody studies. In these trials, aside from the obvious need to assess clinical responses, Investigators must analyze changes in immune responsiveness which results from the immune intervention. Thus, there is a need to collect PBMC from patients at regular intervals both before and following the therapy. These PBMC are cryopreserved to save the PBMC phenotype and function until a later time point when the PBMC can be thawed and assessed to determine if the immunotherapy was effective. In addition, preparation of subsequent doses benefits from a source of readily obtainable PBMC which will respond to in vitro manipulation similar to the day the product was received by the lab. There are few standardized techniques available for cell cryopreservation as it relates to immunotherapy. Most labs have their own protocols which themselves often provide inconsistencies in cell viability and function upon thawing. It is also difficult to evaluate results of immunotherapy trials and make comparisons from lab to lab. The current proposal will focus on cryopreservation medium and compare static vs. controlled rate freezing techniques. In 4 Specific aims, we will examine: 1) optimum medium for cryopreservation. Human serum + DMSO will be compared to Plasmalyte-A, an FDA approved commercial serum free electroyte or rehydration fluid. The comparison of Plasmalyte A to serum is important because if it works, significant cost reduction will occur since the cost per liter is less than 1% of the cost of serum. In addition, this is an FDA approved product which is consistent from batch to batch. The second specific aim will evaluate different methods of thaw. Specific Aim 3 will compare static freeze techniques to controlled rate freezing. The fourth specific aim will monitor Specific aims 1 and 3 by comparing PBMC subset function at various time intervals using assays of cellular immunity. The goal of the proposal will be to develop a strategy for peripheral blood mononuclear cell (PBMC) cryopreservation which can be more UNIVERSITYersally applied. A more standardized approach will allow a more accurate assessment of immne responsivenss which can be compared between studies in different centers. In addition, other clinical studies utilizing PBMC can also benefit from such a study (studies of HIV and transplantation for instance). The 4 specific aims will be done over a two year period of time.
 
R33 CA128625-01A1 2008 HAHN, WILLIAM C DANA-FARBER CANCER INSTITUTE Integrated genomic approaches to identify and validate cancer targets
Most human tumors, particularly those derived from epithelial cancers, exhibit global genomic alterations that make it difficult to identify mutations critical for cell transformation and to define the consequences of specific cancer-associated mutations. Recent advances in technologies to identify structural changes in human cancers now make it possible to consider enumerating all of the genetic alterations harbored by a particular tumor. Despite these advances in annotating structural alterations in cancer genomes, identifying the genes targeted by specific amplification or deletion events and deciphering the function of targeted gene mutations remains a major challenge. Indeed, the parallel development of efficient methods to annotate the function of cancer-associated genes is necessary to distill validated cancer targets from this structural description of cancer genomes. This proposal focuses on the integration of newly developed, high throughput methods to functionally annotate the cancer genome. Specifically, methods to perform large scale loss-of function, gain-of-function, and protein-protein network analyses will be combined in a novel integrated program to identify and validate functionally important cancer genes. Specifically, these studies build upon prior work by our laboratories to develop and implement genome scale RNA interference libraries, complete collections of human open reading frames (ORFs) and comprehensive protein-protein interaction maps. Although the basic tools required to perform large-scale studies are now available, the integration of such whole genome approaches represents an entirely new endeavor that requires the further development of these nascent technologies, the fabrication of comprehensive reagents and the creation of new ways to connect these datasets to achieve a scale beyond what has been previously performed. As such, the overarching goals of this R33 application is to apply these technologies in an integrated manner while simultaneously identifying and validating genes of particular promise for therapeutic targeting. The long-term goal of these studies is to provide a foundation for the expansion of these efforts at genome scale.
 
