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Project # Year
of Award
PI Name(s)
Institution Title
Abstract Text (Official)
R21 CA125284 2007 ANDERSON, KAREN S YALE UNIVERSITY UNIVERSITYersal Technology for Profiling the Dynamics of Normal & Oncogenic Signaling
Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) are essential in the initiation of many protein signaling pathways. Dynamic control of multiple phosphorylation modifications of a single RTK thus can manifest critical control on multiple signal transduction pathways. Alterations of this sensitive dynamic in growth factor networks and aberrant activities of RTKs often have severe biological consequences and are linked to oncogenic processes in many human cancers. Indeed the success of tyrosine kinase inhibitors such as Iressa(tm) and Gleevec(tm) heralds a recent strategy of precisely targeted cancer therapy. An understanding of the molecular mechanisms of these early dynamic events may hold the key to understanding and predicting the nature of oncogenic behavior and for predicting the affects of RTK targeted therapy. An increased understanding of the early temporally regulated states will enable more precise strategies for selectively targeting downstream pathways and may offer a unique approach to cancer therapy based on dynamic rather than static intervention. An integrated platform of novel technologies and approaches are needed to provide temporal resolution of these rapid, early events at a molecular level both in vitro as well as in cell culture. This R21/R33 combined proposal will create a validated general set of innovative and established technologies including rapid reaction methodologies, a novel time-resolved electrospray ionization mass spectrometry (ESI-MS) technique, nanospray ESI-TOF, phosphopeptide mapping using ESI-MS LC/MS/MS and site-specific phosphotyrosine antibody detection that will be applicable for the analysis of a wide range of RTKs, their autophosphorylation patterns and downstream signaling events in the critical subsecond to multisecond time domain. We have chosen a prototypical RTK, epidermal growth factor receptor tyrosine kinase (EGFR) to develop this platform. The epidermal growth factor receptor (EGFR) tyrosine kinase pathway is linked to a large number of cancers and an important molecular target for targeted cancer therapeutics such as the small molecule ATP mimetic Iressa (gefinitib) that has recently been approved by the FDA and underscores the generation of specific kinase targeting as a new paradigm for cancer therapy. The ability to probe the molecular and temporal details of the earliest events of autophosphorylation will reveal signature patterns that will provide a new understanding of the differences between normal and oncogenic forms of EGFR and an expanded functional understanding of the emerging therapeutic class of targeted kinase inhibitors. We believe that a profile of the temporal dynamics of phosphorylation in signaling provides a unique molecular fingerprint or signature for distinguishing normal and cancer cells and the responsiveness to targeted inhibitors. New experimental tools and technologies are needed to distinguish cancer cells from normal cells at a molecular level and evaluate the effectiveness of new cancer therapies. This R21/R33 project describes a strategy to develop novel technologies that will allow us to understand how changes occur at a molecular level in a cancerous cell and a more detailed understanding of how new selectively targeted cancer therapies work.
R21 CA125280 2007 ANDREEV, OLEG A UNIVERSITY OF RHODE ISLAND New Technology for Selective Delivery of PNAs in Cancer Cells In Vitro and In Viv
Many cancers arise from the gradual accumulation of genetic changes in cells. Gene therapy approaches as well as techniques for recognizing cancer cells with abnormal genes or elevated levels of certain mRNAs include the design and delivery into cancerous cells of antisense and antigene oligonucleotides or their synthetic mimics such as peptide nucleic acids (PNAs). PNAs are highly stable, resistant to nucleases and proteases, and bind RNA and DNA targets in a sequence-specific manner with high affinity. One of the main obstacles for gene therapy is a lack of technology for selective delivery of gene agents into cancer cells in vivo. Here we propose a new technology for selective delivery into cancer cells of PNAs targeting mRNAs involved in tumor growth and metastasis. It is well established that tumors develop a hypoxic and acidic extracellular environment, especially in the earlier stages. We designed a short peptide that is soluble in water and able to insert into the membrane as a transmembrane alpha-helix at low pH (< 6.5) but not at normal pH (7.4). The peptide acts as a nanosyringe: it inserts in the membrane at low pH, translocates and releases in the cytoplasm various molecules, including dyes, toxins, and PNAs (Reshetnyak et al., PNAS, 2006, 103, 6460). The fluorescent PNAs are translocated into cells and stain the cytoplasm and nuclei. The mechanism of translocation of pH Low Insertion Peptides (pHLIPs) is based on a protonation of two Asp residues in the transmembrane domain, and this mechanism is fundamentally different from all reported peptide delivery agents. Whole-body imaging revealed that fluorescent pHLIPs accumulate in tumors in mice. The accumulation in tumors occurs because pHLIPs insert in the membrane at low pH while they interact only weakly with the surfaces of cells in tissues at normal pH. The replacement of two Asp residues by Lys or Asn residues eliminates the ability of pHLIPs to accumulate in tumors, which confirms the proposed mechanism of insertion of pHLIPs into cells. Our goal is to develop this nanosyringe technology for selective intracellular delivery of antisense and antigene PNAs into cancer cells in vitro and in vivo. We plan to conjugate various PNAs via disulfide linkages to the end of the peptide that inserts inside a cell. The efficiency of translocation of PNAs mediated by pHLIPs will be tested on different cell lines in vitro and in vivo in mice using fluorescence microscopy, flow cytometry, spectroscopy, whole-body imaging, and by measuring of level of expression of target proteins and rates of cell proliferation and tumor growth. The pHLIP nanosyringe could be a very effective tool for molecular analysis of cancer cells and diagnosis and treatment of cancer.
R21 CA122672 2007 BEEBE, DAVID J UNIVERSITY OF MICHIGAN AT ANN ARBOR Microfluidic Channels for High Density, High Performance Culture Assays
Researchers have demonstrated the potential utility of microfluidics for cell biology and its ability to define the environment. However, the use of microfluidics by biologists is still the exception. With the potential benefits of microfluidics clear, why have microfluidic systems not found more widespread use in cancer research and drug screening applications? Cancer is a complex disease and research into the basic mechanisms of the disease could benefit greatly from the ability to explore more factors more quickly and to utilize functionality (e.g., defined co-culture, precise temporal/spatial environmental control) that is not possible in traditional well culture. We believe there exist several barriers preventing microfluidic culture systems from having a major impact on cancer biology (accessibility, relevance, biological model, efficiency/throughput). Our broad aim is to bridge the gap between the worlds of engineering and cancer biology by understanding the needs/limitations of current cell-based cancer biology research and then providing a technology platform well matched to those needs and capable of addressing emerging needs (e.g., co-culture, 3D culture). Our innovative platform merges simple microfluidic technology (e.g., passive pumping) with existing pipetting methods to provide a range of cell manipulation functionality capable of highly parallel operation. We propose to demonstrate the accessibilty/ease of use of the platform, achieve measureable outcomes in throughput, efficiency, accuracy and robustness, and utilize the system to study growth regulation of mammary epithelial cells. Specific experiments include operational robustness, cell seeding consistency and assay execution. The biological focus of this project will be an extensive 2016 datapoint study exploring the response of the NMuMG cell line and primary mouse mammary epithelial cells (normal or lacking Sdc-1) to a variety of growth factors (EGF, IGF, FGF, insulin, FGF+TGF(3, serum, WntSA) at different concentrations. In addition, we will demonstrate a novel liquid valve to control soluble factor interactions between two cell types to study EMT thresholds. As part of these studies, we will demonstrate a 25X reduction in the number of animals required for typical 24 well plate cell-based studies.
R21 CA122884 2007 BIEBERICH, CHARLES J. UNIVERSITY OF MARYLAND BALTIMORE An Innovative Approach for Profiling Protein Kinase Substrates
Signal transduction is a central component of nearly all biological processes. Protein kinases play critical roles in signaling pathways by phosphorylating proteins involved in signal amplification and in executing the cellular response to extrinsic or intrinsic stimuli. Aberrant kinase signaling plays a role in the etiology of nearly all cancers and in hundreds of other diseases. Systematic approaches to the elucidation of the signaling pathways driven by kinases have the potential to illuminate new points of therapeutic intervention. The identification of physiologic kinase substrates is an important component of this endeavor. Currently, there is a need for technologies that can be widely deployed to approach this problem. We are developing a simple, straightforward technique for kinase substrate profiling that we term the reverse in-gel kinase assay. This assay enables the discovery of kinase substrates in whole or pre-fractionated complex biological extracts. If properly developed and validated, this assay has the potential, through the concerted effort of multiple laboratories, to enable rapid identification of potential physiologic substrates of many kinases.
R21 CA126700 2007 DING, ZHIYONG UNIVERSITY OF TEXAS MD ANDERSON CANCER CENTER Effective Mammalian Two Hybrid Screening Approach
This application, Effective Mammalian Two Hybrid Screening Approach, is in response to RFA-CA-07-001. Identification of novel protein-protein interactions is a fundamental step to understanding protein function and signaling networks allowing efficient implementation of targeted cancer therapy. The majority of protein-protein interactions are currently identified using yeast two hybrid (Y2H), co-immunoprecipitation and mass spectrometry or protein libraries. Each of these approaches has its own set of major limitations, failing to mimic native physiological conditions (Y2H and protein libraries) or to efficiently identify protein interactions on the cytoskeleton or membrane, either due to the location of the interaction (Y2H) or due to difficulties in co-immunoprecipitation of cytoskeletal or membrane proteins. Furthermore, conventional Y2H approaches yield false positive signals with transcription factors precluding screening. Therefore, a novel screening method that efficiently identifies biologically-relevant protein interactions, bypassing the limitations of present screening methods, would have wide applicability. We propose to develop and validate a readily applicable, context-dependent, subcellular localization-, cDNA library- and cell type-independent retrovirus-based mammalian two hybrid (ReMTH) screen method for identification of novel protein-protein interactions, including cytoskeletal and membrane proteins, in allowing native protein folding and post-translational modifications in mammalian cells. The resultant cells will be reagents for the study of the localization and function of the novel protein-protein interaction complex as well as resources for high content drug or siRNA screening. The fully developed technology will identify functional protein-protein interactions more efficiently than present methods and identify interactions not discoverable by present methods, particularly in context-dependent mammalian screens. Furthermore, the proposed ReMTH screen has the unique potential to stabilize or trap transient/weak interactions such as enzyme/substrate interactions, allowing identification of components of signaling pathways and networks in cancers previously undetectable. We have completed an initial proof-of-concept screen in HeLa cells for identification of interaction partners of the oncogene AKT1. We identified a series of previously known AKT1 interaction partners and substrates, as well as novel interaction partners, including cytoskeleton and membrane proteins. We have confirmed that one novel interaction partner, ACTN4, interacts physically and functionally with AKT1. Thus the technology will uncover functional protein-protein interactions not detectable by other approaches and advance our understanding of protein functions and signaling networks in cancer.
R21 CA120566 2007 HUANG, FAQING UNIVERSITY OF SOUTHERN MISSISSIPPI Folate Receptor-Mediated siRNA Delivery to Cancer Cells
Posttranscriptional gene silencing by small interfering RNA (siRNA) has evolved into a powerful tool for down regulation of any target gene(s) with both high efficiency and sequence specificity. In principle, siRNA may become the basis for developing the next generation of antiviral and anti-cancer agents with high potencies and low side toxicities. However, no therapeutically acceptable delivery methods of siRNA are currently available. Although folate receptor (FR)-mediated delivery of functional agents by endocytosis to cancer cells has been shown to be efficient and highly specific towards FR-positive cancer cells, the chemistry of direct coupling between folate and siRNA has not been readily achievable until now. Capitalizing on the recent RNA bio-conjugation methods developed in the Huang (PI) laboratory, this proposed research will develop a novel folate receptor-based siRNA delivery strategy against specific target genes in FR-expressing cancer cells. Integrating the recent advances in such diverse areas as FR-mediated drug delivery and RNA interference (RNAi) with the expertise of the PI in chemistry/RNA and Dr. Quo (co-Pi) in cell biology, we will synthesize folate-conjugated siRNA and investigate their cellular delivery and functions, targeting a cancer cell marker, urokinase plasminogen activator receptor (uPAR). We will test the hypothesis that down- regulation uPAR expression in cancer cells by floate-conjugated siRNA will effectively inhibit the cellular activity of uPAR associated with tumor growth. Both the delivery efficiency and specificity of Folate-siRNA will be analyzed using KB cancer cells (a human nasopharyngeal epidermoid carcinoma cell line). RNAi effects will be assessed by determining the expression of uPAR at mRNA and protein levels and by cellular functional analysis of uPAR. We expect that the delivery of Folate-siRNA against uPAR in KB cells will be more efficient and specific than other current available methods. As a consequence, tumor growth suppression through uPAR silencing is expected. Results from the proposed research will likely lead to the development of general strategies and methods for FR-mediated delivery of siRNA against specific target genes in FR-expressing cancer cells and, therefore, may lead directly to cancer therapeutic applications.
