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Project # Year
of Award
PI Name(s)
Institution Title
Abstract Text (Official)
R21 CA118526-01A1 2006 FARIS, GREGORY W SRI INTERNATIONAL Droplet Cell Array Assays
Understanding the basic genetic and molecular markers of cancer at the cellular level is vital for preventing, diagnosing, and treating cancer. Recent work in our lab has led to new technologies for manipulating small aqueous drops containing biological molecules. We use laser light to induce surface tension gradients, which allows us to selectively move very small individual droplets (mu L-pL), and we have performed simple enzymatic assays with this approach. Because our technique uses laser heating as a basis for droplet control, we believe that it is well suited to genomic analysis methods, such as polymerase chain reaction (PCR), which rely on thermal cycling. We propose to combine our laser-based droplet control techniques with genomic analysis tools to develop a device for screening large numbers of individual cells. To test the capabilities of our apparatus, we propose an R21 project with the following specific aims: Aim 1: Optimize and automate the liquid handling system for genomic analysis. We will choose the optimal materials and reagents for use in our apparatus. We will automate both the droplet delivery and the droplet handling capabilities of our device. Aim 2: Perform a real-time PCR in our droplet-based system. We will test the overall sensitivity and quantify nonspecific amplification in our binary liquid system. Aim 3: Examine single cells within small droplets. This examination will include testing for short- and long-term cell viability and PCR amplification of single genomic material. This research is fully consistent with the goals outlined in RFA-CA-06-002. If this program is successful, we believe that the resulting technology will prove invaluable in helping doctors and scientists better understand the molecular basis of cancer while also providing tools to help diagnose specific variants of disease, plan and assess therapy, and monitor disease recurrence.
R21 CA122864-01 2006 FERRARI, MAURO UNIVERSITY OF TEXAS HEALTH SCIENCES CENTER HOUSTON Nanoparticles for Harvesting and Targeting Angiogenic Proteins
This R21/R33 application entitled "Nanoparticles for Harvesting and Targeting Angiogenic Proteins" has as its hypothesis that development and refinement of surface characteristics of silica chips with nanocharacteristics can enhance sensitivity of mass spectrometry (MS) detection of the low molecular weight angiogenic proteins present in serum and tumors that produced at very early times of tumor development. In addition, refinement of conjugation methods of nanoporous particles will allow selective targeting of endothelial cells in vitro and tumor-associated blood vessels in vivo and in combination with refinement of loading strategies, cytotoxic agents loaded into nanoparticles can selectively destroy these vessels. Our experimental plan is based on our expertise in development and refinement of emerging nanotechnology approaches for protein capture, for selective targeting and loading of silicon nanoparticles. These studies also take advantage of our experience in identification of novel proteins within the vascular endothelial growth factor (VEGF) family of proteins that are essential in the process of tumor-associated angiogenesis. To achieve the goal of developing and refining tools for detection of angiogenic proteins and for selective targeting and destruction of tumor-associated blood vessels, the following Specific Aims are proposed: 1. Develop and refine silica chips with nanocharacteristics to enhance the sensitivity of LC-MS/MS identification VEGF proteins in serum and in skin tumors during skin tumor-associated angiogenesis in vivo; 2. Refine conjugation of silicon nanoparticles to anti-VEGFR-2 receptor antibodies for selective targeting of endothelial cells in vitro and targeting tumor-associated blood vessels in vivo; 3. Determine the ability of silicon nanoparticles conjugated with anti-VEGFR-2 antibodies to be loaded with and to deliver the cytotoxic agent melatin for destruction of endothelial cells in vitro and for destruction of tumor-associated blood vessels in vivo. These studies will provide sensitive nanotechnology tools that are critical in defining the proteome in serum and tumors related to tumor angiogenesis that is currently unexplored. These studies may also provide strategies to selectively target tumor vessels for destruction using nanotechnology approaches.