R33 CA132022-01A1 2008 NETTLES, KENDALL W SCRIPPS RESEARCH INSTITUTE Molecular Analysis of Steroid Hormone Receptors with X-ray Crystallography
The long-term goals of this project are to understand the regulation and role of steroid hormone receptors in cancer development and therapeutics, through our development of a new technology that allows for the rapid analysis of steroid hormone receptors using X-ray crystallography. Specifically, our approach has demonstrated that we can increase both the rate and numbers of hits with crystal structures by at least a thousand fold, and that this allows rapid analysis of the ligand-binding domain of these receptors bound to chemotherapy agents, and pathway selective compounds. The estrogen and androgen receptors (ER and AR) are implicated in the development, diagnosis, and treatment for breast and prostate cancer, respectively. Glucocorticoids have a broader role, as up-front therapeutics for the treatment of several malignancies (e.g., leukemia and hormone-refractory prostate cancer), and as adjuvants that reduce the side effects of other chemotherapy agents. The synthetic compounds that target these receptors have, however, significant problems, including acquired resistance and undesirable side effects. They also display tissue and pathway selective signaling that is poorly understood, at both the molecular and structural level. It is possible to develop tissue and pathway selective compounds that ameliorate some of these problems, but there is very little understanding of the structural basis for such selectivity. The lack of good structural models for tissue selectivity is due to the difficulty in producing crystal structures. The steroid receptor ligand-binding domain (LBD) has proven very difficult to crystallize, due to conformational heterogeneity and protein misfolding. Here we propose to further develop our new technology for molecular analyses of steroid receptors, which we strongly believe will revolutionize the use of X-ray crystallography in both basic research and drug discovery, especially regarding steroid receptors. Specifically, we have identified and generated a series of surface mutations that stabilize the estrogen receptor in the conformations seen with both agonist and antagonist ligands. This advance has allowed us to add compounds in parallel to the purified protein, and to obtain the first structure of an apo steroid receptor LBD. We propose to apply these techniques to apply this high-throughput technology to other steroid receptors implicated in cancer, and to use this approach to define the structural basis through which the glucocorticoid receptor (GR) inhibits the NF-?B oncogenic pathway. We believe that these studies will establish new and robust techniques that will revolutionize the use of X-ray crystallography in defining how small molecules control tissue- and pathway-selective signaling through steroid hormone receptors. This ""class analysis"" approach to studying groups of structures is highly novel, and allows for the incorporation of statistical power into structural analysis. Importantly, this approach will also directly impact the drug discovery process, by rapidly providing structural information that will guide the development of new therapeutics.
 
R33 CA126663-01A2 2008 PASQUALINI, RENATA UNIVERSITY OF TEXAS MD ANDERSON CANCER CENTER Integration of Vascular Genomics and Proteomics for Diagnosis and Therapy of Canc
The integration of transcriptional profiling, cancer cells targeting and molecular-genetic imaging into a single platform would have many biological applications and the potential to improve the practice of medicine. We have designed and validated a new hybrid viral system composed of genetic elements from adeno-associated virus (AAV) and phage (termed AAVP). These ligand-directed particles enable targeted systemic delivery and imaging of transgene reporters. Experimental non-invasive monitoring of reporter trans-activation, may be followed ex-vivo and in vivo. Targeted molecular imaging would represent a major advance in the management of prostate cancer. The overall goal in this project is to combine the homing and tumor transducing capabilities of AAVP with cancer-specific promoters, as a way to develop improved tools for tumor monitoring based on the transcriptional activity of selected markers. These promoters will be identified by the large-scale evaluation of the transcriptome of cancer cells and tumor vasculature, using extensive searches in public databases, as well as by the construction and large-scale sequencing of SAGE libraries, using modern low-cost high throughput pyrosequencing approaches. The identified upregulated genes will be validated in two independent sample sets, and the promoters of confirmed upregulated transcripts will be identified, certified and cloned into AAVPs. With the definition of a reliable set of genes we expect to have comprehensive panel of tumor markers covering most of the gene expression variability seen in distinct patient tumor samples. The concept of establishing a panel of multiple markers is in line with the much heralded era of personalized medicine, and should launch, for the first time, a system for imaging temporally and spatially, in vivo, the transcriptional profile within tumors. The combination of a vector displaying peptides designed to target tumors, which also carries a suicide/reporter transgene (HSVtk) under the control of a tumor-specific promoter should enable transcriptional imaging and tumor growth suppression in a very specific fashion. The imaging and treatment possibilities of this vector reinforce the idea of drug-diagnostic co-development that, if preceded by an individual evaluation of gene expression (in the urine of patients with prostate cancer, for instance), could lead to a personalized diagnosis/treatment based on the up-regulated markers of an individual patient. This personalized imaging test merged with a companion drug comes together with the drug-diagnostic co-development concept recently put forward by the US Food and Drug Administration. Under this vision, the transcriptional imaging provided by a specific tumor gene will also trigger tumor apoptosis, allowing imaging as well as treatment and monitoring of disease progression. Our Specific Aims are: (i) To identify and to validate transcripts upregulated in prostate cancer using large scale transcriptome analysis. (ii) To combine ligand-directed targeting of AAVP to transcriptional targeting, and (iii) To evaluate the efficiency of imaging in vivo the expression of prostate cancer specific transcripts by using transcriptome-directed promoters cloned into RGD/GRP78-targeted AAVP constructs. The concept of establishing a panel of multiple markers is in line with the much heralded era of personalized medicine, and should launch, for the first time, a system for imaging temporally and spatially, in vivo, the transcriptional profile within tumors.
 