R21 CA125337 2007 KAUFMAN, DAVID G. UNIVERSITY OF NORTH CAROLINA at CHAPEL HILL Identification of Areas of Oxidative Damage in Human Genomic DNA
While considerable attention has been given to mapping sites of carcinogen-induced mutations in a number of cancer related genes, less is known about the distribution of the original DMA damage that might have given rise to mutagenic lesions. The goal of this R21 project is to develop and validate methods that would detect sites of DNA damage and allow us to determine the distribution of such lesions both globally and at specific locations in genomic DNA. It is our hypothesis that the distribution of DNA damage is not random, but is influenced by the structure and functional properties of genomic DNA. To test this hypothesis, we need to demonstrate that the methods that we are developing will enable us to visualize the distribution of DNA damage sites and relate the damage sites to specific genomic locations. Once we have demonstrated the general feasibility of this approach, we will perform experiments to evaluate the distribution of DNA damage in specific functional targets. We have demonstrated the ability to perform each of the following research steps necessary for the proposed analysis: (1) We have been able to prepare DNA for fiber analysis using molecular combing; (2) We localized a specific DNA sequence among an excess of genomic DNA using fluorescence in situ hybridization (FISH) on combed DNA fibers; (3) We visualized the distribution of apurinic/apyrimidinic (AP) sites in DNA using either an aldehyde reactive probe tagged with biotin or a fluorescent probe; (4) We were able to visually identify AP sites within regions undergoing replication; and (5) We identified specific regions undergoing replication (all described in the Preliminary Studies section). In this study, we propose to show that it is feasible to combine these techniques in a novel way to establish whether certain areas of the genome, for example sites that are being replicated, are more sensitive to DNA damage than other genomic regions (or, the same sites in the absence of DNA replication). To accomplish this goal, we will determine the sensitivity of detection of AP sites in combed DNA using DNA fibers with a known number of AP sites. We will then determine the background distribution of AP sites in untreated and treated DNA. Finally, we will determine whether it is possible to detect visual signals from all three techniques (AP sites, FISH, DNA counterstain) in the same experimental samples. ASSESSMENT:
Gene profiling technology has enabled analysis of the transcriptome and proteome of tumor cells. This information has provided useful information with regard to molecular mechanisms that define the enhanced survival and proliferation of MM cells. However, an equally, if not more important, goal is to define those proteins that participate in signaling pathways active in MM cells and their supporting stroma. Enzymes that phosphorylate tyrosine, serins and threonine residues on other proteins play a major role in signaling cascades that determine cell cycle entry and survival in MM and the stromal cells that support them. In particular, knowing the signaling pathways that are active in MM cells and their supporting stroma will provide critical information for understanding MM cell survival in the BM. We have developed and are applying to purified cells a novel array-based strategy that allows the simultaneous detection of phosphorylation for 1152 different kinase substrates. Here we propose to apply this emerging technology to the analysis of phosphorylation-based cell signaling pathways in MM and their supporting stroma. This R21/R33 Phased Innovation application will be pursued in two phases. In the R21 phase of this application Aims 1 and 2 will validate that PepChip technology can be applied to MM cells and their microenvironment to reveal signaling alterations in MM. In Aim 3 of the R33 phase we will use PepChip technology to identify kinome alterations within the MM patient population that are correlated with clinical parameters such as relapse and chromosomal abnormalities associated with poor prognosis. In Aim 4 we will utilize an in vivo model that supports the growth of primary patient isolates in RAG2XGammaC mice to determine the effect of therapeutics on the kinome of MM cells. This study will be pursued in the following.phased R21/R33 format: R21 Phase: Aim 1: Define the kinome of MM cells and normal plasma cells. Aim 2: Define differences in the microenvironmental kinome of MM and normal BM. R33 Phase: Aim 3: Identify kinome alterations in MM correlated with clinical parameters of disease. Aim 4: Identify kinome alterations in MM cells in response to therapeutics in vivo.
Gene profiling technology has enabled analysis of the transcriptome and proteome of tumor cells. In fact, massively parallel analysis of the Non-Hodgkin Lymphoma (NHL) transcriptome has revealed discrete gene profile signatures for the major subtypes of NHL, diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). This information has provided useful insights into molecular mechanisms that promote enhanced survival and proliferation in NHL. However, an equally, if not more important goal, is to define those proteins that participate in signaling pathways active in lymphoma cells and non-malignant cells that infiltrate these tumors. Enzymes that phosphorylate tyrosine, serine and threonine residues on other proteins play a major role in signaling cascades that control cell cycle entry, survival, angiogenesis and the immune response. Defining how these signaling pathways are altered in NHL B cells and non- malignant cells present in these tumors will provide critical information for understanding how lymphoma survives, proliferates and interacts with other cells at the tumor site. We are applying to purified tumor cells a novel array strategy that allows the simultaneous detection of phosphorylation for 1152 different kinase substrates. Here we propose to apply this emerging technology to the analysis of phosphorylation-based cell signaling pathways in NHL. This proposal will be pursued in two phases. The R21 phase will consist of two aims that will validate PepChip technology can identify kinome alterations in primary DLBCL (Aim 1) and FL (Aim 2) isolates. The R33 phase will consist of two aims that will define kinome alterations in DLBCL (Aim 3) and FL (Aim 4) that correlate with clinical parameters. In Aim 5 we will utilize in vivo models of FL and DLBCL to determine whether inhibitors specific for deregulated kinases inhibit growth of NHL tumors in immunodeficient mice. This study will be pursued in the following phased R21/R33 format: R21 Phase: Aim 1: Define the kinome of DLBCL B cells and non-malignant cells present in these tumors. Aim 2: Define the kinome of FL B cells and non-malignant cells present in these tumors. Aim 3: Identify kinome alterations in DLBCL correlated with clinical parameters of disease. R33 Phase: Aim 4: Identify kinome alterations in FL correlated with clinical parameters of disease. Aim 5: Determine whether deregulated kinases contribute to NHL growth in vivo.
R21 CA125510 2007 LINDSAY, STUART ARIZONA STATE UNIVERSITY-TEMPE CAMPUS Mapping epigenetic modifications at the nanoscale: Aptamers for microscopy
We propose to develop a technique for mapping post-translational chemical modifications of histones in chromatin by direct imaging of single molecules. Our approach is to use a new imaging mode for the atomic force microscope (AFM) that allows chemical composition to be read directly and at the same time as a high resolution molecular image is acquired. In this way, we will observe both the pattern of post-translational modifications, such as acetylation, and the consequent local modifications of chromatin structure. The mode requires that a cognate ligand be tethered to the AFM probe. Antibodies have not proved adequate for this purpose, so we propose to develop synthetic ligands that are (a) highly reproducible (b) chemically simple and stable and (c) capable of recognizing small chemical modifications. Specifically, we propose to: 1. Develop DNA aptamers that are highly specific for acetylated histones. 2. Test the new aptamers in the simultaneous topography and chemical imaging mode of the AFM ('recognition imaging') using artificial acetylated histone H4 arrays developed in the Peterson lab at the UNIVERSITY of Massachusetts Medical School. 3. Carry out an initial recognition imaging study of promoter chromatin extracted from a mouse cell line at the Georgel lab at Marshall UNIVERSITY. The proposed work will pave the way for a more ambitious project aimed at analyzing chromatin from one (or a few cells) by exploiting the very small sample requirements of atomic force microscopy.
R21 CA125693 2007 LINK, DARREN R RAINDANCE TECHNOLOGIES, INC. Exon Specific Sequencing of Whole Genomic DNA
The long-term objective of this research is to provide a means by which to sequence 10OOs of different exons simultaneously from a genomic DNA sample with 30 to 50 times coverage of each exon. This will provide researchers with significant new information about the genetic makeup and diversity of pre- cancerous, early-cancerous, and cancerous cells. RainDance Technologies (Guilford, CT) has developed microfluidic technology to manipulate sub-nanoliter volumes of reagents at rates exceeding 10A9 droplets per day. We propose to use this technology to process genomic DNA for the sequencing of 1000s of exons from individual samples. We will use a single microfluidic chip for multiple processing steps. The first on-chip processing step is to combine genomic DNA on a droplet-by-droplet basis with a member of a library of 1000s of primer pairs at rates of 1000s per second. The primer pairs ensure that a single exon is amplified in a given droplet during a polymerase chain reaction (PCR) step; the product of this step is captured on a bead for sequencing. The final on-chip step is a quality control step where beads with less than 10 million copies of an exon are removed before the remaining beads are sent for sequencing on the 454 Life Sciences (Branford, CT) instrument. Successful completion of this project will allow one pooled-primer-pair-library emulsion to be used to economically sequence thousands of individual exons from an almost unlimited number of genomic, (including tumor), DNA samples.
R21 CA126761 2007 MUNN, LANCE MASSACHUSSETS GENERAL HOSPITAL Microfluidic Device for Isolating Rare Blood Cells
Isolation of rare cells from peripheral blood is a difficult and tedious step in the diagnosis and treatment of cancer. Examples include leukocytes, which are of interest in leukemia, circulating endothelial cells, which can serve as a surrogate marker for solid tumor treatment, and circulating cancer cells, which may provide a tool for diagnosis and prognosis. In most patients, each of these cell types is outnumbered by red blood cells by a factor of at least 1000. Therefore, isolation of these cells from a sample of whole blood is the required first step of many clinical and basic research assays. We recently described a microfluidic device that takes advantage of plasma skimming and leukocyte margination - intrinsic features of blood flow in the microcirculation - to enrich nucleated cells such as leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels manufactured using standard photolithography and silicone molding techniques, and requires only pressure-driven flow to operate. Its initial channel is designed to enhance lateral migration of spherical cells, which, once near the wall are easily extracted through small extraction channels. In our preliminary design, a single pass through the device produces 34-fold enrichment of the leukocyte-to-erythrocyte ratio. We propose to further develop the microfluidics to provide simple, efficient and inexpensive technology for use as an initial stage in lab-on-a-chip analyses. Its integration into microanalytical devices that require rare cell enrichment will provide less expensive, more reliable, clinical assays that are also convenient and portable for point-of-care testing. Specifically, we will maximize the purity and efficiency of nucleated blood cell isolation and compare the performance with traditional separation techniques. When fully-developed, this technology will be a necessary and integral component of any microfluidic device analyzing nucleated blood cell populations by eliminating the need for preliminary blood processing steps.