R21 CA114160-01A1 2006 FRANKENBURG, SHOSHANA HADASSAH-HEBREW UNIVERSITY MEDICAL CENTER Targeted antigen delivery for cancer immunotherapy
Metastatic melanoma patients currently have a dismal diagnosis, and treatment at the metastatic stage is generally ineffective. The long-term objective of this research project is to develop a novel treatment and vaccination approach for cancer in general, and melanoma in particular, based on immunotherapy. Ex vivo antigen delivery for immunotherapy is laborious and expensive, and is thus not affordable to many of those in need. The investigators propose to develop an antigenic entity that can be applied on the skin, with direct antigen delivery to skin dendritic cells and without the need for in vitro cell manipulations. Thus, the major practical objective of this study is to establish the proof of principle that topically delivered tumor associated antigens can elicit effective anti-tumor responses, and can be used for cancer immunotherapy. Specific Alms The study will be based on two antigenic proteins derived from melanoma: the first is a hydrophilic recombinant gp100 protein, and the second is a multiepitope polypeptide that comprises 3', repeats of 4 HLA-A2 melanoma peptides derived from 3 different melanoma proteins. In order to allow and to improve topical transdermal delivery, the antigens will be genetically fused to potential carrier molecules. One of these is E. coli heat labile enterotoxin, a molecule recently shown to act as carrier and adjuvant. Another is a novel haptotactic C-terminal fibrinopeptide (Haptide). During the first phase of the project, R21, the new antigenic entities will be cloned, expressed, and purified. Novel in vitro models using human skin will be used to evaluate transcutaneous passage of molecules, Langerhans' cells activation and mobilization, and stimulation of specific cytotoxic T cells. The rationale for the milestones that will determine continuation to the second phase, R33, is based on the efficacy of antigen delivered transcutaneously to stimulate the immune system in human in vitro models, and will allow for the selection of the molecules that will be further evaluated in depth in vivo models. In the R33 phase, specific immune responses of splenic T cells from vaccinated mice will be evaluated, tumor models will be established in mice, and the response to vaccination will be determined. Finally, the most effective molecule/s will be produced under GMP or GMP-like conditions for phase I/II clinical trials in a subsequent study. Public Health The success of this project would allow topical application of an immunostimulant for treatment of melanoma and other cancers and would thus significantly simplify treatment, eliminating the need for hospitalization and even day-care and without the need for a specialized laboratory. As a result, one could treat a much larger number of patients, with the potential to clinically evaluate new antigens and immunotherapeutic modalities, improving the life quality and expectancy of metastatic melanoma patients
R21 CA122630-01 2006 GHOSH, INDRANEEL UNIVERSITY OF ARIZONA Direct Detection of Hypermethylation in Cancer
The long-term objective of the proposed research project is to provide a robust, sensitive, and rapid method for the direct detection of CpG island methylation in the promoter region of specific genes implicated in cancer. Cytosine methylation occurs at CpG dinucleotides in 70-80 percent of the human genome, most often in repetitive genomic regions. On the other hand, CpG islands, defined as short sequences with statistically high CpG content, present in the promoter region of many genes (60 percent) are primarily protected from methylation in normal tissues. These CpG islands have been found to be methylated in cancer, leading to transcriptional repression. Recent experiments provide strong correlation between CpG hypermethylation at promoter sites of numerous genes and the incidence of cancer, thus making specific promoter hypermethylation a valuable marker for early detection. Current methods for detection of specific CpG island methylation rely on extensive bisulfite treatment of methylated DNA followed by PCR-based amplification, sequencing, or microarray techniques. These current methods, though powerful, are also laborious, time-intensive and expensive for characterizing known sites of hypermethylation. Towards the goal of rapidly determining promoter CpG hypermethylation, we will apply our newly developed technology called SEquence Enabled Reassembly (SEER) of proteins. The SEER system allows for the recognition of specific sequences of double-stranded DNA that result in the concomitant assembly of functional protein reporters (green fluorescent protein and beta-lactamase). In the proposed detection of specific CpG hypermethylation, we will target CpG islands utilizing the methyl-CpG binding domain (MBD) of the MBD2 protein, while targeting the correct promoter sequence utilizing a designed zinc-finger. Our approach has the potential to provide a sensitive turn-on sensor for directly reporting upon CpG methylation at known promoter sites. This approach, if successful, will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing. We will provide proof of concept by 1) designing and optimizing turn-on biosensors for detecting specific methylation events in model DNA constructs; and 2) designing and testing biosensors that target promoter regions of genes (BRCA1, CDH1, p15, p16, MGMT, GSTp1) implicated in cancer.
