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R21 CA138397-01A1 2010 ANDERS, JOHN C; KANIGAN, TANYA SHARLENE; WILLEY, JAMES C. (contact) UNIVERSITY OF TOLEDO HEALTH SCIENCES CAMPUS Implementation of innovative RNA sample quality control methods
Molecular diagnostic tests comprising transcript abundance (TA) measurement have great potential to better predict cancer risk, prognosis, and the optimal therapeutic for each individual. However, this potential is limited by the status of clinical sample collection and processing methods. Methods typically practiced today yield clinical samples with wide variation in RNA quality. Efforts to improve these methods are limited in part by inadequate means to measure RNA quality. Further, use of existing banks of biospecimens with variable RNA quality is limited due to inadequate RNA quality control (QC) measurement methods. This prevents establishment of appropriate cut-off criteria by which to determine which samples will yield reliable reverse transcriptase polymerase chain reaction (RT-PCR) results. Sources of inter-sample variation in RNA quality include RNA integrity, genomic DNA (gDNA) contamination, and substances or methods that either a) interfere with RT efficiency, and/or b) carry over to cDNA and cause gene-specific inhibition of PCR efficiency. In pilot studies, quantification of ACTB cDNA molecules/ng RNA controlled for RNA degradation over wider scale than that measured by electropherogram, quantification of CC10 gene gDNA in RNA controlled for gDNA contamination, and measurement of transcript abundance of each gene relative to known number of respective internal standard (IS) molecules controlled for PCR inhibitors. In this proposed project, more comprehensive investigation for the best tests for RNA integrity will be developed and these will be implemented, along with the tests for gDNA contamination and RT inhibition on the novel Standardized NanoArray PCR (SNAP) platform. These methods then will be applied to guide development of improved methods for biospecimen collection. The specific aims are: Aim 1. Establish robust tests to measure RNA integrity, gDNA contamination, and RT inhibition along with accompanying reference materials Aim 2. Use the robust RNA QC methods to establish cut-off thresholds for reliable measurement of transcript abundance based diagnostic tests by RT-PCR or microarray and guide improvements in methods for collecting and processing clinical biospecimens. Aim 3. Demonstrate the feasibility of implementing the RNA QC tests either alone or together with disease specific assays on commercial platforms. Successful completion of the proposed project will enable identification of tissue sample collection and processing methods that are more likely to yield RNA samples suitable for molecular diagnostic studies. Further, it will enable more accurate determination of which clinical biospecimens collected according to present methods and/or in existing tissue banks will yield reliable results for promising transcript abundance based diagnostic tests. PUBLIC HEALTH RELEVANCE: Sequencing of the human genome has opened opportunities for advances in diagnostics based on transcript abundance measurement. This promise has been hampered by lack of adequate tests for RNA quality. Development of improved RNA Quality Control tests will enable development of improved biospecimen collection methods and improved methods to determine which RNA samples have sufficient quality to yield reliable transcript abundance values. These advances will lead to more reliable testing required for personalized medicine in the field of cancer diagnosis and management.
 
R21 CA137687-01A1 2010 BARRETT, MICHAEL THOMAS TRANSLATIONAL GENOMICS RESEARCH INSTITUTE High Definition Clonal Analyses of Archival Pancreatic Adenocarcinoma Samples
Genomic instability appears to cooperate with Darwinian selection to promote cancer formation through a process in which genomic aberrations occur at accelerated rates, and those alterations that provide a selective growth advantage lead to clonal evolution and expansion. Consequently the patterns of genomic aberrations in cancers can vary extensively, even in tumors arising in the same organ site. A fundamental hypothesis of current cancer genome efforts is that tumor cells become differentially resistant or sensitive to available clinical interventions according to selected aberrations present in each sample and the pathways that they target. Thus the identification of selected aberrations in patient samples will help develop novel therapeutic targets that can be advanced for improved more personalized approach to the treatment of cancer. Recent advances in genomic technologies provide highly detailed analyses of samples of interest. For example oligonucleotide CGH arrays can distinguish single copy changes across an entire genome at intragenic mapping resolution with probe error rates of < 5%. A challenge in studying complex tissues is the presence of admixtures of cells and the polyclonal nature of human neoplasias. Furthermore, in addition to masking critical genomic aberrations, the presence of clonal mixtures of neoplastic cell populations in biopsies makes it prohibitively difficult to discern which genomic aberrations occur concurrently and to comprehensively define the genomic contexts of patient samples. Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of clinically annotated samples with patient follow-up data including diagnostic and therapeutic outcomes. As such, these samples represent highly desirable and informative materials for the application of high definition genomics that could improve patient management and provide the molecular basis for the selection of personalized therapeutics. However a major limitation to the use of these samples for high resolution genomic analyses to date is the highly variable quality of the DNA extracted from samples of interest. Flow cytometry has been used to identify and isolate neoplastic clones from primary biopsies in a variety of tissues using objective quantifiable markers. Once identified individual populations can be flow purified to greater than 95% purity for subsequent molecular analyses. We have recently developed methods that adapt single parameter flow cytometry of fresh frozen samples to high definition clonal aCGH analyses of pancreatic cancer. The overall objective of this application is to extend these methods by developing and validating multiparameter flow cytometry assays that are compatible with high definition array CGH analyses and next generation sequencing of clinical samples. These will include diploid and aneuploid cell populations from formulin fixed paraffin embedded (FFPE) samples of pancreatic adenocarcinomas.
 
R21 CA143351-01 2010 BURKE, PETER J. UNIVERSITY OF CALIFORNIA IRVINE Nanoelectrode arrays for study of the molecular mechanisms, triggers, and inhibitors of apoptosis
Mitochondria are the central regulator of apoptosis, a process initiated by the activation of the mitochondrial permeability transition pore (mtPTP), an aggregate of several mitochondrial proteins. When this pore opens, the critical membrane polarization of the mitochondrial inner membrane disappears and ions equilibrate between the matrix and cytosol resulting in mitochondrial swelling. This leads to release of the contents of the mitochondrial intermembrane space into the cell cytosol, including a number of cell death promoting factors killing the cell. The mtPTP can be activated by uptake of excessive Ca++; increased oxidative stress; decreased mitochondrial membrane potential, and reduced ADP and ATP. It is generally agreed upon that repression of apoptosis is one of the fundamental steps in tumorigenesis. Cancer cells acquire unresponsiveness to apoptosis facilitating signals, thus enabling uncontrolled proliferation. For this reason, the induction of apoptosis is one of the modes of actions of chemotherapeutic compounds. In order to allow further high throughput studies of the biochemical facilitators and inhibitors, of apoptosis, and to determine if changes in individual mitochondrial membrane potential P are important to cellular metabolism, we need to develop a system to monitor P in individual mitochondria. To accomplish this objective, we propose to extend studies that have monitored the action potentials in neurons using an array of parallel electrodes to which the mitochondria are adhered. Our thesis is that a nanoelectrode technology can be developed to capacitively measure membrane potential across the mitochondrial inner membrane phospholipid bilayer without actually penetrating the membrane. We propose to develop nano-electrical transduction sensor arrays with sufficiently high spatial and temporal resolution to monitor the charge changes on the surface of a mitochondrion sized lipid vesicle and the individual mitoplast. With this technology, we will then interrogate the regulation of P in normal and cancer cells. Several key features on mitochondrial metabolism are now recognized as important to the alteration of cancer cell mitochondrial function: changes in the Akt signal transduction pathway, induction of hexokinase II, alteration an adenine nucleotide translocator (ANT) isoform expression, down regulation of the SOC2 cytochrome c oxidase (complex IV, COX) assembly factor, mutation in mitochondrial DNA (mtDNA) genes, and modulation of the mitochondrial permeability transition pore (mtPTP) and its interaction with the pro- and anti- apoptotic Bcl2 family proteins. While all of these are important factors in the alteration of cancer cell metabolism, they still fall short of explaining the near UNIVERSITYersal alterations in mitochondrial function observed in cancer cells. A high throughput technology to monitor P in mitochondria will allow further studies of these issues in cancer biology.
 