R21 CA128696 2007 NILSEN-HAMILTON, MARIT IOWA STATE UNIVERSITY Drugcarts to Combat Drug Resistance
The current proposal focuses on the development of an aptamer-based reagent for prostate cancer therapy. The proposed reagent, called a Drugcart (Drug carrying aptamers for receptor targeting), is a unique design composed of aptamers with two binding specificities in a single stranded nucleic acid sequence. The Drugcarts will have the following functions: 1) encase and solubilize hydrophobic drugs, 2) target the drugs to the appropriate cells, 3) release the drugs at the target cell surface, and 4) pick up other drug molecules to be shuttled into the cell membrane. For this proposed work, we will link the PSMA aptamer to an aptamer that recognizes PD173955, a drug with similar actions as imatinib mesylate (Gleevec). PD173955 will be further modified to the form of a prodrug with covalently linked peptides that are susceptible to cleavage by the serine protease, prostate specific antigen (PSA). Thus, further specificity for prostate cancer cells will be achieved by the requirement for PSA to cleave the drug and make it permeable to the cell. To develop this promising new drug delivery molecule, we have assembled a team of experienced scientists with expertise in biochemistry, molecular biology, cell biology, cancer therapy, and organic synthetic chemistry. The team has already synthesized a PD173955 analog, isolated two PD173955-binding RNA aptamers, and is ready for the next stage of reagent development as defined by the following specific aims: 1 Synthesize PD173955 derivatives that can be cleaved by PSA to form a permeable PD173955 product. The synthesized derivatives of PD173955 will be tested for their cell permeability, susceptibility to cleavage by PSA, and increased permeability of the product PD173955 after PSA cleavage. 2 Create Drugcarts that bind PSMA and PD173955 for delivering PD173955 to cells. Stabilized RNA Drugcarts containing the PSMA and PD173955 aptamers will be synthesized. Binding activity and stability will be evaluated. 3 Establish that the Drugcarts specifically promote PD173955 uptake by cells that express PSMA on their surfaces. Optimized Drugcarts will be used to deliver the PD173955 prodrug to prostate cancer cells. The specificity of this delivery mechanism for cells that express PSMA and to promote cell death will be investigated.
R21 CA128622 2007 REICH, DANIEL H JOHNS HOPKINS UNIVERSITY Magnetically patterned co-cultures for cancer studies
Adhesive interactions between cancer cells and their surrounding microenvironment play a critical role in regulating the growth, development, and invasion of solid tumors. Direct adhesive contact between cancer cells and tissue stromal cells are thought to contribute to the survival, proliferation, and migration of cancers. However, the contribution of such heterotypic cell interactions to the regulation of cancer cell function remains unclear because these interactions have been difficult to manipulate experimentally. This project will combine the expertise of two investigators to develop a novel system to juxtapose two cell types in micropatterned culture such that high fidelity heterotypic cell interactions can be introduced in a controllable fashion in large numbers. The approach involves the use of magnetic nanowires, microfabricated arrays of magnetic traps, and engineering the adhesiveness of these traps to cells. The long-term goals of this project involve the development of a method to identify heterotypic cell interactions that regulate the function of cancer cells. As a proof-of-concept, this novel approach will be used to identify cancer cell lines that increase proliferation in contact with one stromal cell, the endothelial cell. Specific Aim 1 will be to optimize and characterize the parameters that affect the yield and total number of cells trapped on the arrays that experience heterotypic cell interactions. Specific Aim 2 will be to determine the effect of direct contact with endothelial cells on the proliferation of a model carcinoma cell line. Specific Aim 3 will be to demonstrate the use of this system with biochemical assays and to lay the groundwork for mechanistic studies of heterotypic cancer-stromal cell pair interactions that modulate cancer proliferation. This research will apply recent advances in magnetic micro- and nanotechnology to develop a new approach to study the biology of cancer. The devices to be developed will enable studies of the interactions of cancer cells with surrounding healthy tissue that have not been possible previously. The increased understanding that this new approach will bring will have the potential to provide important input into efforts to control the growth and spread of cancer.
R21 CA125291 2007 RESING, KATHERYN A UNIVERSITY OF COLORADO AT BOULDER New Methods for Phosphopeptide Identification
New mass spectrometers capable of data dependent data acquisition and new database search algorithms have enabled proteomics profiling of complex samples by multidimensional LC/MSMS, where proteins are proteolyzed, separated chromatographically, and identified in a high throughput manner by peptide MSMS sequencing. An important goal is to identify phosphoproteins in complex mixtures and map their sites of modification by profiling phosphopeptides. Protein phosphorylation events are prevalent in cell regulatory and signaling pathways, and aberrations that lead to changes in phosphorylation are underlying causes of cancer and many other human diseases. Thus, the ability to profile phosphopeptides and monitor their changes in abundance is of key importance for cancer treatment and diagnosis. However, technical methods to achieve phosphoproteomics profiling have proven very difficult due to the chemical properties of the phosphate, the large database size when searching a protein database allowing variable phosphorylation on Ser, Thr, and Tyr, and the resulting low sensitivity and specificity of current scoring methods. In order to match MSMS spectra to phosphopeptide sequences with greater accuracy, it is critical to develop a greater understanding about the MS behavior of phosphopeptides and chemistry of gas phase fragmentation, and evaluate the factors that interfere with their detection and identification. Therefore, studies in this proposal will improve the ability to identify phosphopeptides from high resolution MSMS spectra. In Sp. Aim 1, we will rigorously compare the performance of the most promising MS protocols currently available for phosphoproteomics. In Sp. Aim 2, we will improve computational technologies for automated searching of phosphopeptides by positive ion MSMS, by incorporating information about their unique chemistry into scoring algorithms. In Sp. Aim 3 we will test the performance of negative ion MSMS for phosphopeptides, which is very promising but relatively unexplored, and compare it against the most promising positive ion methods, asking whether preferential ionization of phosphopeptides by negative ion MSMS alleviates the need for enrichment. Our optimized methods will be applied in future studies to identify signal transduction targets and biomarkers in melanoma and prostate cancer, and will be widely useful to basic researchers and clinician scientists in the cancer research community. Dysregulation of protein phosphorylation in response to signal transduction pathways is commonly observed in diseases such as cancer and as a side effect of drug treatment. Therefore, methods that screen for changes in protein phosphorylation under disease conditions would reveal targets that can be used to design new therapeutic or diagnostic methods. Our proposal will rigorously compare MS protocols for phosphoproteomics and develop improved experimental and computational approaches to improve the sensitivity and specificity of this experiment. These methods will be applied in future studies to identify signal transduction targets and biomarkers in melanoma and prostate cancer, and will be widely useful and freely available to basic researchers and clinician scientists in the cancer and biomedical research community.
R21 CA125313 2007 ROGERS, RICK HARVARD UNIVERSITY Sample prep methods to allow automated 3D analysis of microvessel morphology
Abnormalities of vascular morphology resulting from angiogenesis, the formation of new blood vessels, are characteristic of cancerous tumors and are associated with a switch from quiescent to aggressively invasive, metastatic behavior. Thus angiogenic activity, as reflected by morphological microvascular change, is a critical point of assessment in cancer research. Visualization and quantification of microvascular attributes permit monitoring of disease progression and response to therapy, yet there are no currently available sample preparation methods to produce data that are of capillary-resolution and suitable for automated 3D quantitative analysis. Specimen preparation techniques for vascular visualization thus far have primarily been developed to meet the requirements of individual studies and have not addressed the need for standardized quantitative analysis across labs. Routine vascular metrics, such as density, often still rely upon time-consuming manual counting from 2D paraffin sections by multiple observers, in part because of the lack of 3D image data of sufficient quality to allow automated quantification. Moreover, one of the principal techniques allowing the collection of 3D quantitative vascular data, corrosion casting, destroys surrounding tissue, eliminating the possibility of simultaneous probing for molecules of interest. Our goal is to create and refine specimen preparation techniques that allow collection of high-resolution 3D vascular image data within tumors and the supporting peri-tumoral tissue, while simultaneously allowing labeling of related molecules of interest. The methods will be optimized to produce vascular image data that will allow automated quantitative analysis, thus introducing a more rapid, standardized approach for studies of microvascular changes associated with tumor growth and treatment. Methods appropriate to animal models and to fixed archived tissue will be developed toward the ultimate goal of extending these techniques to human biopsy tissue. Our methods will combine and optimize protocols used in vascular biology and adapt procedures developed in other fields, such as non-mammalian developmental biology, that though relevant, have not previously been employed in this realm. We propose to modify these existing specimen preparation techniques, and in concert with optical clearing methods to minimize problems associated with tissue opacity, produce 3D renderings of the microvascular architecture of whole-mount specimens, while permitting labeling of other molecules of interest. Our preparation techniques will employ combinations of: 1) casting of the vasculature by filling with a contrast agent; 2) marking of functional blood vessels by fluorescently labeled lectin administered i.v.; 3) marking of other structures of interest by whole-mount labeling with antibodies or nuclear dyes; and 4) tissue clearing to minimize light scattering for imaging by confocal microscopy so that data can be collected from deep within peri-tumoral and viable tumoral tissue.
R21 CA125473 2007 SAVRAN, CAGRI PURDUE UNIVERSITY WEST LAFAYETTE Label-free detection of cancer markers using aptazyme-based amplification
Detection of cancer markers in a fast and sensitive fashion is important in determining the type and stage of the disease and hence can facilitate early detection of cancer. Today, most methods used to detect biomolecules utilize fluorescent and radiolabels, which increases the time, cost and expertise required for assays. Micro- and nanotechnology has enabled researchers to develop label-free detectors that offer scalability (down in size and up in number of sensors) for detecting multiple analytes. However, the majority of micro/nanosensors have not yet achieved the sensitivity levels of label-based detection schemes. This proposal explores a novel set of methods to enhance the sensitivity of fluorescent/radio-label-free platforms to enable detection of cancer markers in clinically significant conditions and concentrations. The specific aims of the proposal focus on adapting allosteric nucleic acid catalysts (aptazymes) to 1) a nanomechanical cantilever biosensor, which is a model label-free platform which has attracted an immense amount of academic attention in recent years but has not received much clinical acceptance due to insufficient sensitivity, and 2) a bead-based diffraction biosensor, which is a much newer detection fluorescence-free platform. Aptazymes that can covalently bind to immobilized substrates on a solid surface (upon activation by target proteins) will be developed based on their functionality on a surface (Aim 1) and adapted for operation with the cantilever and the bead-based diffraction biosensor (Aim 2). For the cantilever sensor, large aptazymes (constructed either by introducing additional nucleotides or by conjugating aptazymes to micron-sized particles) activated by target molecules will ligate to the functionalized cantilever surface and yield an amplified signal. For the diffraction biosensor, magnetic beads functionalized with aptazymes will be used to selectively capture target molecules from complex mixtures. The capture will activate the aptazymes and allow their ligation (and hence the ligation of the beads) to a solid surface that is functionalized in an alternating pattern. The binding of the beads will result in a solid diffraction grating that generates very sensitive signals. Further signal enhancement in both platforms will be achieved by performing rolling circle amplification. It is expected that detection of proteins via aptazymes will generate far greater signals in comparison with those generated by direct detection of non-covalent protein-receptor binding. Two different cancer-relevant proteins will be used as model targets against which aptazymes will be developed: basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). It is expected that adapting these new aptazymes to the nanomechanical cantilever sensor and the new bead-based diffraction biosensor will enable 50 fM and 500 fM detection limits for bFGF and VEGF. The developed strategy will be tested in biologically significant media such as serum and cell lysate (Aim 3). The aptazyme development will be performed at The UNIVERSITY of Texas at Austin while all experimental work will be undertaken by Purdue UNIVERSITY. Many cancers can be treated if detected early. Detecting cancer markers in body fluids is a promising way to achieve early detection. This project will constitute a milestone in development of sensitive but relatively simple platforms for detection of cancer markers.