R33 CA126659-01A1 2008 POOLE, LESLIE B WAKE FOREST UNIVERSITY HEALTH SCIENCES Proteomic Profiling of Cancer-Related Redox Signaling Pathways
It is widely appreciated that reactive oxygen species (ROS) play a major role in the initiation of cancer, and they are also implicated in many cancer therapies, such as ionizing radiation, cisplatin and taxanes. More recently, it has been discovered that cancer cells produce ROS as signaling molecules that promote proliferation. Unfortunately, the molecular details of how redox regulation affects cell signaling events are far from clear. New experimental and computational technologies that we have developed are uniquely suited to identifying the molecular targets that are modified by ROS, either as a result of ROS damage or ROS signaling. With the reagents and methods that we have recently developed, we can now evaluate the ""redox profile"" of cell populations by targeting uniquely reactive cysteine sulfenic acid (Cys-SOH) groups, the initial intermediates generated following reaction of activated protein thiolate groups with hydrogen peroxide and peroxynitrite (and perhaps other ROS). In this R33 application, our labeling technology will be further developed for quantification and multiplex analysis, so that it will have broad applicability in: 1) the investigation of basic mechanisms of ROS damage and ROS signaling; 2) molecular profiling to stratify patients with cancers that are sensitive to ROS-generating therapies; and 3) the development of novel cancer therapies based on the inhibition of ROS-dependent proliferative signaling. The following Specific Aims are proposed: 1) to develop reagents and methods of use for additional new, multicolor fluorescently-labeled Cys-SOH reagents for multiplex analysis of samples; 2) to develop quantitative mass spectrometry methods, which have some major advantages over gel-based methods (including direct readout of protein identity and numerous posttranslational modifications); and 3) to use the new quantitative methods to detect and identify Cys-SOH modified proteins generated during ROS-dependent signaling in HEK-293 cells and ovarian cancer cells. Taken together, the approaches developed in Specific Aims 1 and 2 will provide new tools for the research community to use to study the mechanisms of redox regulation and signaling. In Specific Aim 3, these tools will be used to determine the targets of ROS in the regulation of cell proliferation and apoptosis. First we will continue our study of NF-?B regulation in HEK-293 cells in response to cytokine (TNF-?) and tumor promoter (TPA) stimulation. Second, we will use ovarian cancer cells treated with cisplatin or taxane to determine which protein oxidations are critical to regulating survival and apoptosis. Besides providing specific information about the mechanism of redox regulation and signaling, these biological experiments will allow us to further refine our reagents and methods to make them most useful to the cancer biology community. These approaches to detecting functional oxidative modifications to cellular proteins hold promise in identifying specific protein targets that mediate the actions of anticancer drugs, e.g. through their effects on cell cycle arrest, cell division or apoptosis. An outgrowth of these studies could also be the development of new anticancer drugs and the ability to predict efficacy of a given drug in the treatment of individual patients.
 
R33 CA132073-01 2008 VIDAL, MARC DANA-FARBER CANCER INSTITUTE Application of Technologies for Interactome Network Analyses of Cancer Mutations
The molecular mechanisms underlying cancer have been mainly studied one or a few ""cancer genes"" at-a-time. However, it is thought that combinations of mutated or aberrantly expressed tumor suppressor- and oncogenes may be responsible for advancing cells through most steps of tumorigenesis. Many cancer causing mutations are disrupting interactions and these alterations are often directly related to the mechanism of pathogenesis. Thus, altered protein-protein interactions may directly point to a mechanism for cancerogenesis. More importantly, since it is becoming increasingly clear that genes and their products interact in complex biological networks with local and global properties, it is possible that perturbations of these networks contribute to cancer formation. We propose that a further understanding of the mechanisms involved in cancer, and the development of new therapeutic strategies, can be gained by i) studying genes and their products in the context of the molecular networks in which they function, and ii) investigating how such networks are altered in tumor cells compared to their unaffected counterparts. In addition to the information available from several drafts of the human genome sequence, genome- wide experimental strategies have been developed that will help us understand the effects of cancer mutations in the context of molecular networks: i) protein-protein and DNA-protein interaction networks or ""interactome"" networks are being mapped at an increasing pace, producing datasets with ever increasing quality and decreasing costs, and ii) large numbers of cancer-associated mutations are being discovered in the context of the human cancer genome project. Here we propose to develop a genome-wide application for a new technology platform that we have recently initiated to systematically study the effects of cancer-associated mutations on the physical and functional interactions mediated by the products encoded by cancer genes in the context of global interactome models. Our specific aims are to apply our experimental and computational technology platforms to: i) clone large numbers of cancer-associated missense or single amino acid change (SAC) alleles, ii) identify and characterize the interaction properties of large numbers of SAC alleles, iii) analyze the effects of SAC alleles on the local and global properties of interactome networks.