R21 CA137681-01A1 2010 CAI, DONG ; CHILES, THOMAS CRANE (contact); NAUGHTON, MICHAEL JOSEPH BOSTON COLLEGE A Novel Nanocoaxial Biosensor for Detection of Cancer Biomarkers
Many cancer therapies are limited and usually ineffective in their success once a tumor has spread beyond the tissue of origin. For many cancer diseases, five- and ten-year survival responses can approach 90% when detected at an early stage, whereas it may drop to 10% or less when detected at a late stage. Tumors release or ""shed"" cellular materials and as a consequence many of these so-called ""biomarkers"" can be found in the blood and other fluids. Clinical measurement of biomarkers offers the promise of a noninvasive and cost effective screening for early detection of cancer. Measurement of a single cancer biomarker however, is usually not sufficient to achieve the sensitivity required for accurate early-stage cancer screening. Rather, the simultaneous measurement of a panel of biomarkers will be necessary to reach this goal in an overall clinical screening program. Concomitant with the pace of discovery of novel cancer biomarkers, comes a need for low cost, real-time, ultra-sensitive, multiplex biomarker detection. The research described herein will develop and test proof-of- concept of a novel 3-dimensional carbon nanotube-centered ""nanocavity"" array (nanocoax) for the quantitative detection of multiple cancer biomarkers. The sensor unit both constitutes a nanoscale capacitor and forms a nanoscale coaxial transmission line built around an aligned internal conductor that, in turn, can be coupled to a biomarker recognition component. In contrast to nanosensors built around 2-dimensional array architecture, the nanocoax design will enable unprecedented sensitivity, selection, combined with proofreading capability for the simultaneous detection of several distinct biomarkers, due to the platform design, which allows 8 2 for a high site density of discrete sensors/chip (10 /cm ). Moreover, the nanocoax biosensor will incorporate a non-optical design based on dielectric impedance spectroscopy detection, enabling label-free measurement and eliminating the need for any sophisticated optical instrumentation. The proposed study will incorporate as a ""model"" biomarker, the ovarian cancer biomarker CA125, together with a panel of putative ovarian cancer biomarkers that have recently shown promise for early stage ovarian cancer detection. The specific aims are three- fold: 1) to optimize fabrication and evaluate performance of a single nanocavity; 2), demonstrate proof-of-concept for detection of ovarian cancer biomarker CA125; and 3) demonstrate proof-of- concept for detection of multiple ovarian cancer biomarkers.
 
R21 CA147831-01 2010 CHIU, DANIEL T UNIVERSITY OF WASHINGTON High Sensitivity Detection and Isolation of Circulating Tumor Cells
In recent years, correlation between the number of circulating tumor cells (CTCs) in peripheral blood and survival in metastatic cancer patients has been reported. These reports demonstrate that the number of CTCs can provide an early and reproducible indication of disease progression and treatment efficacy, and establish CTCs as an emerging and important marker of disease status. The detection of CTCs thus may represent an early indication of micro-metastasis caused by the shedding of tumor cells from aggressive tumors into blood. The ability to isolate CTCs and subsequently profile the biochemical changes that have occurred in these cells will greatly enhance our understanding both of cancer metastases and in assessing the metastatic risks of cancer patients. It is, however, difficult to detect and study CTCs. The principal difficulty originates from the rarity of CTCs, especially in peripheral blood where they are the most accessible from a clinical perspective. To address this challenge, we propose to develop a sensitive technique for the isolation of CTCs from blood. Our approach is based on aliquoting blood into nanoliter-volume droplets, high-sensitivity and high-speed optical detection of a CTC within the nanoliter-volume droplet in a flow-through format, sorting of the droplet that contains the CTC, and isolation of the CTCs contained within the sorted droplets. Our specific aims are: (1) Develop a microfluidic device for aliquoting blood into nanoliter-volume droplets and an optical system for the high sensitivity flow-through detection of a fluorescently labeled CTC contained in the nanoliter aliquot of blood. To increase the sensitivity and throughput of our method, we will also develop and employ conjugated-polymer dots (CPdots)-tagged antibodies as highly fluorescent labels of CTCs. CPdots are orders of magnitude more fluorescent than dyes or quantum dots, thereby allowing us to detect CTCs with a high signal-to-noise in fast flow. (2) Develop an appropriate microfluidic-based sorting technique for sorting the droplet containing the detected CTC. (3) Develop a method to remove CTCs from the sorted droplets and from other blood cells present in the droplets. PUBLIC HEALTH RELEVANCE: Circulating tumor cells have been advocated as a marker of cancer prognosis because these cells are the ones that get lodged in organs to give rise to vascularized macrometastases. Here, we propose to develop a highly sensitive technique for the detection and isolation of circulating cancer cells from whole blood. The availability of this tool will allow for a better understanding of the makeup and physiology of these cells, which in turn will facilitate the development of more accurate prognostic markers as well as more effective therapies to eradicate these cancer cells.
 
R21 CA147912-01 2010 DIEHL, MICHAEL RICHARD RICE UNIVERSITY Multiplexed Reiterative Immunofluorescence Analyses via Engineered DNA Circuitry
The evaluation of the spatial distributions of molecular marker levels in cells and tissues via immunohistological analyses constitutes a vital component of the diagnosis, prognosis and clinical management of human diseases including cancer. Nevertheless, immunohistological methods remain substantially restricted by the fact that only a few molecular markers can be examined on a single specimen. Considering that the size of clinical specimens can be small and that the number of informative molecular markers can be large, the types and number of molecular and cellular analyses that are actually performed on individual samples are frequently compromised. These issues limit current efforts to personalize the clinical management of cancer via molecular marker analyses. Furthermore, the present need to utilize multiple tissue sections or aspiration biopsies to examine multiple markers limits the ability to fully characterize individual rare cells and cellular niches. This project will surmount these problems by developing a new multiplexed and reiterative immunofluorescence imaging method called DNA-Catalyzed Molecular Biomarker Imaging and Amplification (DC-MBIA). Employing principles from the field of DNA-nanotechnology, DC-MBIA enables (1) the selective fluorophore labeling of multiple molecular probes (e.g., unique DNA-conjugated antibodies that direct nucleotide sequence-specific reactions of fluorophore-bearing DNA-complexes), (2) the stoichiometric amplification of fluorescent signals in the local proximity of a molecular marker, and (3) the removal of fluorophores from a sample via exceptionally-mild processing conditions. In this way, DC-MBIA permits fluorophore reutilization on a single specimen; the same types of fluorescent dye molecules can be selectively exchanged between molecular markers, and hence, distinct fluorescent channels of a microscope can now be used multiple times to image several sets of molecular markers, even if the markers of interest are present at low levels. While building the necessary infrastructure to facilitate this advance, this project will evaluate and optimize protocols for DC-MBIA to facilitate multiplexed and reiterative marker analyses. Here, the short-term feasibility goal is to demonstrate a minimum four-fold enhancement in the number of molecular markers that can be examined on a single specimen over that of current technologies (i.e., several tens of markers imaged, with line of sight to resolve hundreds). PUBLIC HEALTH RELEVANCE: The proposed project will create a new molecular probe technology that allows large numbers of molecular biomarkers to be examined on a single clinical biopsy, and hence, will improve the utility of biospecimens for the early detection and clinical management of cancer. Furthermore, the proposed technology will surmount current technological barriers that prohibit characterization of rare cells and low abundance markers within a single specimen, which in turn will lead to a better understanding of the molecular and cellular-level changes that occur within tumors, and assist in the future discovery of new targetable cancer markers.
 
R21 CA138331-01A1 2010 HAKANSSON, KRISTINA UNIVERSITY OF MICHIGAN AT ANN ARBOR Novel Approaches for Structural Determination of Cancer Stem Cell Glycans
Pancreatic cancer is expected to cause >32,000 deaths in he United States this year. This high mortality is largely due to lack of reliable methods for early tumor detection, and lack of treatment options that produce a cure. One complication with traditional cancer treatments is that they can easily miss the small subset of cells, termed stem cells, which have been shown to be responsible for a tumor's ability to proliferate. Aberrant protein glycosylation is linked to the onset and progression of cancer. Particularly, acidic glycans with sialic acid and sulfate groups are often altered. Interestingly, recently identified cell surface markers of pancreatic cancer stem cells are glycoproteins. However, due to tremendous analytical challenges associated with structural determination of labile, heterogeneous and branched glycans, detailed cancer-associated glycan structures, which are required for generation of novel diagnostics and therapeutics, including cancer vaccines, are scarce. Mass spectrometry (MS) can provide sensitive and accurate glycan analysis. However, a major challenge in acidic molecule MS is low ionization efficiency. A second challenge is the determination of saccharide branching and specific linkage. Also, sialic acids and sulfate groups are extremely labile, further compromising ionization and rendering sulfate localization difficult. This application focuses on developing novel MS approaches for identifying and structurally characterizing glycans uniquely expressed by pancreatic cancer stem cells (potential biomarkers). Specifically, zirconia and titania surface chemistry will be utilized to enrich sialylated and sulfated glycans from complex mixtures, thereby greatly improving their detection by nano-scale normal phase liquid chromatography Fourier transform ion cyclotron resonance (FTICR) MS. For structural determination of these glycans, we will utilize metal-assisted electron capture dissociation, electron detachment dissociation, and vacuum ultraviolet photodissociation, respectively, to increase sugar cross-ring cleavage, which provides linkage information and therefore allows determination of branched structures, and to determine sulfate location. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a major death cause in the United States with a five-year survival rate of < 5%. This research focuses on developing novel approaches for improved detection and structural determination of carbohydrates (sugar molecules) present on the surface of pancreatic cancer stem cells, that is, a subset of cells within a tumor that is responsible for its ability to grow and propagate. Carbohydrates are known to be altered in cancer and therefore constitute promising targets for cancer vaccine development.
 