The tools and resources are now available for the development of screens for genes that enhance metastasis in vivo. Such screens will identify new targets for drug development that may complement therapies currently under development or in clinical use. In addition, the information provided by such screens will aid in reducing the number of potential targets that are identified through microarray studies by indicating which gene products are likely to be driving metastasis. We propose to combine high throughput gene expression and suppression technologies with in vivo metastasis methodology to accelerate in vivo screening by a factor of up to 50. The screen will indicate the contributions of proteins to the steps of primary tumor growth, intravasation, and lung colonization. The first aim will focus on production of pools of cells expressing or suppressing selected proteins. The second aim will evaluate detection technologies for measuring construct distributions in a pool. The third aim will determine the appropriate formation of pools to be screened. The fourth and fifth aims will perform an initial screen and validate candidates identified in the screen. This R21 application will enable the development of the screen and demonstrate feasibility; providing a paradigm for evaluating gene function for the most critical feature of tumor cell malignancy - the ability to metastasize. ASSESSMENT:
R21 CA125285 2007 SHEA, LONNIE D NORTHWESTERN UNIVERSITY Transfected cell arrays for cancer research
A major challenge in cancer biology is to understand how genotypic abnormalities or mutations will alter cellular responses, such as cell cycle regulation and drug resistance, involved in cancer progression. Our long-term goal is to investigate the early events in cancer progression and how they impact clinical outcome from the perspective of transcription factor (TF) activity, which relates to the activity of the signaling pathways. We hypothesize that cell-based assays that report on the activity of various signaling pathways within the cancer cells will link genotype to phenotype. The objective of this proposal is to develop a transfected cell array for high throughput analysis of TF activity in cancer cells, with breast cancer serving as a model. Our hypothesis and proposed research are based on the following observations: a) cDNA or protein microarrays cannot predict cellular activity, as activity is based on a specific protein conformation or state and cellular localization, b) cell-based assays provide the physiological context in which to examine cellular activity, c) TF activity is widely used to measure cellular activity using a two-plasmid system: one measures the activity of a specific TF and the second normalizes for the transfection efficiency: Based on these observations, the experimental objectives of the proposal are to develop a high throughput system to quantify activity of many TFs. The specific aims of the proposal are: Specific Aim 1: Create a transfected cell array with 100 spots capable of localized, efficient transfection of breast cancer cell lines. We will a) develop the procedures to deposit lipoplexes in spots while retaining their bioactivity and b) quantify the percentage of transfected cells and transgene expression in the spot. Specific Aim 2: Validate the transfected cell array for quantifying TF activity by a) determining ratio of the two plasmids and positional consistency between array spots and b) quantifying TF activity non-invasively using an imaging technique that enables following the TF activity over time. Specific Aim 3: Compare the TF activity for cells differing by one or more genotypic mutations. We will characterize TF activity for a) mammary epithelial cells immortalized by TERT (76NTERT) or by mutant p53 (76Ndel239) and b) primary tumor and malignant tumor cells, which were established from the same patient. ASSESSMENT:
R21 CA128671 2007 SOPER, STEVEN ALLAN LOUISIANA STATE UNIVERSITY A&M COLLEGE BATON ROUGE Genotyping Molecular Signatures of CRC using Polymer Microchip-CE and FSCE
Colorectal cancer (CRC) represents the third-leading killer amongst all cancer-related diseases in the US. While a number of biomarkers and technologies have been evaluated for the management of this disease, few have emerged for use by clinicians in battling CRC, with the predominant screening methods still consisting of monitoring blood in the stool and/or colonoscopy. In this R21 application, research will focus on developing micro-electrophoresis that can monitor the absence/presence of molecular biomarkers originating from nuclear DNA. The molecular assays to be investigated require high-resolution electrophoresis for reading out the results. DNA is typically separated by electrophoresis in a viscous sieving matrix using fused-silica capillaries or microchips (¨-CE). ¨-CE is particularly attractive because it can be integrated to front-end processing steps to provide automated sample processing in a closed architecture free from contamination and envisioned for point-of-care testing. The matrix is loaded into the device using high pressure, which can be time and energy-intensive, must be reloaded between every run, and is expensive. The elimination of sieving matrices and the development of free-solution electrophoresis to sort DNA would drastically decrease the cost associated with molecular assays as well as simplify system set-up and reduce run time. Since both the charge and the friction scale linearly with chain length, the electrophoretic mobility of DNA in free-solution does not change with increased chain length. In order to run free-solution electrophoresis of DNA, the DNA must be conjugated to an uncharged perturbing entity or ""drag-tag"" producing Free-Solution Conjugate Electrophoresis (FSCE). In this application, FSCE with ¨-CE devices that are fabricated in polymers using replication technology will be used for several different molecular assays, including Ligase Detection Reactions (LDR) for scoring the presence of known point mutations in K-ras oncogenes and an Endo V/LDR assay, a mutation scanning assay to score the presence of sporadic p53 mutations. In addition, microsatellite instability (MSI) will also be evaluated using ¨-CE FSCE. MSI is undertaken using a panel of markers from nuclear DNA that are PCR amplified and analyzed via electrophoresis. Comparisons of electrophoretic mobilities of diseased tissue versus normal tissue provide an indication of MSI status and can be used as a prognosticator for determining effective therapies for treating CRC patients.
R21 CA125514 2007 STEINMAN, RICHARD A UNIVERSITY OF PITTSBURGH AT PITTSBURGH A Nucleosomal Biosensor for Identification and Isolation of Nuclear Hormone Recep
Nuclear hormone receptors (NHRs) modulate transcription by binding small lipophilic ligands and have a profound impact on normal cellular function and on development. Aberrant NHR function occurs frequently in cancers. This has prompted the development of therapeutic ligands targeting NHR receptors (e.g. tamoxifen in breast cancer, ATRA in promyelocytic leukemia). New natural or synthetic ligands for nuclear receptors are likely to include useful anticancer agents. We propose an innovative, high-throughput approach to identify functional ligands of nuclear receptors. We hypothesize that an in vitro nucleosome- based biosensor can identify and distinguish ligands that activate or inhibit nuclear receptor activity. The overall goal of our work is to develop the capacity to screen large chemical libraries rapidly for functional ligands to known or to orphan nuclear receptors through the use of such a biosensor. We have developed unique tools to facilitate our pursuit of these goals. These include reagents and instrumentation that enables us to follow nucleosomal remodeling at both the single molecule and population level in real-time. This R21 application focuses on the estrogen receptor (ER) signaling pathway as a model to demonstrate the power and sensitivity of this assay. In Aim One, we construct a nucleosome in which fluorophore-tagged DNA containing an estrogen response element (ERE) is wrapped around histones so that energy is transferred from the donor fluor (Cy3) to the acceptor fluor (Cy5) (fluorescence resonance energy transfer, or FRET). In Aim Two, we measure the ability of estrogen receptor agonists to specifically remodel ERE-containing nucleosomes, manifested as a loss of FRET. Nucleosome remodeling will be studied as a function of ERE sequence, ligand (agonist versus antagonist) and tissue source of cell extracts. A cell extract-free biosensor will also be developed utilizing only recombinant proteins. In Aim Three, we will adapt the nucleosomal biosensor to a high throughput format for screening libraries and validate the system using ER agonists, antagonists and nonligands. The biosensor developed in this project should enable rapid screening of libraries of putative ligands for chosen nuclear receptors. Moreover, it will enable rapid dissection of protein or DNA components of NHR pathways. This biosensor should be more rapid and versatile than cell-based reporter gene assays and more informative than assays that measure only ligand/receptor binding.
R21 CA128720 2007 WANG, NICHOLAS J UNIVERSITY OF CALIFORNIA-LAWRENC BERKELEY LAB Nano-scale detection of the disease proteome
We propose to work collaboratively with investigators funded through this R21 mechanism to develop a new protein detection technology, the nano-capillary immunoassay. This technology will result in significant advances in protein detection and analysis with utility for both basic research and as a clinical diagnostic device. Our group, consisting of researchers at Lawrence Berkeley National Laboratory and Cell Biosciences, Inc., has the multidisciplinary expertise that this project requires. We will use CD44 in breast cancer as our model system to develop and optimize the nano-capillary immunoassay to reach optimal sensitivity (fg/¨L), accuracy, and reproducibility. CD44 is an ideal model protein that will, if we are successful, demonstrate the potential of the technology to identify multiple splice variants and post-translational modifications (e.g., glycosylation, phosphorylation, and proteolysis) in cancer cell lines, tumor samples, and blood serum. Through this funding we will engineer improvements in protein capture chemistry, analyte detection, background reduction, and temperature control to increase sensitivity. We will also develop a new detection system to enable multiplexed analysis of several analytes in a single assay. Standardized protocols will be developed for optimal protein extraction from a range of biologic samples coupled with full automation resulting in high throughput and reproducibility. These approaches will yield a systematic and quantitative approach to protein analysis. This technology fulfills multiple goals of the RFA. 1) The nano-capillary assay offers unbiased detection of protein isoforms and post-translational modifications using a single detection reagent that will produce more complete protein profiles than can be achieved with standard protocols. 2) It will advance our knowledge of the molecular nature of cancer and provide a platform for the discovery of new biomarkers. 3) It has the potential for identifying undiscovered forms of existing biomarkers that may enhance their application as clinical diagnostics. The ultimate goal is to develop a technology to propel cancer biomarker discovery and enable early detection of breast cancer.
The detailed cytogenetic analyses of human tumors and cancer tissues, in particular, have revealed the presence of structural chromosome abnormalities in most of cases. The underlying changes are often specific for the type of tumor or cancer and the cells' altered phenotype. Furthermore, investigators could demonstrate a correlation between the type or extent of chromosome changes, disease progression, and outcome. However, present laboratory techniques for the screening for structural abnormalities are very limited with respect to the detection of small, 'cryptic' translocations. For example, Giemsa (G)-banding of metaphase chromosomes or the fluorescence in situ hybridization (FISH)-based techniques of whole chromosome painting (WCP) and Spectral Karyotyping (SKY) analysis typically miss translocations that involve segments of less than 10 megabasepairs (Mbp), i.e., about the size of a chromosome band. We postulate that small cryptic translocations exist undetected in the genomes of individuals with a normal phenotype or diseases such as mental retardation, impaired fertility, precancerous lesions, or early stage tumors. Knowledge about structural alterations and chromosomal imbalances might help clinicians make more accurate predictions regarding the onset and course of a disease. This R21 project will investigate the feasibility to rapidly and inexpensively screen the human genome for the presence of occult cryptic translocations (OCTs). Specifically, we will develop and test FISH assays using collections of validated chromosome-specific bacterial artificial chromosomes (BACs) for the detection of OCTs in human cancer cells. With BAC probes spaced on average 0.9 Mbp apart and covering the entire euchromatic part of the human genome, we expect our 'BAC-FISH' assay to lead to greatly increased sensitivity compared to WCP or banding tests. Our innovative assay for sensitive genome-wide screening for translocations will be developed with cancer cell lines for which limited information about structural abnormalities is available. At the end of this project, we will be well positioned to conduct a larger study of the frequency of OCTs in the normal population as well as tumor cells and to offer BAC-FISH screening service and reagents to research and clinical laboratories for collaborative studies.
R21 CA128695 2007 XU, BAOGANG JONATHAN VANDERBILT UNIVERSITY MEDICAL CENTER Identification of Breast Tumor Secretome Changes Throughout Tumor Progression
Innovative techniques which can continually identify vast proteome changes during tumor progression in live animals are in demand. The reciprocal effects of tumor-host interactions in the microenvironment are critical for tumor growth, invasion and metastasis. In addition to the accumulation of mutations in tumor cells, the effects from the tumor microenvironment also contribute to tumor growth and progression. The complex tumor microenvironment is virtually impossible to reproduce in vitro. A sampling technique that can continually collect proteins from mammary tumor interstitial fluid in vivo throughout tumor progression is highly desirable. This fluid represents the changes not only in the tumor cells but also within the microenvironment. It is also becoming increasingly clear that the behavior of tumors cannot be fully understood through the analysis of individual genes or proteins. Comprehensive identification of differential protein expression is needed. In this project, in vivo microdialysis combined with quantitative proteomic techniques is proposed to systematically identify the temporal secretome changes during breast tumor initiation, progression and metastases in live mouse models. Preliminary data has demonstrated the feasibility of obtaining reliable protein profiles from breast tumor interstitial fluid using in vivo microdialysis and different proteomic techniques. High-throughput identification and quantification of secretory proteins from a mouse mammary fibroblast cell line using a proteomic approach is also shown. With this innovative sampling technique and advanced proteomic platform, the current study will focus on the identification of breast tumor secretome changes temporally regulated by TGF-¨ and MMP 2 during tumor progression and metastasis. Unique breast cancer mouse models previously developed in our labs provide us with valuable experimental settings. Mice with conditional knockout of Tgfbr2 (MMTV-Tgfbr2MGKO) and knockout of MMP 2 (mmp2-/-) all crossed to the MMTV-PyVmT mammary tumor model will be used. The secretory proteins associated with TGF-¨ and MMP 2 during tumor progression can then be characterized. Additionally, the inter-relationships of TGF-¨ and MMP 2 will be assessed. MMP 2 activity at different mammary tumor progression stages in wildtype MMTV-PyVmT live animals will also be directly measured using microdialysis and fluorogenic substrates. In conclusion, successful implementation of this innovative technology will accelerate our understanding of basic tumor biology raising the possibility for advances in the clinical treatment of breast tumor.