R21 CA123006-01 2006 HSI, ERIC CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE-CWRU Characterization of Methods for Preservation of Phosphoproteins in fixed tissues
Protein phosphorylation is an important mechanism of regulating protein function and activity that depends on a competing system of kinases and phosphatases. It is a dynamic processes that is altered in many disease states. For example, activated tyrosine kinases are central to the pathogenesis of chronic myelogenous leukemia (BCR-ABL1) and gastrointestinal stromal tumors(KIT). Detection of phosphoproteins (PPs) in fixed tissues by in situ immunohistologic methods may have diagnostic, prognostic, and therapeutic implications for cancer patients. Initial studies have shown that PPs are quite labile. Little is known regarding methods to preserve phosphorylation status in tissues. The purpose of this application is to develop optimal tissue handling methods that will be suitable for detection of PPs in fixed tissues, keeping in mid practical limitations in the clinical setting. To this end we intend to 1) develop a quantitative immunofluorescence (IF) method using quantum dots to quantitate PP status in fixed cell blocks; 2) characterize optimal fixation conditions (time, fixative, requirement of phosphatase inhibitors) in murine xenografts of human cell lines as a controlled model of available control material that is assayed both by quantitative Western blot and IF; and 4) show proof of principle of in a murine model of BCR-ABL1 containing cell line xenograft treated with imatinib mesylate (IM) and bone marrow biopsies from patients suspected of chronic myeloproliferative disorder harboring the JAK2 V617F mutation. Phospho-STATS is known to be increased in both these systems. Decreased expression by phospho-STAT5 immunostaining in IM-treated xenografts and increased expression in JAC2 V617F+ bone marrow megakarycotyes is expected in optimally handled tissues. This application has relevance in the diagnosis, prognosis, and therapy of malignancies and other diseases that have altered PP levels as part of their pathogenic pathways. It will define tissue handling conditions that adequately preserve in vivo PP status for subsequent diagnostic and prognostic testing. Furthermore, control material with defined relative expression levels of many PPs will result from this application and allow laboratories to assess performance of their individual assays.
R21 CA114043-01A1 2006 JU, JINGFANG UNIVERSITY OF SOUTH ALABAMA Novel Method for Isolating Actively Translated mRNAs
Transcriptional regulation has been the main focus for gene regulation in the past. However, a tremendous amount of evidence from recent studies also indicates that translational regulation plays a key role during development, cell cycle control, and mechanisms related to acute drug resistance. Gene expression analysis on actively translated mRNA transcripts provides a unique approach to study post transcriptional regulation. Previous studies have relied on a traditional sucrose gradient ultracentrifugation procedure to isolate polysome complexes and requires a large amount of cells (up to 500 million cells). As a result, this still remains a major bottleneck for the investigation of post transcriptional regulation with limited quantities of clinical samples. Therefore, there is an urgent need to develop a novel approach to isolate actively translated polysomes from a small number of cells (10 to 500 cells). The new approach will allow us to systematically study potential translational regulation with limited clinical samples. It has been shown that actively translated mRNAs are associated with multiple units of ribosomes and the newly synthesized polypeptides are closely associated with molecular chaperones such as hsp73. These molecular chaperones assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones will provide the anchor to separate actively translated mRNAs associated with polysomes from free mRNAs. Affinity antibody capture beads will be developed to capture hsp73 chaperones associated with the polysome complexes so that all polysomes can be separated from monosomes and free mRNAs. The isolated actively translated mRNAs will be used for high throughput gene expression analysis. The specific aims of the proposed project are: 1.) Develop antibody conjugated affinity capture magnetic beads and conditions to capture actively translated mRNAs associated with the polysome complex from a small number of cells. 2.) Validate the antibody affinity capture approach for polysome isolation by comparing with traditional polysome isolation protocols via quantitative RT-PCR gene expression analysis. 3.) Identify potential translationally regulated genes that are responsible for determining chemosensitivity during 5- fluorouracial (5-FU) treatment from human colon cancer samples.