R21 CA132741-01A2 2010 HANSEN, KIRK C UNIVERSITY OF COLORADO DENVER Methods for the Analysis of Tumor Extracellular Matrix
Recent key studies have shown that the composition of the extracellular matrix (ECM) can determine metastatic outcome, specifically whether a tumor cell will transition from a non-invasive to an invasive phenotype. However, the study of ECM interactions has been limited to reductionist methods that focus on one or a few ECM proteins. To date, the extracellular biomolecules that are responsible for influencing this change in cell phenotype are largely unknown. A better understanding of the ECM role in promoting tumor cell metastasis will be facilitated by a more global characterization of ECM composition. Proteomic approaches to study ECM have been hindered by the proteolytic and solubilization resistant properties of highly cross-linked matrix components, making study by more traditional proteomic techniques difficult, if not impossible. A major barrier to progress in this field is the lack of suitable sample preparation methods for effective molecular characterization of ECM. The focus of this grant is the optimization of ECM sample preparation methods. To develop effective sample preparation methods we will establish a reproducible source of ECM. A three- dimensional cell culture model will be used to evaluate the tumor promotional attributes of ECM secreted by two pairs of isogenic human mammary epithelial cell lines. The first areas of sample preparation development will be in the optimization of cell removal from its underlying ECM, with emphasis on eliminating contaminate intracellular proteins that co-purify with the ECM. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) profiling and Western blots will be used to evaluate the level of cellular contaminants. Next, ECM solubilization strategies and effective cleavage methods will be explored using as endpoints the number of distinct ECM protein identified and percent sequence coverage. Utilization of a label-free mass spectrometry based quantification method will provide relative ranking of the strategies and assist in further method optimization. We hypothesize that protein-protein crosslinks influence the effectiveness of matrix sample preparation methods for ECM. The two major classes of proteins that catalyze the cross-linking of ECM proteins show aberrant expression and activity in neoplastic cell lines and various cancerous tissues. We will use recombinant human tissue transglutaminase and its inhibitors to determine how crosslinking influences cell removal, solubilization and digestion efficiencies with emphasis on protein quantification. Cancer cells deposit ECM proteins into their microenvironment that can program non-metastatic cells to a phenotype consistent with metastasis. The development of the proposed cell culture model and state-of the- art ECM specific proteomic techniques will advance our ability to explore and characterize the role of the extracellular matrix in metastasis. These studies will be the foundation for translational experiments, clinical investigations and should ultimately lead to biomarkers and therapeutic approaches that will improve patient care and survivability. PUBLIC HEALTH RELEVANCE: Despite the fundamental role that the cell microenvironment plays in tumor progression, methods to study the underlying molecular mechanisms are lacking. This work is aimed at developing the sample preparation methods necessary to characterize the matrix component of the cell microenvironment so that we may ultimately develop new therapeutic strategies and identify early diagnostic markers of cancer.
 
R21 CA147985-01 2010 HULKOWER, KEREN ISAAC (contact); VOGT, ANDREAS PLATYPUS TECHNOLOGIES, LLC Transformative HTS cell migration assay for rapid screening of cancer therapeutic
The goal of this project is to develop a 384-well cell migration assay suitable for high throughput screening (HTS) of chemical libraries for cancer therapeutics. Current cell migration assays are not feasible in HTS settings; they are not robust, reproducible or cost-effective to perform. Fundamental changes to Platypus Technologies' OrisTM 96-wellcell migration assays, as described in this proposal, will make the proposed HTS 384-well assay compatible with automated liquid handling systems and high content screening (HCS) platforms. These enablements will dramatically improve testing throughput (allowing increased numbers of compounds to be tested) and efficiency (reducing hands- on time) while markedly reducing the cost per assay. The availability of a 384-well HTS cell migration assay that requires minimal numbers of cells, minute volumes of test compounds and reduced operator time will transform drug development research for cancer therapeutics. The proposed assay, offering transformative improvements over existing technologies, will be amenable to primary screens for cell migration inhibitors and will also permit subsequent secondary screens in the same assay wells for multiplexed probing of inhibitor effects on target molecules, viability and morphology using high content screening instruments. This HTS assay is based on the innovative use of a temporary cell exclusion zone comprised of a non-toxic, biocompatible polymer that will be deposited in a defined central area at the bottom of a tissue culture well. Cells are seeded and attach at the perimeter of the well, and the polymeric exclusion zone dissolves to reveal a zone that is now permissible for cell migration. The first generation OrisTM Cell Migration Assay currently provides 96 wells, populated with silicone stoppers to create exclusion zones, for investigating the effects of cell movement modulators. Using the Oris"" assay, we have demonstrated measurable migration of A-549 cells and reported z-factors of > 0.46 to support claims of assay robustness. We have shown that the assay is suitable for testing modulators of cell motility. We have further shown the ability to collect multiple pieces of information from a single test well (i.e., high content screening capable) and data analysis compatible with fluorescence microplate readers and imaging platforms. Finally, we provided recent data to support the creation of a dissolvable polymeric exclusion zone that eliminates the need for a silicone stopper and makes the assay highly amenable for use with automated liquid handlers employed by HTS laboratories. It appears that the polymer completely dissolves as evidenced by the full migration of cells into the previously restricted area and has no obvious deleterious effects on cell viability or test compounds. These data strongly support the feasibility of modifying the current, stopper-based Oris"" 96-well cell migration assay into a 384-well, high throughput cell migration assay. In this 2-year project, we propose to develop a 384-well cell migration assay that allows for greater amounts of both primary and secondary data to be obtained from a single assay well by using multiplexed staining techniques with different fluorophor conjugates. The assay will be validated in collaboration with Dr Andreas Vogt at the UNIVERSITY of Pittsburgh's Drug Discovery Institute, a HTS facility. The assay will enjoy a wide range of compatibility with a variety of HCS platforms for quick data retrieval. Our intended product will dramatically streamline the drug discovery process to facilitate rapid screening of molecular libraries for development of therapeutics that block cancer cell metastasis.
 
R21 CA140089-01A1 2010 JI, HANLEE MT. SINAI SCHOOL OF MEDICINE Mutation Analysis Of Paraffin-Embedded Tumors With Next Generation Sequencing
Creating a ""personalized"" cancer therapy strategy requires decoding the genome sequence of the individual's tumor for genetic errors, also known as mutations. In essence, this is like troubleshooting the ""operating system"" of a computer by searching for errors by scanning through every line of code that may be relevant. In the case of cancer, the relevant errors may influence drug response, tumor growth and ultimately prognosis. Generally, this has been difficult to accomplish because tumors stored in paraffin, the preferred method for all clinical laboratories, are extremely difficult to work with using the methods of current molecular analysis. Therefore, the development of novel molecular diagnostics which could help patients has been extraordinarily limited. We are developing an approach which will enable highly sophisticated ways of determining which mutations contribute to cancer development, its aggressiveness and its response to therapy. By making use of novel molecular assays integrated with what is commonly referred to as ""next generation"" sequencing, we are developing technologies which will enable any research group to conduct large scale analysis of cancers at a fraction of the time and cost currently required using archival material. In addition, these technologies will enable much more sensitive detection of mutations than is feasible with current sequencing methods. The radically improved approaches we are developing will substantially accelerate the identification of mutations which contribute to prognosis (e.g. survival, recurrence of cancer) and prediction (e.g. response to specific targeted therapies). Thus, large scale studies of thousands of individuals can now be accomplished using archival material which is commonly available and associated with critical clinical information that will lead to improved DNA diagnostics. PUBLIC HEALTH RELEVANCE: We are developing an approach which will radically improve and expand our ability to detect genetic errors in mutations from archival tumor material. Current methods are expensive, difficult to conduct, inefficient, lack sensitivity and are limited to extracting only small portions of genetic information from tumors. Our approach will dramatically increase any scientist's ability to scan specific DNA sequences for the genetic errors which influence cancer patient prognosis and their response to specific targeted therapies.
 