R21 CA116103 2007 XU, XIAOWEI UNIVERSITY OF PENNSYLVANIA Ultrasensitive FACTT assays for serum biomarkers
Serological tests for circulating tumor markers (biomarkers), such as PSA and AFP, are simple and cost-effective diagnostic and prognostic tests. However, only a handful of useful serum tumor marker tests are available clinically. Recent clinical proteomics indicates that specific biomarkers or protein signatures are present in the serum even during early cancer development. However, these biomarkers are present in the serum in low levels and likely beyond the detection limit of conventional ELISA. The principle investigators have invented a new antigen detection and quantification method termed Fluorescent Amplification Catalyzed by T7 RNA polymerase Technology (FACTT). FACTT uses similar principles as ELISA but coupled with the linear amplification ability of T7 RNA polymerase. It is a high throughput isothermal quantitative antigen detection assay and the preliminary data showed that FACTT increased the detection limit of ELISA by three or more orders of magnitude. FACTT assays are developed using 96 well or 384 well plates and can be easily used in clinical chemistry laboratory. As a proof of principle, this application will focus on developing FACTT assays to detect circulating biomarkers associated with malignant melanomas, because melanomas have cell lineage specific markers that can be targeted, but useful serological tests are lacking. The Pis plan to: 1) Set up and optimize FACTT assays to known melanoma markers (tyrosinase, TRP-1 and Melan-A) using pure proteins or cell lysates as template; 2) Optimize the FACTT assays in serum samples and test robustness and reproducibility of these assays; and then quantify tumor markers in sera collected from patients with measurable metastatic melanoma. 3) Validate specificity of the FACTT assays and establish normal ranges of the analytes in large cohorts of control population. The detection of circulating biomarkers in the early stage of tumor progression using FACTT assays would allow clinicians to identify high risk patients, to stratify patients for specific treatment, and to monitor treatment efficacy and disease recurrence. Early detection of metastatic melanoma might spur adjuvant therapies that could help eradicate metastases before they become clinical evident. With the emerging treatment options for melanoma such as Braf inhibitors and immunotherapies, there is a strong need for ultrasensitive assays to detect early melanoma metastasis. ASSESSMENT:
Currently, there are no effective treatments for disease recurrence following allogeneic hematopoietic stem- cell transplant (HSCT). T therapy can target malignancies using mechanisms independent of chemo- radiotherapy, with non-overlapping and generally mild toxicities. Thus, we are investigating adoptive immunotherapy as a strategy to augment the graft-versus-tumor (GVT) effect after allogeneic HSCT. However, this approach has been limited by problems delineating immunogenic epitopes for a large number of HLA alleles, T tolerance to leukemia-associated antigens, and the difficulty of manufacturing patient- specific T cells in a timely manner. As an alternative strategy, we propose to use T cells genetically modified to express a chimeric antigen receptor (CAR) specific a desired tumor antigen independent of MHC. To target B malignancies, we have designed a CAR which re-directs the antigen-specificity of T cells to the B cell lineage-restricted cell-surface molecule CD19. CD19 is expressed on the majority of B-lineage leukemia or lymphoma cells, but is absent on hematopoietic stem cells and non-hematopoietic cells. Genetically modified CD19-specific T cells are activated via chimeric CD3-^ upon CAR binding CD19, resulting in antigen-dependent cytokine production, killing and proliferation. These preclinical data were used to open a Phase I clinical trial (BB-IND 11411) infusing autologous CD19-specific T cells (expressing the first-generation CAR) in patients with relapsed follicular lymphoma. In this grant, we propose a new clinical trial to infuse pre-prepared CD19-specific T cells derived from umbilical cord blood (UCB) in patients with relapsed B-lineage leukemia/lymphoma after allogeneic HSCT. Significantly, this trial will be (i) the ""first-in- human"" infusion of CD19-specific T cells after allogeneic HSCT, (ii) the first to infuse T cells expressing a second-generation CAR capable of providing a fully-competent T activation signal (through chimeric CD3-¨; and CD28), and (iii) the first to image distribution of genetically modified T cells and their activation status in vivo by positron emission tomography (PET), an example of radio-gene-therapy. We hypothesize that the a priori generation of banks of a homogenous population of UCB-derived HLA-unmatched CD19- specific T clones will permit infusion of T cells in a safe and timely manner in a patient population with little chance of survival. T isolation, genetic modification, and expansion will follow Standard Operating Procedures at MDACC, and T doses will be manufactured in our Good Manufacturing Process (GMP) facility in accordance with quality control/assurance standards mandated by the FDA for a master cell bank. Lav language: T cells will be developed which can destroy B-lineage disease.
R21/R33 CA126674 2007 GARRAWAY, LEVI ALEXANDER DANA-FARBER CANCER INSTITUTE High-throughput oncogene mutation detection in human cancer
Cancer represents a disease of the genome; each tumor harbors a distinct set of mutations that activate oncogenes and inactivate tumor suppressor genes. In the era of targeted therapeutics, it is expected that cancer treatment decisions will increasingly be made based on tumor genetic composition as opposed to tissue of origin. However, most molecular diagnostics that detect cancer gene mutations are expensive, informative for only a single genetic locus, and adversely affected by degraded or stromally admixed genomic DNA. Thus, despite the promise of somatic cancer genetics, at the present time it remains impractical to identify critical oncogene mutations on a large scale and in a manner compatible with routine clinical use. To address these limitations, this application aims to adapt a high-throughput, mass spectrometry- based genotyping technology to detect somatic mutations in a large panel of cancer genes.' In the R21 phase, a platform based on SequenomTM iPLEX genotyping will be developed that interrogates over 600 point mutations (or small insertions/deletions) across 50 oncogenes and selected tumor genes. This platform will also be optimized for cancer gene mutation detection in genomic DNA from paraffin-embedded tumor tissue. In the R33 phase, test the feasibility of this mutation detection approach will be demonstrated in a study of a large and diverse tumor collection. Here, high-throughput oncogene mutation detection will be performed on nearly 2,700 frozen and paraffin-embedded tumors spanning many lineages, including several that have not undergone prior genomic characterization. The ability to perform high-throughput mutation detection in clinical tumor samples will enable unprecedented molecular analyses applicable to molecular epidemiology and translational oncology, including patient stratification for targeted cancer therapeutic trials. These studies therefore offer immense potential to benefit investigators and patients alike on the path to rational cancer therapeutics.
R21/R33 CA125297 2007 KOPELMAN, RAOUL UNIVERSITY OF MICHIGAN AT ANN ARBOR Nanobiophotonics Enabled Tumor Surgery and Intraoperative PDT
Improvements in the treatment of brain tumors have produced little impact on outcomes over the past three decades. Still, survival for both pediatric and adult brain tumors is known to be maximized by radiographically complete surgical resection. Unfortunately, even with the best microsurgical technique, resection may leave behind residual, MRI-demonstrable tumor. Nanoparticle assisted neurosurgery, or the application of nanotechnology to enhance neurosurgical technique, is proposed here to maximize surgical efficiency by visibly delineating neoplastic tissue and mediating adjuvant photodynamic therapy. Multifunctional tumor targeted nanoplatform (TNP) will be used to delineate tumor, thus enabling maximal surgical resection, while minimizing adjacent tissue damage. In addition, the same multifunctional nanoparticles will be used intraoperatively to mediate photodynamic therapy to eliminate occult or unresectable tumor. The surgical exposure, created during resection, will provide a corridor for the efficient delivery of visible light necessary for photodynamic therapy. The TNP will consist of a slowly biodegradable polyacrylamide core containing optical dye and photosensitizer molecules. The nanoparticle size has been designed to allow extravasation across the areas of blood brain barrier breakdown within tumors, while minimizing passage across an intact blood-brain barrier. The localization of nanoparticles at tumor sites will be optimized by coating the nanoparticles with tumor-homing F3 peptide. Previous work demonstrated the high therapeutic index and satisfactory bio-elimination of similar nanoparticles capable of enabling PDT without causing collateral damage to adjacent neural tissue. The ability of multifunctional nanoparticles to enable intraoperative optical delineation and photodynamic therapy (PDT) will be initially developed in vitro and will later be refined for preclinical use in several animal models of glioma. In summary, this proposal introduces a novel approach to brain tumor therapy, through an extension of the capabilities of biophotonic nano-devices previously developed in our laboratories. Importantly, the proposed research will be carried out by a diverse group of investigators with expertise ranging from biophysics to neurosurgical oncology. The ultimate goal is to apply advances in nanotechnology to address the challenges in the surgical and adjuvant therapy of brain tumors.
R21/R33 CA126667 2007 MINDERMAN, HANS ROSWELL PARK CANCER INSTITUTE CORP Clinical application of multispectral imaging flow cytometry
Cellular homeostasis and reactive changes depend on signal transduction between the cytoplasm and nucleoplasm. In cancer, aberrant signal transduction is commonly observed and novel therapeutics targeting these signaling cascades are currently evaluated in clinical trials or have already secured a position in the therapeutic arsenal. Aberrant signal transduction is often associated with abnormal intracellular distribution of specific signaling intermediaries thus their subular localization could potentially be used as a parameter of treatment response. Traditionally, intracellular localization of molecules have been studied by molecular techniques or by immunological labeling techniques studied with (confocal) microscopy. These approaches have the disadvantage of lacking specificity for target cell populations or the ability to evaluate large cell populations required for statistically robust analyses. The ImageStream 100 multispectral imaging flow cytometer (IS 100) has been developed to produce high resolution brightfield, darkfield, and fluorescence images of cells prepared in suspension at rates up to 100 cells per second. The IDEAS(tm) analysis software quantifies over 200 morphometric and photometric parameters for each cell based on its imagery, including parameters that measure subular location of probes. The aim of this application is to evaluate the potential of multispectral imaging flow cytometry to evaluate target cell-specific, therapy-induced changes in nuclear-cytoplasmic distribution of specific signaling pathway intermediaries as response parameters in clinical samples. The proposed studies will ultimately be conducted in conjunction with a phase I clinical trial in patients with acute myeloid leukemia (AML) undergoing therapy with the macrolide rapamycin (sirolimus) which is known to affect the function (and cellular localization) of nuclear factor-KB (NF-icB). During the R21 phase of this application, system hardware, software and sample preparation conditions will be optimized to efficiently and accurately determine NF-icB translocation events using cell line model systems. Applicability of detecting other translocations of proteins associated with NF-icB signal transduction and rapamycin action, will also be assessed and if successful will lead to testing of additional hypotheses in the R33 phase of this application. The R33 phase will apply this technique in the analysis of patient samples and will test the hypothesis that cellular distribution of NF-icB can be used as a determinant of response in AML patients treated with rapamycin and will also test additional hypotheses generated in theR21 phase. Lay Summary: By analyzing images of cancer cells at a high rate the ImageStream cytometer can provide specific information on the success or failure of specific cancer treatment responses. The aim of the proposed studies is to determine necessary adjustments to this instrument's hardware and analysis software to accurately and efficiently use it in a clinical research setting.
R21/R33 CA122904 2007 YEE, CASSIAN FRED HUTCHINSON CANCER RESEARCH CENTER Identification of T Cell-Defined Antigens in Ovarian Cancer
Immunotherapy of ovarian cancer represents an emerging modality for the treatment of patients with advanced disease. The majority of patients with disease spread beyond the ovaries initially respond to conventional therapy, but most will eventually relapse with chemo-resistant disease; recurrent ovarian cancer is generally incurable. Immune-based therapy represents a potentially effective strategy in the treatment of early relapse and residual disease. Unfortunately, a major obstacle to advancing the understanding of ovarian tumor immunity and the application of immunotherapeutic strategies for the treatment of patients with ovarian cancer has been the paucity of immunogenic target antigens. A rational and comprehensive evaluation of target antigens expressed by ovarian cancer cells would be desirable and would permit a molecular analysis of the profile of cancer cells from an immunologic perspective. Experimental hurdles associated with identifying T cell- defined antigens include: 1) the use of tumor cells (typically poor antigen presenting cells) as stimulator cells; 2) the difficulties associated with the isolation of tumor-reactive T cells; and 3) the identification of tumor antigens recognized by T cells. Each of these obstacles is addressed in turn through the application of emerging technologies developed in our laboratory and that of our collaborators. These have led to improved strategies for the generation of tumor-reactive antigen-specific T cells using opsonized tumor cell- derived antigens, the direct isolation of T cell clones and expansion for characterization and screening, and the use of a positional scanning synthetic combinatorial peptide libraries to identify T cell targets, and the use of high-throughput genetic screening to identify functionally enhancing altered peptide ligands. The results of these studies are expected to yield a diverse panel of well-defined tumor antigens that have been characterized for their immunogenicity, restricting allele, and prevalence of expression - features that factor into the consideration of candidate antigen targets for cellular immunotherapy. We anticipate that these studies will be essential for advancing research in understanding the immunobiology of ovarian cancer for tracking such responses in patients as a means of early detection and for targeting therapies by tumor vaccination or adoptive T cell therapy trials. Relevance of Research to Public Health (lay language): Most patients with ovarian cancer die of disease that is resistant to chemotherapy. The immune system may be used to treat patients with recurrent ovarian cancer. One of the major obstacles to this strategy is that very few targets are known which can be targeted by the immune system. We have developed a method to efficiently identify immune targets for ovarian cancer and enhance their ability to stimulate cancer immunity.
R33 CA122892 2007 BERTICS, PAUL JOHN UNIVERSITY OF WISCONSIN MADISON Molecular Analysis Using Liquid Crystal Technology
Cancer is an area where technological breakthroughs have resulted in major advances in our understanding of the molecular basis of disease. Knowledge concerning the role of key signaling proteins in cancer has prompted attempts to develop anti-cancer agents that are directed against signaling targets such as the epidermal growth factor receptor (EGFR). Considerable research has centered on antagonizing the tyrosine kinase activity of the EGFR, and EGFR mutations have recently been identified that correlate with tumor shrinkage following treatment with kinase inhibitors. However, clinical trials in non-small cell lung cancer (NSCLC) have revealed highly variable levels of effectiveness in cancer patients to kinase antagonists such as gefitinib or erlotinib. Moreover, little is known regarding the precise mechanisms of action of these anti-cancer agents in human tumors, primarily due to the difficulty in performing relevant molecular assays on limited tissue material. Uncertainty about how these agents affect EGFR status/signaling in human tumors has made clinical development of these drugs extremely difficult. Accordingly, the present project seeks to further develop a technology based on a new class of exquisitely sensitive tools that use nanostructured surfaces and liquid crystals (LCs) to amplify and image molecular interactions. Our preliminary data show that we can: a) fabricate surfaces with nanometer-scale topographies, b) identify EGFR expression and phosphorylation status in cell preparations using LCs, and c) detect inhibition of EGFR phosphorylation status and kinase activity in cell extracts following exposure to EGFR antagonists. Our goals are to refine, validate, and implement this methodology to allow high throughput screening on limited clinical specimens, and to ultimately use this technology to assess which tumors are most likely to respond to EGFR antagonists. The proposed Aims are: 1) Optimize the torque balance method for utilizing LC-based assays to report EGFR expression and phosphorylation status in cell preparations in a highly sensitive, reproducible and quantitative manner; 2) Using LC technology, evaluate and refine the concept that small numbers of cells are sufficient to report the expression, phosphorylation status and tyrosine kinase activity of the EGFR in a sensitive and quantitative fashion. It is anticipated that this research will provide a powerful and novel tool for the study, diagnosis, and treatment of cancer.
R33 CA126966 2007 LIU, EDISON TAK-BUN GENOME INSTITUTE OF SINGAPORE Pair-end-ditag technologies for the complete annotation of fusion genes
We have developed a new technology called Gene Identification Signature Pair-end-diTag (GIS-PET), which captures precisely and joins the 5' and 3' most 18 base pair of any clone insert in a library. The resultant ""ditags"" are signatures of the boundaries of any cDNA or genomic fragment within the library. When coupled with advanced sequencing technologies, GIS-PET can annotate an entire transcriptome 300-500 fold faster and less expensively than contemporary cloning and sequencing. We have captured the transcriptome snapshot of the MCF-7 breast cancer cell line to >500,000 full length cDNA equivalents and have captured the state of the transcriptome of this breast cancer cell line with unprecedented resolution. With this approach, we found that we can identify, again with precision, candidate fusion transcripts where the beginning of one gene is fused with the end of another. These fusion transcripts are cancer-specific and can function as biomarkers, as monitors of treatment effect, and as targets for new therapeutics. We therefore propose to expand and validate this technology on primary breast cancers and to confirm the significant list of candidate fusion transcripts already identified in the MCF-7 cell line. We will clone and sequence all the fusion transcripts accessible to these technologies and will assess if their presence in primary breast cancers is associated with cancer behavior and clinical outcome. We also propose to optimize the GIS-PET technology to genomic DNA so as to map the precise location of genomic rearrangements in cancer cells. When put together, the combined power of the technology can uncover the complete rearrangement map of any cancer genome. Because we have brought this technology beyond conceptualization, we are submitting this application as an R33. Relevance to public health: Cancer is a genetic disease and the understanding of the genetic abnormalities in cancer has been shown to uncover new biomarkers and new treatments in the management of the disease. We have developed a new technology that can map the consequences of all the important abnormal rearrangements in a cancer cell. We propose to perfect this technology and to apply it to human breast cancer tissues.
R33 CA128726 2007 MACBEATH, GAVIN HARVARD UNIVERSITY Quantitative Interaction Networks for Tyrosine-Phosphorylated Proteins
Intracellular signaling networks that involve protein tyrosine kinases are critical in the control of most cellular processes, including growth, adhesion, migration, differentiation, and apoptosis. Misregulation of these networks results in a variety of human diseases, including cancer, diabetes, and immune deficiency. Many of the proteins in these networks contain Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains, which recognize tyrosine-phosphorylated proteins in a sequence-specific fashion. In this proposal, the molecular recognition properties of virtually every SH2 and PTB domain encoded in the human genome will be investigated with respect to physiologically-relevant ligands using protein microarray technology. Recombinant SH2/PTB domains will be arrayed in the wells of microtiter plates and subsequently probed with 258 fluorescently-labeled phosphopeptides representing experimentally-verified sites of tyrosine phosphorylation on human receptor tyrosine kinases, as well as with 604 peptides representing sites of tyrosine phosphorylation on downstream proteins (nonreceptor tyrosine kinases and SH2/PTB-containing proteins). By probing the arrays with eight different concentrations of each peptide, equilibrium dissociation constants will be determined for the binding of each peptide to each protein (~140 active SH2/PTB constructs). This effort will produce high quality, quantitative protein interaction networks which will reveal individual connections between signaling proteins, as well as how network connectivity changes with protein concentration. We have previously proposed that the extent to which a protein becomes more promiscuous when overexpressed contributes to its oncogenicity, and the study described here will generate the quantitative data needed to investigate this hypothesis further. In addition, the information revealed by our systematic efforts should prove invaluable to cell and cancer biologists who study tyrosine kinase-mediated signaling, to computational biologists who study molecular recognition, and to systems biologists who seek to model signal transduction networks. As such, we intend to make our data easily accessible though an interactive web site in formats suitable both for broad computational studies and for more focused hypothesis-driven inquiries. It is our hope that the studies described here will shed light on how signaling proteins are integrated into complex networks and how we can intervene most effectively when these networks go awry.
R33 CA114151 2007 MILOSAVLJEVIC, ALEKSANDAR BAYLOR COLLEGE OF MEDICINE Multiplexed Methods for the Study of Chromosomal Aberrations in Cancer
The general aim of the present application is to recruit newly available genomic technologies such as Bacterial Artificial Chromosome (BAC) library preparations, finished sequence of the human genome, and next- generation sequencing technologies developed by the 454 Life Sciences Corporation in the fight against cancer. This application aims to develop the Barcoded Tag Pooled Genomic Indexing (BT-PGI) method for highly multiplexed BAC-based mapping of chromosomal aberrations in cancer. The BT-PGI method achieves throughput and resolution by combining two types of multiplexing -- the pooling of BAC clones by the PGI method and the parallel sequencing of short mappable sequence tags by the newly developed 454 sequencing technology. A commercially available BAC library obtained from the well-established MCF-7 breast cancer cell line will be used for'testing and validation of these methods. Specific rearrangements detected in MCF-7 will then be measured in other breast cancer cell lines, and in breast cancer tissue. The biological function of these rearrangements will be tested by cloning and expression in both immortalized and transformed breast epithelial cells. Biological assays will depend upon the rearrangement or breakpoints of interest, but will focus on proliferation, survival, invasion and transformation. The improved understanding of chromosomal rearrangements will open new opportunities for charting the progression of cancer, understanding relevant molecular mechanisms, identifying biomarkers for informed therapy, and identifying targets for therapeutic intervention in breast cancer and other tumors.