R21 CA143101-01 2010 KASSIS, AMIN I HARVARD UNIVERSITY (MEDICAL SCHOOL) Novel Blood Assay for Lung Cancer Detection
Every year, close to one million new cases of lung cancer (LC) are diagnosed. In the USA, it is estimated that approximately 213,000 new cases were identified in 2007 (American Cancer Society). Within two years of diagnosis, most of these patients (~160,000) will die. The diagnosis of this disease is usually subsequent to routine chest X-ray and/or CT and MRI and is confirmed upon biopsy and functional (fluorine-18-labeled deoxyglucose) PET imaging. Treatment of lung cancer can involve a combination of surgery, chemotherapy, and radiation therapy as well as newer experimental methods. However, the five-year survival rate of patients with lung cancer is approximately 15% and has not changed over the past several decades. Thus, it is imperative to develop highly specific, sensitive, and accurate methods for screening and early detection of occult disease. Tumors originate from normal cells upon the accumulation of genetic and epigenetic alterations. These transformed cells acquire new ""cancer-specific"" molecular fingerprints (DNA/RNA/protein/lipid) that give them unique phenotypes. We postulate that the false-positive and false-negative rates obtained in various blood-based, tumor-specific-signature assays are a consequence of the same Achilles' heel: they all depend on population-derived average signature profiles obtained from the blood of ""healthy"" controls, i.e. the baseline/background signature(s) is/are NOT specific to the genetic makeup of the individual being tested. In this application, we propose to (i) develop a highly innovative WBC-based assay that (a) will accurately predict the presence of primary and/or metastatic LC lesions in an animal, and (b) is independent of population-derived average signatures of ""healthy"" controls; and (ii) examine the capacity of the assay to detect the response of tumors to therapy as well as the presence of LC in animals before the tumors are diagnosed by external imaging modalities. We anticipate that the WBC-based blood assay eventually will (i) be useful in the detection of occult lung tumors with high specificity, sensitivity, and accuracy; (ii) enable the early diagnosis of tumor presence in individuals who are not known to have the disease or who have recurrent disease; (iii) move meaningful intervention to a much earlier position on the path of tumor progression, thereby forestalling the development of metastatic disease; (iv) monitor the response of tumors to routine (e.g., surgery, chemotherapy) and experimental treatment(s); and (v) allow tumor detection, diagnosis, and treatment to be closely coupled (i.e., personalization of LC therapy).
 
R21 CA143177-01A1 2010 LEE, CHENG S UNIVERSITY OF MARYLAND COLLEGE PARK CAMPUS CITP-Based Selective Tissue Proteome Enrichment
In contrast to UNIVERSITYersally enriching all analytes by a similar degree, the result of the capillary isotachophoresis (CITP) stacking process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins will drastically reduce the range of relative protein abundances within complex tissue proteomes, and greatly enhance the proteome coverage using the CITP-based proteomic technology. Our proposed research efforts therefore aim to fully characterize, develop, and exploit the use of this differential concentration effect to achieve comprehensive proteome analysis of clinical specimens with limited sample availability. The laser capture microdissection (LCM) process provides a rapid and straightforward method for isolating selected subpopulations of cells for downstream biochemical and molecular analyses. On the basis of cell enrichment, LCM also serves as a targeted sample fractionation approach toward the reduction in protein complexity and relative abundance. The proposed coupling of tissue microdissection for diseased cell enrichment with CITP-based selective analyte concentration not only presents a synergistic strategy for the detection and characterization of low abundance proteins, but also offers a novel biomarker discovery paradigm for enabling the identification of tumor-associated markers, exploration of molecular relationships among different tumor states and phenotypes, and a deeper understanding of molecular mechanisms that drive cancer progression. PUBLIC HEALTH RELEVANCE: By combining our unique bioanalytical capabilities with the expertise of Dr. Zhengping Zhuang at the National Institute of Neurological Disorders and Stroke (NINDS) in tissue microdissection and neuropathology, the proposed research represents a synergistic effort toward the development, evaluation and validation of a novel biomarker discovery paradigm for enabling the proteomic analysis of tumor cells and their micro-environment in support of cancer research, diagnosis, and treatment.
 
R21 CA137703-01A1 2010 LIU, GUODONG NORTH DAKOTA STATE UNIVERSITY Development of a Hand-Held Cancer Biomarker Monitor
The project will develop a Hand-Held cancer biomarker monitor based on imunochromatographic technology and nanoparticle based electrochemical immunoassay for rapid, sensitive, and low- cost detection of cancer biomarkers in human blood samples. Breast cancer biomarkers including carcinoenbryonic antigen (CEA), CA15-3 and human mammaglobin will be used as model biomarkers to demonstrate the proof of principle. The principle of the proposed device is based on nanoparticle-powered multiplex bioelectrochemical immunodetection and immunochromatographic separation technique. The multiplex capabilities and significant signal amplification of electrochemical immunoassay are realized conveniently by the use of multiple metallic phosphate nanoparticle labels. Integrating with immunochromatographic separation technology, the new biosensor microanalysis device will provide a portable, sensitive, simple, and low-cost tool for the rapid detection of multiple breast cancer biomarkers. The complete assay time will be less than 10 minutes and the detection limits are 10 times lower than the cutoff values. The device will be used to detect the samples from breast cancer patients and the results will be validated with traditional enzyme-linked immunosorbent assay (ELISA). If the project is successful, the proposed device will be an effective and innovative tool in aiding early cancer diagnosis, monitoring response to therapy and providing real-time prognostic information in patients with cancers. The developed device can also be applied for the detection of other cancer biomarkers. PUBLIC HEALTH RELEVANCE: Cancer is a major and increasing public health problem worldwide. In US, it is the second leading cause of death, just behind heart disease. It is estimated around about 565,650 Americans are expected to die of cancer, more than 1,500 people a day (Cancer Facts and Figures, 2008). The key to decrease the death rate is to detect the cancers as early as possible. However, the majority of patients are diagnosed as having cancer at a late stage. For example, 72% of lung cancer patients, 57% of colorectal cancer patients, and 34% of breast cancer patients in the US are diagnosed at late stage. Traditionally, the diagnosis, screening and staging of cancer, as well as the evaluation of response to therapy have been primarily based on mammography, magnetic resonance imaging (MRI) and biopsy. These methods, although having a high detection rate, are expensive, time-consuming, invasive and uncomfortable, and have significant limitations for predicting a given tumor's potential for progression and response to treatment. There is, therefore, a need for an inexpensive, noninvasive, quick and simple tool with a high sensitivity and specificity for the detection of cancer biomarkers in aiding early cancer diagnosis, monitoring response to therapy and providing real-time prognostic information in patients with cancers. The proposed research for developing a hand-held cancer biomarker monitor is greatly needed for rapid, sensitive, low- cost, and multiplex detection of cancer biomarkers in human blood. Breast cancer biomarkers including carcinoenbryonic antigen (CEA), CA15-3 and human mammaglobin (HMAM) will be used as models to demonstrate the proof of principle. If the project is successful, the developed device can also be applied for the noninvasive monitoring of other tumor biomarkers in biological fluids such as blood.
 
R21 CA151197-01 2010 MITRA, ROBI D WASHINGTON UNIVERSITY HIGHLY PARALLEL EDMAN SEQUENCING OF INDIVIDUAL PEPTIDE MOLECULES
Most biological processes are executed by proteins, but no method currently exists to accurately measure protein abundance and post-translational state proteome-wide. To redress this deficiency, we propose Digital Analysis of Proteins by End Sequencing (DAPES), a method that sequences many individual peptide molecules in parallel using Edman degradation. DAPES will be cost-effective, highly sensitive, and quantitative. DAPES is based on two innovations - 1) the use of dye-labeled antibodies to inexpensively and robustly detect single peptide molecules; and 2) a strategy that uses a UNIVERSITYersal set of ~20 antibodies to sequence peptide molecules. Our previous work, in which we used fluorescent antibodies to detect and quantify protein levels by single molecule counting, demonstrates that this approach is realistic and powerful. PUBLIC HEALTH RELEVANCE: Most cellular functions are performed by proteins, yet current methods are unable to accurately quantify protein levels and post-translational state in a comprehensive manner. This shortcoming is preventing a quantitative understanding of normal cellular processes, the mechanisms by which they fail, and how these failures lead to disease. To redress this deficiency, we propose to apply recent advances in single-molecule imaging to the field of protein detection. By sequencing single peptide molecules in parallel we will develop a protein analysis tool with unprecedented sensitivity, dynamic range, and utility.
 
R21 CA147967-01 2010 POPESCU, GABRIEL UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN Label free imaging of blood smears and tissue biopsies
This exploratory project is aimed at investigating the potential of newly developed optical interferometric technique that we developed- Spatial Light Interference Microscopy (SLIM)-- for characterizing blood smears and tissue biopsies. SLIM is label-free and uniquely sensitive to refractive index modifications brought to the biospecimen by disease development . If successful, in the long-run, i.e. 5-10 years period, we anticipate that this label-free approach to imaging biospecimens will provide faster access to clinically-relevant data, as it bypasses most of the histology preparation process, e.g. staining. Further, due to its exquisite sensitivity to structural changes in the tissue, SLIM will provide a greater wealth of information than common histology, which should aid the pathologist in reaching a faster, more accurate diagnosis. The exploratory stage project will have the following specific aims: develop advanced, high-throughput instrumentation, perform proof-of principle studies on blood smears with focus on several specific diseases, and image biopsies of normal and three types of cancers (breast, prostate, and lung).
 
R21 CA147978-01 2010 RICHARDSON, ADAM DAVID SANFORD-BURNHAM MEDICAL RESEARCH INSTIT Functional Metabolomics and Metabolic Flux Analysis in Cancer
The mechanistic and transformative role of metabolism in tumor initiation and progression is a research topic of increasing importance. Isotopomer-based functional metabolomics and metabolic flux analysis are the most direct and informative approaches with which to study cellular metabolic processes. However, for a number of reasons, the robust isotopomer methodologies developed in bacterial systems are not commonly used in mammalian cancer research. This research proposal aims to evaluate and address the major issues confronting the application of isotopomer tracing in mammalian cells, including the use of non-glucose substrates, sample size and throughput, and the relevance of key tumor model systems. We will build on our recent development of a glucose-based metabolic model for human tumor cells to improve our detection capabilities, expand our isotopomer model and determine the extent of conservation between in vitro and in vivo models, all with the overall goal of facilitating the broader use of functional metabolomics in cancer research. This will directly support the both NCI's goal of funding research with a high potential for positive patient impact, and the IMAT program's goal of supporting the development of transformative technologies. Specifically, we are asking three interrelated questions. (1) How can we conserve the isotopic and chemical information provided by isotopic labeling while significantly decreasing sample size and increasing throughput? (2) Which metabolic fluxes can be quantified by tracking various metabolic substrates, and how is this information best incorporated into a comprehensive flux model? (3) To what extent does tumor cell metabolism in two-dimensional tissue culture reflect tumor metabolism in vivo? To answer these questions, we will (1) compare the isotopic information gained from GCMS analysis with that from NMR analysis and determine the how much information is conserved between the two methods; (2) perform a systematic evaluation of the utility of each of these compounds in mapping tumor cell metabolism in order to determine which metabolic pathways are most accessible to each precursor; and (3) compare the metabolic program of two-dimensional tissue culture, three-dimensional tissue culture and mouse xenograft models to determine which metabolic activities are conserved between systems and which vary with the microenvironmental conditions.
 
R21 CA143349-01 2010 RIETHMAN, HAROLD C. WISTAR INSTITUTE Technology for detection and quantitation of telomeric DNA aberrations in cancer
Telomeric DNA abnormalities are a critical and UNIVERSITYersal aspect of carcinogenesis. The gradual replicative loss of telomeres ultimately results in telomere dysfunction and plays a role in many age-related diseases, including cancer. Sporadic telomere deletion events are a UNIVERSITYersal cell-intrinsic mutational mechanism that can lead to dysfunctional telomeres and chromosome instability; the frequency of these events is believed to depend upon factors such as DNA replication fork stalling and other DNA replication stress, oxidative damage, and homologous recombination events such as unequal sister chromatid exchange (SCE) and T-loop recombination. Sporadic telomere deletion events occur at a very low frequency in normal cells; an increased frequency of these events (and hence dysfunctional telomeres) may be among the very first mutational events in carcinogenesis. Repair of dysfunctional telomeres can result in telomere-telomere fusions, telomere- chromosome arm translocations at sites of internal double-strand DNA breaks, and additional DNA rearrangements as a consequence of repeated fusion-breakage-fusion cycles that result in genome instability and help drive cancer progression. Eventually, activation of telomere maintenance mechanisms (either telomerase-based or ALT-based) are believed to help stabilize dysfunctional telomeres and permit rapid growth of tumor cells. It is impossible to measure accurately the global frequency of sporadic telomere deletion events and telomere fusions with current technology. As a consequence, telomere mutational data are totally absent from the high- throughput datasets being acquired from tumor samples as part of the Cancer Genome Atlas, and telomere mutational data gleaned from a few labor-intensive studies of telomere function in cellular and organismal cancer models are incomplete and biased. Our lab has focused upon detailed analyses of human telomeric DNA structure and variation; here, we propose to use this knowledge to develop a UNIVERSITYersal, high throughput assay for detection and quantitation of telomeric DNA mutational events in humans and to obtain proof-of-principle data for its utility. The method couples the physical enrichment and purification of telomeric DNA with quantitative analysis of the telomeric genome fraction by high-throughput paired-end sequencing. It is designed to detect and quantitate single-allele-resolution ultrashort (TTAGGG)n tract profiles, telomere fusions, and subterminal DNA breakage-rejoining events. In addition, it is amenable to future refinement to permit miniaturization and multiplexing of the assays. The quantitative, single-allele-resolution measurements of telomere length and instability will permit unprecedented insights into the role(s) telomere loss and telomere fusion play in carcinogenesis, including mechanistic insights into molecular events mediating these processes and translational insights for the potential prognostic and tumor stratification applicability of the method.
 
R21 CA151139-01 2010 SIEGMUND, KIMBERLY D UNIVERSITY OF SOUTHERN CALIFORNIA Applying Molecular Phylogeny to Predict Clinical Outcomes in Cancer
Our goal is to investigate the utility of cancer molecular phylogeny, a novel technological concept, to predict clinical outcomes. Rather than use novel high-throughput technologies to identify a molecular marker or signature to predict survival, we propose to use a molecular measure of cancer age. Estimation of cancer age is complicated by the fact that we do not observe the initial transformation and clonal expansion. What we do observe is a population of cells that are descendants of the original transformed cell. What we can measure is the molecular diversity of the population. Population genetics dictates that cells from an older population will show more diversity than cells from a younger population. Thus a molecular measure of tumor diversity should capture tumor age. We propose that tumor age will help predict patient outcomes. Thus, we challenge the current paradigm of finding a common pathway of cancer development that leads to poor survival. Instead we propose that older tumors are more diverse, regardless of the sequence of mutations accumulated, and that it is this diversity that makes them resistant to chemotherapy and prone to dissemination, thus leading to poorer outcomes. We propose to calibrate this novel technology using cell lines, in order to characterize the association between diversity and number of passages of cell division at a large number of (epi)genomic regions. We hypothesize that this basic, but presently unstudied, tumor characteristic of aging, will predict clinical outcome. We propose to demonstrate this for the special case of cancer patients with Stage III colon cancers. If successful, the approach has promise for predicting clinical outcome for solid tumors of many different cancer sites (e.g. breast, lung, prostate). This application has two Specific Aims: Aim 1: Characterize 70 somatic cell cancer molecular clocks to measure progression histories; Aim 2: Test whether the timing of metastases correlates with Stage III colorectal cancer disease-free survival. PUBLIC HEALTH RELEVANCE: Our hypothesis is that older cancers are more diverse cell populations and more resistant to treatment than younger cancers. However, this simple hypothesis has never been tested because we do not observe the initial transformation and clonal expansion of a cancer, and therefore cannot measure tumor age directly. We resolve this fundamental challenge by apply concepts from the field of population genetics to infer tumor age. In this project we apply tools for molecular phylogeny to predict clinical outcomes in cancer research.
 
R21 CA143275-01 2010 SOOD, ANUP GENERAL ELECRIC GLOBAL RESEARCH CENTER Releasable Antibodies for Multiplexed Analysis of Cancer Biomarkers
This proposal seeks to overcome the limitations of current and emerging multiplexed technologies that are critical to improving cancer diagnosis and treatment. These technologies utilize classical antibodies for biomarker detection, and as such are hampered by scarcity of species in which the antibodies are raised, steric hindrance, harsh multiplexing conditions, and permanent modification of the cell or tissue sample. To overcome these limitations and simultaneously enable multiplex analysis of living cells, GE will develop labile antibodies with controllable affinity. Specifically, for demonstration purposes, existing antibodies against HER2 will be modified and the resulting conjugates will be prepared at varying degrees of modification and characterized by UV-vis spectroscopy, LDS-PAGE and MALDI-TOF-MS. These conjugates will then be evaluated for their capability to bind HER2 using fixed SKOV-3 cell pellets by fluorescence microscopy and the binding kinetics will be quantified via surface plasmon resonance (Biacore). The resulting information will be used to prepare improved anti- body conjugates for validation of the technology's multiplexing capabilities. Specifically, sequential detection of HER2 and Ki67 biomarkers will be performed in clinically-relevant human breast cancer samples using primary antibodies from a single species. The specific aims of the work will be to prepare and characterize a library of modified anti-HER2 antibodies, identify and optimize lead antibody conjugates, and to validate the multiplexing capabilities of the lead antibody conjugates on clinically-relevant tissue samples. Successful development of the proposed technology will allow unlimited multiplexing with the primary antibodies from the same species; detection and quantification of multiple closely-spaced biomarkers in the same tissue sample; assessment of biomarker spatial information; correlation of biomarkers to each other, to disease progression, and to treatment response; and the opportunity to perform multiplex analysis on living cells in addition to fixed tissue. PUBLIC HEALTH RELEVANCE: The ability to screen tissue samples or individual fixed or living cells for biomarkers in a multiplexed fashion is critical for enhanced cancer diagnostics and the development of more targeted therapies. The proposed project will achieve this by chemically modifying antibodies targeted against disease biomarkers to achieve controllable affinity, which will enable efficient binding and release. These antibodies will preserve the integrity of the tissue or cell sample while enabling potentially unlimited multiplexing capabilities without restrictions due to limited antibody-species availability or overlapping biomarker epitopes.
 