R33 CA120726 2007 MOORE, PATRICK S. UNIVERSITY OF PITTSBURGH AT PITTSBURGH Emerging Technologies Applied to the Discovery of Human Tumor Viruses
Infection contributes to ~20% of human cancers worldwide. The list of known carcinogenic infectious agents, however, is surprisingly short and no new human tumor viruses have been discovered over the past decade. Current tumor virus discovery methods are not comprehensive and are likely to miss discovery of new families of agents. Negative studies using these techniques do not rule out the presence of a tumor virus and may miss a previously unknown agent. We propose modifying long Serial Analysis of Gene Expression (SAGE) as an unbiased means for virus discovery using in silica digital transcript subtraction (DTS). We show here a practical method to perform transcriptome-wide in silico subtraction of short transcript tags, allowing discrimination between human and nonhuman sequences. Once a candidate tumor virus sequence is found, it can be used as a start point for viral genome walking and characterization. To demonstrate the feasibility of this method, we performed pilot studies of DTS on a tumor cell line infected with latent KSHV virus. DTS rapidly and uniquely identified 5 KSHV transcripts de novo comprising 0.44% of the total cell transcriptome. The technique was quantitatively reproduced by spiking KSHV-infected cell line RNA into uninfected human tumor tissue RNA. We also identified practical cut-off levels that distinguish most human polymorphisms from viral SAGE tags using DTS. Finally, we performed pilot DTS on 3 squamous cell conjunctival carcinoma (SCCC) tumors, an immunodeficiency-related malignancy. In silico subtraction of 108,000 SAGE tags generated 46 candidate sequences, including 12 high probability tags that are being evaluated as possible SCCC agent sequences. We show that this technique is surprisingly immune to RNA degradation so that it can be used on rare or archival materials in which partial RNA degradation has occurred. We seek phase II R33 funding to perform DTS on 4 SCCC samples, extending our pilot studies into a full analysis of SCCC at the 5-10 transcripts per million level. This will allow us to either identify or exclude a likely tumor virus causing this immunodeficiency-related cancer. This also completes development of DTS technology, allowing us to fully optimize its performance for application to other suspected infectious tumors ASSESSMENT:
R33 CA125520 2007 ROTHSTEIN, RODNEY J. COLUMBIA UNIVERSITY HEALTH SCIENCES Using synthetic dosage lethality to screen for novel anti-tumor targets
The significant challenge in cancer therapy is to selectively kill cancer cells while not harming normal cells. Novel therapeutic targets are needed to develop new cancer drugs that will achieve this goal. Cancer cells often increase expression of specific genes due to mechanisms such as translocations or gene amplification. New therapeutic targets could be found by identifying genes whose function is required only when a specific gene is over-expressed. In yeast this type of interaction where a non-essential gene becomes essential when a second gene is over-expressed is termed synthetic dosage lethality (SDL). SDL interactions involving genes over-expressed in cancer cells could identify partner genes that are only essential in specific cancer cells. Drugs developed to inhibit function of the normally non-essential genes should then selectively kill cancer cells and not cells from normal tissue. Thus the problem becomes one of identifying cancer-related SDL interactions. To speed the identification of such SDL interactions, we propose to use yeast as a model system. In our approach, we create a yeast cell that over-expresses the yeast ortholog of a gene that is over-expressed in human cancer. Since many essential functions are conserved between yeast and humans, we select genes that have a functional ortholog in Saccharomyces cerevisiae starting from a list of interesting candidate genes that are over-expressed in tumor cells. To search for the SDL non-essential gene, we are using the 4827-member yeast gene disruption library. We have developed a novel method to introduce any plasmid of interest into this library via a process we term plasmoduction. We have shown that this method can be used to screen the entire library and uncover new genetic interactions. We will develop this technology to increase the throughput for identifying yeast SDL interactions. We will then show that human orthologs of these interacting genes define a similar interaction in human cells. Finally, we will determine if these interactions can be exploited to selectively kill cancer cells. The application can be divided into the following specific aims: 1. We will increase the throughput for measuring SDL interactions eventually permitting the screening of approximately 125 genes/year (>600,000 interactions/year). At first we will concentrate on genes that are involved in cell cycle regulation, checkpoints, DNA replication and recombination that are over-expressed in tumors. By over-expressing the yeast orthologs of these genes and screening the yeast deletion library, we will define all potential SDL interactions within the set of 4827 non-essential gene disruption strains. Candidates from this screen will then be used for experiments described in Aims 2 and 3. 2. Yeast SDL partners that have clear orthologs in human cells will be tested by establishing cell lines that over-express the human ortholog. of the query gene. To validate the SDL interaction, expression of the target gene will be reduced by siRNA in these cell lines and cell survival will be assayed. 3. SDL interactions that are validated in human cell lines will then be tested in cancer cell lines that exhibit over-expression of the query gene. Expression of the target gene will be knocked down by siRNA to determine the effect on cell viability.
R41 CA128782 2007 Cao,Han Bionanomatrix, Inc. Continuous Chromosome Sorting With Micro/Nanofluidics
Single molecule analysis of long, genomic DNA will provide greater knowledge of genomic structural aberrations/variations and improved understanding of their association with cancer. The long-term goal of this project is to develop a fully integrated chip and reader capable of single molecule analysis of large native state genomic material. The anticipated embodiment will permit direct visualization and analysis of chromosomal and megabase fragments of DNA extracted directly from a sample (possibly a single cell) with sub-kilobase resolution. Furthermore, the chip will accommodate massively parallel analyses of individual DNA molecules in a high- throughput manner thus providing statistically relevant data in a timely fashion. As most disease related loci are located on specific chromosomes, it is of great value to be able to pre-sort them prior to further single molecule level analysis in nanofluidics. In order to help reach this objective, we propose investigating the possibility of integrating microfluidic particle sorting technology developed under Prof Sturm at Princeton University with BioNanomatrix's nanofluidic DNA analysis technology. A micro/nanofluidic standardized platform based on continuous sample analysis in massive parallel fashion could dramatically reduce the cost and serve as a basis for consistent, high-throughput genomic analyses in future patient care.
R42 CA116048 2007 Rampersaud,Arfaan Columbus Nanoworks, Inc. Commercialization Of Magnetic Nanobeads For Cancer Cell Enrichment
Columbus NanoWorks (CNW) proposes research leading to the commercialization of custom magnetic nanoparticle reagents for the detection of cancer cells from various sources such as bone marrow and peripheral blood. Cell separation is becoming increasingly commonplace for researchers and clinicians who isolate rare cells from complex cell populations to understand how cells function in different environments or to diagnose disease occurrence, recurrence or progression. Complex mixtures and searches for small variations in cell samples require new techniques that provide exquisite sensitivity with high throughput; therefore, advances in cell separation technology must allow the researcher to find low percentage cells of interest, as well as purify them to allow for accurate assessment of biological relevance. In Phase I research CNW demonstrated the manufacture of magnetic nanoparticles that were uniform in size within a range of 50 to 70nm, had a ligand density of 2 to 5 ug/ml Fe particles and elicited a magnetophoretic mobility of immunomagnetically labeled cells an order of magnitude greater than commercially available magnetic reagents. The aforementioned criteria defining magnetic particles achieved in the Phase I application, allowed the enrichment of 1 tumor cell in 107 total cells with an average recovery of 74.1% of spiked tumor samples, as well as an average 3.21 log10 depletion of contaminating cells in the flow-through magnetic sorter known as the Quadrupole Magnetic Sorter (QMS). Phase II research thus calls for the design and commercialization of magnetic reagent kits specific for QMS applications involving cancer cell enrichment. CNW will (1) scale up processing efforts to obtain a 50 fold increase over current process techniques; (2) establish procedures and practices which assure consistent control of nanoparticle size and magnetic properties; (3) develop protocols for CNW cell enrichment kits for use in the commercial magnetic flow sorter, QMS; and (4) beta testing of protocols using breast cancer patients and head and neck cancer patients. The proposed Phase II end items are customized magnetic reagent kits specific for the QMS system that will allow the enrichment of cancer cells from various blood sources such as bone marrow and peripheral blood. Specifications of the particles will provide a greater than 70% recovery of desired cells, as well as a 4 log10depletion of contaminating cells. The resultant enriched fraction using CNW nanoparticles in the QMS system will provide a quality-purified product for further downstream processing and subsequent molecular analysis. The goal is for CNW customized reagents to become an integral part of gold standard testing in the current pathology repertoire. Columbus NanoWorks is a magnetic nanoparticle reagent company that provides reagents for magnetic cell separation. In this application we will develop magnetic reagent kits as commercial reagents for the Quadrupole Magnetic Sorter (QMS) system. In the past, commercial magnetic nanoparticles used in the QMS system have provided sub-par results with respect to purity and recovery of desired cells. Columbus NanoWorks demonstrated in their Phase I application that reagents custom designed for the QMS were able to achieve an average 3.21 log10 depletion of contaminating cells as well as an average recovery of 74.1% of tumor cells from control samples. We believe that CNW's custom reagent kits for the QMS system will provide more predictable, reproducible results from sample to sample.
R43 CA125605 2007 Bogen,Steven Medical Discovery Partners, Llc Isolation Of Circulating Tumor Cells From Peripheral Blood
The aim of this application is to develop a new enabling technology for isolating circulating tumor cells (CTCs) from blood. During the last decade, there have been numerous publications describing the presence of CTCs in the blood of cancer patients. However, the methods have been insufficiently sensitive to serve as a cancer screening test to detect otherwise occult solid tumors. The only FDA- cleared CTCs test is approved solely for clinical staging, in identifying patients with many CTCs. Therefore, there is a need for a more sensitive CTCs sample preparation technology. We have developed the only antigen-dependent, negative selection technology for isolating CTCs from peripheral blood. Our technology is distinguished from existing methods by a uniquely high CTCs recovery rate, generally >90%, and a 5-6 log order enrichment. We attain these high rates through several sample preparation design innovations that minimize CTCs losses. For example, we do not introduce an external surface, such as a paramagnetic bead, that may interfere with downstream analysis. We have overcome the problem of limited EpCAM expression on CTCs associated with positive selection cell separation methods by designing ours as a negative selection technology. Our technology also is not dependent upon arbitrary physical distinctions between CTCs and blood cells, such as cellular density or sensitivity to chemical lysis. The technology is also unlike flow sorting, in that only basic laboratory instrumentation is required. Our method also avoids centrifugation, a procedure which, in our hands, consistently resulted in unacceptable CTCs losses. In this Phase I project, we will complete the development of our technology and test it with both model blood systems and patient samples. In Specific Aim 1 we will synthesize new reagent conjugates for removing leukocytes and erythrocytes from whole blood. We will also characterize the parameters of CTCs enrichment and evaluate entirely new methods of forming the reagent conjugates for scale-up to clinical trials (in Phase II). In Aim 2, we will characterize the efficacy of new cellular preservatives for maximizing CTCs viability during specimen transport. There is a dearth of information in the published literature on CTCs preservation in blood samples. In Aim 3, we will validate the assay with clinical samples from patients with prostate cancer and characterize whether it is substantially better than existing methods. If successful, this technology will establish a platform for detecting cancer at an earlier point in time, when surgical intervention may be curative. The best prospect for curative treatment of solid tumors is surgical excision, provided the tumor can be detected early, before it spreads. Unfortunately, all too many cancers are detected clinically only after they have spread. We propose to develop a novel technology for detecting extremely rare circulating tumor cells in blood, so as to facilitate early detection and curative surgical treatment.
R43 CA126647 2007 Sun,Ye Sci-Tec, Inc. Enzymatic Luminescence Microrna Assay
The goal of this project is to develop a new, highly sensitive, and cost-effective RNA Enzymatic Luminescence Assay (qELA) for high-throughput detection and quantification of microRNA in biological samples. The assay implements the same detection concept known from pyrosequencing, yet expands pyrosequencing detection methodology for highly sensitive and accurate quantification of small RNA molecules. The proposed assay has unique sensitivity and dynamic range and is expected to outperform real-time PCR in applications for analysis of small RNA molecules. The qELA requires less expensive reagents and equipment than RT-PCR and microarrays and can be used in a number of commercial assays for application in life sciences research, drug discovery, and clinical diagnosis.
R43 CA126726 2007 Medghalchi,Susan Fasgen, Inc. Fatty Acid Synthase Phosphorylation As A Cancer Biomarker
During the last decade, increasing interest has developed in fatty acid synthase (FAS) as a potential diagnostic and therapeutic target for human cancer. These notions are based on two fundamental observations: [1] FAS is highly expressed in most common human cancers, and [2] pharmacological inhibition of FAS leads to apoptosis of human cancer cells in vitro and in vivo. FAS is the enzyme which catalyzes the de novo synthesis of fatty acids predominantly from dietary carbohydrates. In addition to its expression in human cancers, our collaborators have found that FAS circulates at high levels in the blood of colon, breast, lung, ovarian, and prostate cancer patients compared to normal subjects. Recent data have emerged which directly impact FAS as a biomarker for cancer: [1] FAS elevations have been found to occur in the blood of obese subjects with non-alcoholic steatohepatitis, and [2] FAS derived from tumor cell lines is phosphorylated on threonine residues while FAS from non-transformed cells is not phosphorylated. These findings will enable the development of a diagnostic ELISA serum test for human cancer based on phosphorylated FAS which would not react with FAS derived from normal tissues such as liver. The goals of this Phase I SBIR are to determine that FAS derived from human cancer is selectively phosphorylated and detectable in the serum of cancer patients but not in the sera of obese subjects. This tumor selective phospho-FAS would form the basis for the development of a cancer-selective FAS ELISA assay for the Phase II application. The early diagnosis of cancer enables more effective therapy and enhances patient survival and quality of life. Fatty acid synthase is present at high levels in most common human cancers including colon, lung, prostate and breast cancer. The goal of this proposal is to advance the development of a blood test for cancer based on the identification of FAS which will broadly identify the presence of most human cancer.
R43 CA128207 2007 Rush,John Cell Signaling Technology, Inc. Finding Acetylated-Lysine Sites In The Cancer Proteome
Recent studies indicate acetylated-lysine is an under-appreciated post-translational modification with a potentially prominent role in cancer biology. However, despite advances in proteomics, it is still difficult to find lysine acetylation sites in proteins. Although IMAC can be used to isolate phosphorylated peptides in bulk, there is no bulk purification technology for acetylated peptides. The long-term goal of this project is to develop and commercialize a proteomic method for isolating, identifying, and quantifying lysine acetylation sites. This method will contribute to the development of drugs that affect protein acetylation levels in cancer by elucidating their mechanisms of action. It will also identify new acetylation sites that could become targets for cancer diagnosis and treatment. During Phase I we will establish the method and its tools, using an immunoaffinity approach with an antibody specific for acetylated-lysine. We will first screen several acetylated-lysine monoclonal antibodies to select the one that performs best as an immunoprecipitation reagent and that best recognizes acetylated-lysine peptides in a sequence-independent manner. After choosing one antibody for further study, we will isolate and identify acetylated-lysine peptides from several cancer cell lines. Modified peptides will be isolated by a variation of the immunoaffinity technology we developed for phosphorylation profiling and will be identified by commonly practiced liquid chromatography-tandem mass spectrometry methods. To demonstrate that we can evaluate the relevance of the sites we find, we will collect and present information about known and novel human acetylated-lysine sites in an easy-to-use web-based format, as we have done for phosphorylation sites in PhosphoSite(r). This project has the potential to vastly increase the number of known acetylated-lysine sites and to greatly stimulate the newly emerging fields of lysine acetylation and acetylation biology.
R43 CA128752 2007 Kravitz,Rachel Neoclone Biotechnology International A Novel Platform For Mining The Repertoire Of Antigen-Specific B Cells
Cancer remains a significant threat to the health and economy of today's society. Advances in cancer research are key to cancer biomarker discovery, development of rapid and reliable methods of cancer-specific diagnosis, and development of new treatments. Multi-analyte detection platforms such as protein microarrays have potential for fast, high-throughput analysis of complex samples for small molecules and proteins of interest. The next few years will likely yield rapid advancement in the use of these platforms for cancer biomarker discovery and early-stage diagnosis of different cancers. Despite the advancement of multi-analyte platforms, however, the overall performance and usefulness of these approaches depends heavily on the quality of the monoclonal antibodies used to capture and detect molecules of interest on the microarray surface. Successful microarray development requires screening numerous monoclonal antibodies for affinity, specificity, cross-reactivity, and platform compatibility. For a given diagnostic cancer target, dozens of monoclonal antibodies may need to be screened and, thus the techniques used to generate the monoclonal antibodies must be able to generate a panel of highly diverse target-specific monoclonal antibodies. Because of limitations in current monoclonal antibody development methods, such panels of target-specific monoclonal antibodies often are not available and are prohibitively expensive to make. The overall objective of this Phase I application is to generate a unique technology for mining the clonal repertoire of B cells from immunized mice and generating panels of antigen-specific monoclonal antibodies against targets of interest. NeoClone proposes to adapt its successful ABL-MYC retroviral technology to develop a novel in vitro platform for the clonal expansion, selection, and transformation of antigen-specific B cells. This platform will enable unprecedented clonal diversity, generating panels of monoclonal antibodies that can be tested for specific epitope identification, affinity, and platform compatibility. In addition, the in vitro methods proposed here will allow for the selection of rare clones, thereby enabling NeoClone to develop antibodies against important targets where traditional methods have failed. The rapid growth of new platforms for detecting rare targets in complex samples demands the parallel development of new technologies for generating affinity reagents against those targets. Any new technology must produce reagents with high affinities, high selectivity, and high clonal diversity. NeoClone's unique in vitro ABL-MYC technology can meet these requirements. Monoclonal antibodies are essential for helping to discover cancer in the body, especially in early stages (biomarkers), develop ways to diagnose cancer, and to find new treatments for the disease. NeoClone proposes to develop a new way to create large numbers of high-quality, diverse, cancer-specific monoclonal antibodies both quickly and cost-effectively. This technology can potentially advance new methods for diagnosing cancer, such as antibody microarrays, which need high-quality cancer biomarker-specific antibodies to move into the clinic.
R43 CA128769 2007 Sheldon,Edward Chembionics, Inc. A Rapid, Sensitive, And Low-Cost Gene Expression Profiling Technology For Cancer
Current methods of cancer classification and diagnosis utilize mostly histological and morphological properties of the tumor, leading to generic and often unsuitable treatments for patients. Recent genomic advancements in oncology have established the ability to perform gene expression profiling of cancers, which can guide health workers towards a more patient-specific, accurate, and more successful treatment. The majority of cancer-based gene expression profiling is done using fluorescently labeled nucleic acid microarray technologies that are expensive, slow, and sometimes unreliable due to low sensitivity and specificity. Here, a completely new gene expression technology is proposed that, while obtaining high sensitivity and reliability, is designed to make gene expression profiling more accessible even to smaller clinical or hospital labs by utilizing a rapid and inexpensive system based on robust, electronic detection technology. The proposed gene expression profiling system will achieve these characteristics in a new type of microarray. This project will utilize two main thrusts to achieve these goals: 1) a new label-free, electrochemical-based hybridization sensor, and 2) confinement of each sensor using microactuators. The new sensor relies on an array of specific capture probes bound close to electrodes and surrounded by microactuators that assure confined analysis. The recognition of the right target is achieved by flow-through hybridization and an enzymatic cascade detection system enabling (i) linear amplification of the target above each sensor and (ii) transduction of this specific recognition into an electrical signal, which will be measured using a simple amperometric device similar to a glucometer. This new sensor will avoid the need for expensive nucleic acid labeling and optical/fluorescent detection equipment. The proposed device is well suited to micro-scale systems that can be operated as portable instruments at the bedside. The proposed microfluidic confinement of each sensor will eliminate sensor cross-talk and will increase sensitivity by confining enzyme products to small volumes. Objectives of this second thrust will be to obtain optimal fabrication and operation conditions to make an actuator of the desired size operate in the same conditions as the sensor. The overall goal of the Phase I and Phase II project is to validate the system and perform clinical testing of an integrated micro-scale gene expression profiling system for accurate genetics-based cancer diagnosis. In Phase I this system will be evaluated in terms of sensitivity and robustness using gene expression profiling based on several characteristic cancer genes by detecting small quantities of reconstituted cDNA or RNA from tumor cells. Clinical evaluations are envisioned to be performed in the Phase II project.
R44 CA128858 2007 Dolinger,David Iquum, Inc. Rapid Point-Of-Care Assay For Detecting High Risk Human Papillomavirus
We propose to develop a highly sensitive and rapid point-of-care nucleic acid test based on IQuum's lab-in-a-tube (Liat(tm)) platform for the detection and genotyping of high risk Human Papillomavirus (HPV) from cervical swab samples. The Liat HPV Assay will utilize the Liat Analyzer to enable the minimally trained personnel to perform the HPV test at a hospital, clinic or physician's office in 1 hour. By enabling effective and sensitive single visit HPV testing, we expect that the Liat assay will allow clinicians to identify more women at risk for cervical cancer and provide immediate and effective intervention, thereby significantly lowering the incidence of cervical cancer. The Liat HPV Assay overcomes the limitations of current HPV tests, whose long turnaround time and technical complexity requires testing to be performed in centralized laboratories, resulting in missed opportunities for disease prevention due to significant patient loss to follow-up and lack of penetration into resource-poor regions in the US and abroad. In Phase I of this Fast Track project, we intend to prove the feasibility of developing a 1 hour sample-to- result Liat HPV Assay by developing melting-point PCR assays for HPV-16 and HPV-18 and Liat-based extraction for HPV nucleic acids from HPV-containing cervical cell lines. In Phase II, we propose to expand the assay developed in Phase I to include the majority of high risk HPV types that are found in cervical cancer, develop Liat sample preparation for cervical samples, as well as integrate all assay components, including internal controls, into a single Liat tube for multiplex HPV detection. We further intend to characterize and validate the Liat HPV Assay in a pre-clinical study. At the conclusion of this Fast Track project, we will be prepared to submit the Liat HPV assay for FDA- approved clinical study. The assay will be among the first point-of-care nucleic acid tests, and will provide significant benefit to public health.
R44 CA130026 2007 Levenson,Richard Cambridge Research And Instrumentation Cellularly Resolved Molecular Pathway Assessment In Biopsies Via Spectral Imaging
This is a Fast-Track application to provide reliable, cellularly resolved molecular pathway assessment in cancer biopsies to assist pharmaceutical drug development and provision of patient-specific prognosis and therapy guidance (""personalized medicine""). The organizing theme is that the appropriate unit of analysis should be the individual cell as opposed to averaged tumor extracts. To this end, novel technologies will be coupled with careful methods-development. Spectral imaging and advanced image analysis tools will permit multi-target immunohistochemical (IHC) and/or immunofluorescence (IF) detection, at the cellular and subcellular level in intact tissue sections. CRi-developed image processing and machine-learning tools provide automation and sophisticated quantitation options. Multiplexed staining protocols will yield independent, potentially stoichiometric labeling with combined IHC and IF. The sensitivity of all potential markers to variations in tissue handling will be carefully assessed; some may be robust and suitable for archival tissue studies, others will be too labile. Ultrasound-assisted fixation will be tested for its ability to preserve such labile epitopes for use in prospectively acquired tissues. Four or more pathway-related proteins will be detected in tissue sections, on a cell-by-cell basis, even if co-localized and with spectrally overlapping labels. The coordinated subcellular location of the pathway molecules will be also tracked, with simultaneous assessment of cell-surface receptors (e.g., EGFR, VEGF, Her2-neu), downstream signaling proteins and phosphoproteins (e.g., pAKT, pERK), nuclear proteins (e.g., ER, Ki67), and novel players such as protein-folding mediators (e.g. BIP1). The project will combine optimized tissue protocols, multiplexed IHC/IF reagent kits, and unique machine- learning image analysis that can be used to automate region-detection and label-quantitation. All these depend on CRi's multispectral imaging approaches for assessing multiple analytes on a cell-by-cell and cell- compartment basis in tissue sections. Our collaborators will provide small-animal tumor models for early methods development, multiplexed immunohistochemical labeling of pathway proteins in clinical cancer biopsies, access to archived and prospectively acquired tissues from pathway-targeting clinical drug trials, highly informative archival tissue microarrays, access to validated, activation-specific antibodies, ultra-fast tissue fixation, and biostatistics support. The ""deliverable"" will be a suite of products suitable for clinical use that can provide much-needed valid information on single-cell-based pathway status in an intact tissue context to support pharmaceutical drug development efforts and provide molecularly focused patient care. Significance and lay narrative: The ability to quantitatively evaluate multiple molecular targets and pathways on a cell-by-cell basis, in a single preparation of clinical tissue, is missing from the current toolbox of personalized medicine, which lacks good means of matching novel drugs and drug candidates to specific patients. Conventional molecular pathology methods are typically limited to a single immunohistochemical (IHC) test on a given tissue section or to expensive and time-consuming proteomics or expression-array approaches (which cannot directly report out pathway activation status in cancer cell populations and subpopulations). Multiplexed IHC (including immuno-fluorescence) combined with optimized sample handling protocols to retain pathway proteins and advanced image analysis will enable the unambiguous detection of active signaling pathways, benefiting pharmaceutical research in the selection of patients for better targeted trials and in the monitoring of response, and clinical practice for diagnosis, therapy selection, and monitoring response (i.e., theranostics).