R21 CA151128-01 2010 SOUSA, RUI J. UNIVERSITY OF TEXAS HEALTH SCIENCES CENTER HOUSTON Development of imaging reagents to monitor GTP levels and GTP/GDP ratios in vivo
Cellular processes intimately tied to cancer and to a variety of other human pathologies, are globally regulated or coordinated by GTP and ATP, molecules that represent the energy currency of the cell and which are also the most common allosteric modulators of protein function. Methods to measure the levels of these molecules in vivo, with high temporal and spatial resolution, would be invaluable in understanding how variations in GTP and ATP concentrations regulate and coordinate cellular metabolic processes, and in elucidating the role played by disruption of cellular metabolism in cancer and other diseases. Such methods-luminescence by insect luciferase and fluorescence from GFP- ATP binding protein fusions-exist for ATP. The luciferase assay is widely used and has led to important recent discoveries regarding the role of variations in ATP levels in the cell cycle and human disease, including cancer. However, no equivalent methods exist for GTP, even though GTP, through its action on numerous G-proteins, arguably plays a larger regulatory role in the cell than ATP. To address the need for such technology we will: (1) Engineer insect luciferase so that it will specifically use GTP, rather than ATP, to generate light, and (2) Engineer GFP-G-protein fusions that will exhibit altered excitation spectra upon binding GTP. These genetically encoded sensors will provide two complementary methods for monitoring GTP levels and GTP/GDP ratios inside living cells with high temporal and spatial resolution. PUBLIC HEALTH RELEVANCE: Cancer and many other human diseases, especially those associated with aging, often involve changes in cellular metabolism: alterations in the nature and concentrations of the molecules that supply the energy for cellular activity and dis-coordination in the network of the many cellular processes normally involved in maintaining cellular health. Insight into how metabolic processes are coordinated and regulated in a healthy cell can come from monitoring the levels of regulatory molecules, but this can be challenging to do in living cells. The proposed work will make it possible to monitor levels of GTP, one of the most important of these regulatory molecules, and will therefore create an invaluable tool for cancer and biomedical research.
 
R21 CA147975-01 2010 SZMACINSKI, HENRYK UNIVERSITY OF MARYLAND BALTIMORE Method for Detection of Secreted Proteins in Single Cell Assays
In this application we propose to develop an improved method for detection of proteins secreted from single cells. The proposed method offers several significant advantages over the existing technologies; a simple biochemical protocol, detection of multiple secreted proteins, and real-time monitoring of the secretion process. Our approach is to employ optical amplification of fluorescence signal from a probe bound to the surface in the vicinity of a protein-secreting cell. The optical amplification will be achieved using metallic nanostructures deposited on the surface of a solid support. The method is termed MEFspot (Metal-Enhanced Fluorescence spot). This will avoid the use of more complex enzymatic amplification or biochemical amplifications used in current technologies; enzyme-linked immunospot (ELISPOT) and FLUOROSPOT, respectively. MEFspot represents a significant advance in single cell assays with sensitivity comparable to ELISPOT but utilizes a substantially simpler procedure, capabilities for real-time monitoring, and potential use for quantitative analysis. Specifically, we will demonstrate the application of MEFspot to the detection of cytokines from cells because cytokines play an important role during inflammation and diseases and they are regarded as the best tool to measure the activation of immune cells. It was found that a large number of diseases are associated with quantitative patterns of cytokine production. Cytokines have assumed increasing importance in cancer biology with the demonstration that many can be produced by tumor cells and can influence the malignant process both positively and negatively. The projected work will be accomplished by (1) Developing a procedure for reproducible fabrication of substrates with metallic nanostructures that perform in culture media for a prolonged length of time and result in the desired sensitivity for cytokine detection. (2) Evaluating MEFspot with several cytokines using model cell lines. (3) Demonstrating the multiplexing capability for detection dual (or possibly triple) cytokine secretion from a single T cell. (4) Validating the MEFspot method using clinical cell samples with comparison to the ELISPOT method. Various formats of MEFspot including real-time monitoring of secretion and quantitative analysis of single cell efficiency will be demonstrated. PUBLIC HEALTH RELEVANCE: The development of methods for high-throughput analysis of cell functionalities is highly in demand in current life science research, proteomics and its clinical applications, as well as in drug discovery. We propose to develop a significantly improved method for detection of proteins secreted from single cells. The method includes the optical amplification of signal from bound fluorescent probes allowing for real-time monitoring, multiplexing and quantization. It is anticipated that proposed method will be of broad use in immunology research, and in conjunction with other functional assays for detection of intracellular and blood level of cytokines, provide a better understanding of fine mechanisms of immune response.
 
R21 CA151159-01 2010 TSENG, HSIAN-RONG UNIVERSITY OF CALIFORNIA LOS ANGELES 3D-Nanostrcutured Substrates for Detection of Circulating Tumor Cells
The long-term objective of this application is to develop an integrated technology platform for highly sensitive detection and molecular analysis of circulating tumor cells (CTCs) from whole blood. The unique working mechanism based on the high affinity nanopillar-grafted substrate confers the advantages of enhanced CTC capture efficiency/purity, low operation cost and ease of use to this new technology. The PI's research group has demonstrated that a silicon nanopillar (SiNP)-covered substrate, coated with anti-EpCAM, exhibits outstanding efficiency when employed to isolate viable CTCs from whole blood samples. With a simple stationary device setting and operation protocol, CTCs can be immobilized onto the SiNP substrates because of enhanced topographic interactions between the SiNPs and cell surface components. The clinical studies of this CTC capture technology have been initiated for side-by-side validation with the FDA-approved CellSearchTM assay. In parallel, a quantitative ICC approach for multiparametric molecular profile of individual cancer cells has been established and can be directly applied for molecular analysis of CTCs. These preliminary results constitute a solid foundation for our proposed research. CTCs are cancer cells that break away from either the primary tumor or metastatic site(s) and circulate in the peripheral blood. Enumeration and characterization of CTCs in patient blood provides valuable information for examining early-stage cancer metastases, predicting patient prognosis and monitoring therapeutic interventions and outcomes. Over the past decade, a variety of technologies capable of isolating and counting CTCs have been developed based on different working mechanisms. Some of these technologies have been demonstrated in the clinical setting and allow reproducible detection of CTCs in the patient blood. However, challenges remain in improving CTC capture efficiency, reducing measurement costs and conducting sequential molecular analysis of these cells. Herein, we propose to first perform a comprehensive optimization of the SiNP-based CTC capture technology by (i) exploring the use of polymer-based nanopillars, (ii) altering the dimension and packing density of nanopillars, (iii) enabling a capability to capture a broader diversity of CTCs, (iv) incorporating anti- biofouling function, and (v) integrating a microfluidic chaotic mixer. In parallel, we will carry out optimization of an operation protocol for ICC quantification of 4-protein molecules, including cytokeratin (CK), CD45, androgen receptor (AR) and CD44, in the isolated CTCs. Next, we will use optimal CTC capture conditions to detect CTCs from whole blood samples obtained from prostate cancer patients at different stages. Sequentially, single multiparametric molecular analysis (i.e., CK, CD45, AR and CD44) of the substrate-immobilized CTCs will be carried out using the quantitative ICC approach to unveil the molecular properties and cellular heterogeneity of the CTCs. PUBLIC HEALTH RELEVANCE: The long term objective of this application is to develop a new technology platform for detection and characterization of circulating tumor cells (CTCs) from cancer patient blood. This new CTC-based diagnostic platform offers the advantages of high CTC capture sensitivity, low operation cost and user-friendliness, thus introducing a valuable point-of-care tool for patients with metastatic cancer.
 
R21 CA147993-01 2010 TURK, BENJAMIN E YALE UNIVERSITY Global peptide microarray profiling of tyrosine kinases deregulated in cancer
Protein tyrosine kinases (PTKs) play pivotal roles in human cancer and are the targets of a major class of emerging anti-cancer drugs. In a single tumor, multiple PTKs are active and a substantial number are essential for maintaining the transformed phenotype. Though large numbers of phosphorylation sites have been mapped in cancer cells through mass spectrometry (MS), the identity of the specific kinases that phosphorylate these sites are with very few exceptions unknown. We propose to use emerging peptide microarray technology to identify consensus phosphorylation sequences for the entire set of human PTKs. We will generate a set of mammalian expression vectors producing every PTK fused to glutathione S-transferase. Each PTK will be affinity purified from a mammalian cell overexpression system and subjected to peptide microarray screening. These screens will reveal specific sequences preferred by each kinase at phosphorylation sites in their target substrates. We will use this data to mine phosphoproteomics data from cancer cells to connect known sites of phosphorylation to their respective kinases. Predicted kinase-substrate relationships will be validated through siRNA knockdown of the relevant kinase in cancer cell lines. The broad goal of this work is to elucidate critical connections in phosphorylation networks in cancer cells. These studies will enrich our understanding of the basic mechanisms of cellular transformation and tumor maintenance, provide insight into the mechanisms of action of kinase-targeted therapeutics, and suggest new targets for therapeutic intervention.
 
R21 CA143282-01 2010 WALDMAN, TODD A GEORGETOWN UNIVERSITY Development and Application of Endogenous Epitope Tagging Technology in Human Cel
Project Summary Virtually all oncogenes and tumor suppressor genes will likely be identified over the next decade via next- generation sequencing of human cancer genomes. As these ongoing cancer genome projects move to completion, attention will invariably shift away from the identification of cancer genes and towards determining their functions and the pathways they control. In an effort to develop new technologies for the identification of cancer gene/pathway function, we have recently developed a new technology that makes it possible to quickly and easily identify the interaction partners of endogenous human proteins (i) in human cells, (ii) without requiring high quality antibodies to the individual proteins of interest, and (iii) without the need for ectopic expression of epitope-tagged transgenes. This approach, which we refer to as ""endogenous epitope tagging,"" exploits recent advances in human genomic modification, making it possible to relatively quickly and easily add an epitope tag to the amino or carboxyl terminus of a protein via modification of the endogenous allele of its gene. After growing isogenic sets of parental cells and epitope-tagged derivatives, protein lysates are prepared and immunoprecipitation/mass spectrometry performed. In this way it is possible to identify, in an unbiased way, proteins immunoprecipitated from epitope-tagged cells but not from the otherwise isogenic parental cells that lack the epitope tag, thereby identifying candidate proteins that may interact with the tagged protein. Of note, such an approach has been successfully applied to generate complete interactomes of the lower eukaryote Saccharomyces cerevisiae, but because of limitations in homologous recombination technology has only very recently been applied to human cells. Here we propose to further develop the technology in human cells, enabling more efficient protein production and purification (Specific Aim #1), and to apply the technology to the initial identification of a cancer-pathway interactome (Specific Aim #2). The long-term goal of these studies is to provide a foundation for the eventual expansion of these efforts at the scale of complete signal transduction pathways, and eventually the entire human proteome. PUBLIC HEALTH RELEVANCE: In this application we propose to further develop and apply a new technology that makes it possible to determine the function(s) of proteins that are intimately linked to the pathogenesis of cancer. Such insights would be expected to: i) provide important clues about the potential effectiveness of new anticancer drugs, ii) provide new linkages cancer pathways, and iii) aid in the discovery new cancer-causing genes and proteins. In addition to accomplishing these goals, funding for this project will enable us to demonstrate the feasibility needed to initiate a larger scale approach to the ongoing identification of a dynamic and evolving cancer interactome.
 
R21 CA138366-01A2 2010 WU, MINGMING CORNELL UNIVERSITY A 3D microfluidic platform for quantitative assessments of tumor cell migration
Cancer cell motility, chemotaxis as well as its ability to transmigrate through an endothelium layer play important roles in cancer cells' metastatic cascade. Cancer metastasis is a dynamic and complex process, it involves cancer cells leaving the primary tumor, entering blood circulation, arresting in blood vessel, invading a distant organ, and growing a new tumor. Rather than primary tumors, metastases are responsible for most cancer deaths. Despite their clinical importance, cancer metastases remain poorly understood. Current gene/protein expression profiling work has revealed many molecular factors that are responsible for cancer metastases. Intra-vital cell imaging in animal models has, for the first time, connected the cancer cell metastatic behavior directly with the molecular mechanism in vivo and provided insights into the cancer cell metastatic pathways. Despite of all the advances in our understanding of the cancer metastasis processes, inhibitors derived from these studies have either lacked specificity and/or been ineffective clinically. This is, in part, due to our lack of understanding that cancer cells never act alone. They are actively interacting with the microenvironment via the secretion of chemokines, growth factors, as well as the remodeling of the extracellular matrices (ECM). The understanding of the intricate interactions among different cell types and the extracellular matrices has becoming a critical component towards the eventual understanding of cancer metastases. We propose to bring together expertise on micro-chemical, micro-mechanical engineering and imaging (Dr. Wu), vascular vessel and cancer cell biology (Dr. Swartz) and cancer cell biology (Dr. Yen) to the challenges in both fundamental cancer cell biology and its potential applications in clinical chemotherapy for cancer metastases. Our short term goal is to build a physiologically relevant (3D), mechanically and chemically tunable in vitro model, and to bring the two important steps in cancer metastasis steps, migration and intravasation, under the light. Our long term goal is to find the key molecular players in the tumor cell microenvironment that underlie the cancer cell's metastatic behavior. To achieve this, we propose the following Specific Aims Aim1: To develop a 3D high throughput, hydrogel based, microfluidic in vitro model, with tunable micro- chemical and micro-mechanical environments, for mimicking two important steps in cancer cell metastasis - tumor cell migration within a ECM and intravasation from a 3D ECM through a vascular vessel. Aim2: To develop a computation algorithm, in conjunction with a newly developed 4D imaging technique, to automatically score the tumor cell invasiveness (characterized by cell motility, chemotaxis and cell transmigration rate). This Aim is critical in the realization of a truly high throughput system. Aim3: To assess quantitatively the tumor cell invasiveness in vitro under the influences of various chemokines, growth factors, ECM compositions, as well as the presence/absence of immune cells and stromal cells. PUBLIC HEALTH RELEVANCE: Metastases are responsible for most cancer deaths. The proposed work will use the emerging microfluidic technology in conjunction with a novel 4D alive cell tracking imaging technique to investigate roles of microenvironments in cancer cell invasiveness. Experimental results will advance the basic cancer cell biology; and it will also generate microfluidic in vitro tools that will find direct applications for high throughput cancer inhibitor screening.
 
R33 CA143453-01 2010 BESTOR, TIMOTHY H COLUMBIA UNIVERSITY HEALTH SCIENCES Mammary Carcinoma and Genomic Methylation Patterns
We have developed a new technology that provides whole-genome methylation profiles. The new technology has large advantages in coverage, simplicity, accuracy, and economy over other methods. The approach combines a new method of enzymatic fractionation of DNA into methylated and unmethylated compartments and the application of emerging technologies in ultrahigh throughput DNA sequencing to profile genomic methylation patterns. We will determine sites of demethylation and de novo methylation in the genomes of a panel of cell lines derived from mammary carcinomas and of a large panel of primary mammary carcinomas that are well-characterized in terms of histopathology and expression of molecular markers. Whole-genome methylation profiling of DNA of 2 ductal carcinomas and adjacent normal tissues has been completed, as has a complete methylation profile of the mammary carcinoma cell line MCF7. We will then use fully-implemented computational methods to identify those methylation abnormalities that are most highly correlated with type, grade, and stage of mammary carcinomas. The goal is a set of diagnostic and prognostic criteria that can be used in the development of biomarkers for mammary carcinoma. A diagnostic procedure will be developed that will test for de novo methylation and demethylation of the sequences found to be most often hypermethylated in mammary carcinoma, and a simple and novel method for the evaluation of methylation abnormalities in biopsy DNA is described. Our preliminary results, together with some long-standing enigmas in the literature, lead us to propose that the methylation abnormalities so common in mammary carcinoma may reflect the operation of a system (the methylation suicide system) that kills cells that have lost growth control.
 
R33 CA143531-01A1 2010 CLAFFEY, KEVIN P. UNIVERSITY OF CONNECTICUT SCHOOL OF MEDICINE & DENTISTRY Discovery Platform for Cancer Antigens
Spontaneous breast and other cancers are often complex and heterogeneous. A current driving theme in the field of cancer biology is to refine our methods to characterize patient tumors on a molecular level such that therapies can be tailored to each individual patient. We have developed a method to utilize the patient's own immune response to isolate antibodies that they make against their cancer proteins. In the laboratory we synthesize these antibodies and identify novel cancer proteins targeted by these antibodies. The main goal of this application is to implement a platform-based method to isolate and identify tumor-specific antibodies and their cancer-specific antigens. This platform technology has the potential to yield hundreds of antibodies that can be used to target individual cancers that express the antigen recognized by these antibodies. It is the intention of these investigators to use these reagents to expand our understanding of breast cancer biology, detection of cancer specific biomarkers and as novel therapeutic treatments. These single domain antibodies are infinitely renewable and hold great promise as tools for population based biomarker screening and individualized patient-specific targeted therapy for advanced and metastatic disease. The specific aims of this application will focus on four specific goals: Specific Aim 1- Perform throughput platform screening of at least 40 breast cancer cases representing up to 65 total libraries and identification of an estimated 1000-1500 novel VH antibodies. Specific Aim 2- Identify the cognate antigens for 200-400 antigen driven VH single domain antibodies identified in Aim 1 based upon clonal expansion and somatic hypermutation scores. Specific Aim 3 - Validate identified antigens and their presentation in human breast cancer using multiplex large scale arrays. Specific Aim 4 - Assemble multiplex protein arrays of recombinant soluble domain antibodies and matching arrays of their cognate antigens for use in cancer diagnostic, screening and clinical targeting applications. PUBLIC HEALTH RELEVANCE: Discovery Platform for Cancer Antigens applies a unique biochemistry and molecular biology technology that we have developed that recovers antibodies from patients and identifies cancer proteins that the immune system has targeted as ""abnormal"". The incorporation of this platform will facilitate the development of reagents which will be useful in understanding cancer biology, detection reagents for diagnostic biomarker screening, as well as long-term potential for individualized therapeutics based upon the expression of these ""abnormal"" proteins in any patient. The platform will be applied primarily to breast cancer but holds great promise for applications to most other cancers. Finally, these reagents will be made available to the broad cancer research community to rapidly define their utility and application in multiple diagnostic and clinical therapeutic approaches.
 
R33 CA140084-01A1 2010 DAVISSON, VINCENT JO; ROBINSON, JOSEPH PAUL (contact) PURDUE UNIVERSITY WEST LAFAYETTE Specific Detection of Cervical Cancers Using Cytometry-Based Molecular Diagnostic
Cervical cancer is second to breast cancer as the most common form of malignancy in both incidence and mortality for women worldwide. The population-wide utilization of screening cervical cytology (Pap tests) has been associated with a dramatic decrease in morbidity and mortality from cervical cancer in the United States and in other industrialized nations. Despite this success, the cytologic diagnosis of cervical lesions is plagued by a persistent problem of low specificity for clinically significant high-grade lesions in patients with low-grade cytologic abnormalities. As a result, over four million women each year receive a cytologic diagnosis that requires further evaluation to rule out the possibility of high-grade dysplasia or cancer. In most cases, further evaluation does not identify underlying high-grade lesions in patients with low-grade cytologic abnormalities. Although HPV testing can play an important role for the triage of some patients, it is not useful for several cytologic diagnoses. Complicating the situation is that simple detection of high risk HPVs does not predict an underlying high grade lesion, since infections do not indicate clinically significant cervical lesions. The long-term goal of this project is to apply emerging technology to develop a high-throughput cell-based analysis with suitable specificity to identify high grade premalignant and malignant lesions of the cervical mucosa. The methods to be used in this project will employ, test and validate the approach of cytometry-based molecular diagnostics to detect false negative cervical specimens. Under the guise of the previous grant phase, an application of protein expression of p16INK4a and Mcm5 (cervical cancer biomarkers) with high- throughput flow cytometry and cell sorting has been used to identify and capture the rare cancerous cells in cervical specimens. Furthermore, a multiplexed HPV genotyping assay has been implemented to analyze the rare cells isolated in this approach. Importantly, the work flow has been implemented using common sample preparation with current pathology testing protocols. The technology and methodology being applied in this application will be implemented using an integrated workflow, with substantial automation, to assess feasibility of further translation to accommodate clinical need and to improve the standard of care worldwide. The ultimate goal is to establish a primary assay with potential to supplant slide based cervical cytology with greater sensitivity, less subjectivity, and less labor intensiveness. PUBLIC HEALTH RELEVANCE: This proposed project will apply new technology, for detection of abnormal cervical cells, to clinical samples from previous Pap tests. We will use automated analysis of protein content of cells to isolate abnormal cells, after which automated DNA analysis will determine whether cancer-causing human papillomavirus types are present. These results will be compared to the Pap smear and biopsy results of the same samples in order to determine how well our test can detect early stages of cervical cancer.
 
R33 CA137719-01A2 2010 GASKINS, H. REX ; KENIS, PAUL J A (contact) UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN FRET-based Biosensors to Monitor Redox in Cell Cycle Regulation
Cancer can be viewed as a state in which the balance between cell proliferation and cell death aberrantly favors the former. We and others have discovered that the intracellular redox environment exerts a profound influence on the normal cellular processes that regulate the balance between proliferation and cell death, including DNA synthesis, enzyme activation, cell cycle progression, proliferation, differentiation, and apoptosis. In fact, it could be argued that redox homeostasis is central to the governance of cell fate. Unfortunately, molecular mechanisms mediating redox sensitivity and regulation within cells are still poorly defined. Current pharmacological methods to alter intracellular redox state are limited by (i) their inability to operate independent of global biochemical alterations and cellular toxicity, and (ii) the required significant manipulation of culture conditions that perturb intracellular homeostasis. Our genetic constructs overcome these limitations as they enable real-time and extended assessment of alterations in intracellular redox without cellular disruption. These constructs use fluorescence resonance energy transfer (FRET), a distance- and orientation- dependent energy transfer process between donor and acceptor fluorophores. In these biosensors a change in redox induces a conformational change in the redox-sensitive switch that links the donor and acceptor, changing their distance, which in turn causes a detectable change in FRET efficiency. Here we propose to further define the sensitivity and dynamic range of our FRET biosensors relative to changes in the intracellular redox environment that appear to dictate cell fate. Advantages of this approach include: (1) the ability to quantify the change in redox state; (2) independence of sensor concentration; and (3) the ability to precisely tune the redox sensitivity and range by exchange of the switch or the fluorophore modules in the construct. Aim 1: Define the sensitivity and dynamic range of genetically engineered FRET redox biosensors during proliferation by comparison of nontransformed fibroblasts and isogenic porcine tumor cell lines with respect to the presence or absence of contact inhibition. Specifically, detection of physiologically relevant changes during successive stages of cell growth is proposed. Aim 2: Determine the extent to which the FRET biosensors are sensitive to changes in the intracellular redox environment of isogenic HCT116 p53+/+ and p53-/- cells treated with the chemotherapeutic drugs fluorouracil and doxorubicin in combination with perturbations in glutathione homeostasis. Specifically, the intracellular redox environment will be visualized in response to common chemotherapeutic drugs in combination with agents that modulate biosynthesis or metabolism of glutathione. Aim 3: Create second generation FRET biosensors that permit visual monitoring and dissection of intraorganellar local redox potentials. Specifically, we intend to quantify differences in redox potentials within subcellular organelles that are at a nonequilibrium steady-state with respect to each other in living cells. In sum, the proposed work will provide novel molecular tools that enable in depth examination of the role of redox signaling at the intracellular and intraorganellar level in cancer development. PUBLIC HEALTH RELEVANCE: This project pursues novel molecular tools-redox-sensitive biosensors-that will enable in depth examination of the role of redox signaling in cellular processes related to cancer development. Optimization of these biosensors will enable visualization of local changes in redox potential that might regulate progression through the cell cycle and mediate contact-dependent inhibition of cell growth, the disruption of which is a key hallmark of cancer. Ultimately, the tools will enhance understanding of the extent to which cancerous cells have lost the ability to mount changes in redox potential that accompany normal cell growth versus their sensitivity to these changes.
 
R33 CA138330-01A1 2010 WAKELEE, HEATHER ; WANG, SHAN X. (contact) MT. SINAI SCHOOL OF MEDICINE Cancer Sample Preparation with Micromachined Magnetic Sifter and Nanoparticles
Variations in sample preparation may contribute to major discrepancies in the quantity and type of biomolecules or cells identified by different laboratories, even though the same reagents and biosensors (or biochips) are employed. This is especially hampering our efforts to fight cancer as sample preparation is a prerequisite for cancer biomarker discovery, validation, and their use in molecular diagnosis and treatment of patients. This urgent need for cancer sample preparation requires innovative methods to bring about better and more affordable sample preparation tools. We have formed an interdisciplinary team with expertise from magnetics, nanotechnology, cancer clinical practice and research, biochemistry, and proteomics to work on a novel micromachined magnetic sifter and directly fabricated magnetic nanoparticles. The magnetic sifter can be used to separate and enrich cancer targets; including proteins and rare cells from raw samples. The utilization of precisely dimensioned structures, including the use of sub-nanometer control of quantum magnetic effects will enable high throughput, high capture yield, low impurities, and low cost. The technology is very unique because high magnetic field gradients (>1 Tesla/ 5m) and large flow rates (>1 mL/hour) are enabled by three-dimensional Si-based micromachining. The use of high-moment magnetic nanoparticles with distinctive shapes and controlled magnetic chain formation give rise to unprecedented capture and release efficiencies, cell damage minimization, enhanced characterization, and run-to-run reproducibility. The proposed project is organized in three aims: Specific Aim 1. Magnetic sifter for high efficiency capture and analysis of protein cancer markers and cancer cells: a) Construct a magnetic sifter with a high magnetic field gradient (>1 Tesla/ 5m) and large flow rate (>1 mL/hour) and demonstrate the efficient capture, release and analysis of protein cancer markers. b) Use magnetic sifter to capture and release NCI-H1650 lung cancer tumor cells cancer cells spiked into samples of human blood with an overall capture efficiency of >70%, while keeping cells viable. Specific Aim 2. Functionalization and characterization of novel magnetic nanoparticles for high efficiency high speed magnetic separation. Specific Aim 3. Magnetic sifter for rare cells capture and analysis from human blood. The utility of the grant includes drastically reducing post-separation analysis and enabling new investigations in areas where rare molecules and cell types are currently too difficult to obtain and analyze. Furthermore, separating protein and cancer markers from high concentration impurities in blood, with 1000-fold enrichment, will enable detection of low abundance cancer markers which are difficult to quantify with conventional technologies. We expect that the tools developed under this grant will have a great impact on the cancer research and treatment community.