R21 CA118600-01 2006 LARGAESPADA, DAVID ANDREW UNIVERSITY OF MINNESOTA TWIN CITIES Transposon-based somatic mutagenesis/prostate cancer genetics
Prostate cancer is the second leading cause of male cancer death in the United States and often results in a reduced quality of life for those living with or treated for this disease. Prostate cancer is commonly treated by androgen ablation therapy and although many tumors initially respond to this treatment, many eventually progress to hormone refractory prostate cancer (HRPC). The genetic basis for the transition to hormone insensitivity is poorly understood. We propose to use a mouse model for invasive prostate cancer that results from prostate specific loss of the tumor suppressor gene Pten. This mouse model is relevant to human disease as PTEN expression is lost in many human prostate tumors and the tumors that form in the mice remain partially sensitive to hormone withdrawal. We will use a novel method for cancer gene discovery in mice, the Sleeping Beauty (SB) transposon system, to promote aggressive tumor formation in this model. The SB transposon is a DMA element that is capable of mobilizing and inserting in a different location in the genome. If a mobilized transposon reinserts near a cancer gene, it can promote changes in expression of that gene that promote the transition from a normal cell to a transformed cancer cell. We have previously generated mice engineered with all the components necessary for mobilizing SB transposons in various tissues in the adult mouse. In unpublished experiments, we have successfully used the SB system to identify genes involved in sarcoma and lymphoma formation in mice, and we believe that SB will prove to be equally as successful in prostate tumor models. By using SB to promote HRPC formation we can both identify the genetic changes that cause a tumor to become insensitive to hormone withdrawal and also generate a useful mouse model of HRPC that will be useful for discovery and testing of novel chemotherapeutic agents for advanced prostate cancer. Finally, this approach represents a novel method for the unbiased molecular/genetic analysis of cancer development and could be used widely in the study of important clinical cancer problems.
R21 CA118477-01 2006 O'LEARY, TIMOTHY J. AMERICAN REGISTRY OF PATHOLOGY, INC. Recovery of RNA from Formalin-Fixed Tissues
State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This abstract is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. High-throughput molecular biologic and proteomic methods provide several promising approaches for relating genetic changes, such as mutation or altered gene expression, to metastasis, to treatment outcomes, and to survival. In cancers where the interval between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations would be more readily obtained if formalin-fixed paraffin-embedded (FFPE) tissues could be used instead of fresh or frozen specimens. Large-scale multiplex techniques, such as serial analysis of gene expression (SAGE), and gene chip methods yield experimental results that are somewhat different for FFPE tissue and unfixed tissue. The long-term goal of our research program is to use high-throughput molecular biologic screening methods to identify the molecular and genetic basis of cancer origins and behavior. The objective of this proposal is to identify the formaldehyde-induced chemical modifications that occur to nucleic acids during histologic tissue processing and to develop methods to reverse these modifications. Our centralhypothesis is that formaldehyde adducts and cross-links formed during tissue processing can be sequentially reversed by a series of heating and dialysis steps, carried out under appropriate solvation conditions. We formulated this hypothesis on the basis of preliminary data which show that the reversal of formaldehyde-induced chemical changes in proteins and nucleic acids is relatively facile in aqueous solutions, but less so following dehydration in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for applying high-throughput screening methods to FFPE tissues. This will lead to improved practical interventions for the diagnosis, evaluation, treatment, and prevention of cancer and facilitate the development of therapeutic agents. Our studies are innovative in that we have pioneered a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins and nucleic acids that occur during tissue processing. At the completion of this project it is our expectation to have established a comprehensive understanding of the formaldehyde-induced chemical modifications to mRNA that occur during tissue histology, and methods for optimally reversing these modifications. This knowledge should result in an ability to carry out genomic analysis on FFPE tissue, significantly expanding our capability to conduct genomic research and opening important new areas to practical investigation. PERFORMANCE SITE(S) (organization, city, state) American Registry of Pathology UNIVERSITY of Maryland, Baltimore County (UMBC) 1413 Research Boulevard, Building #102 Department of Chemistry and Biochemistry Rockville, MD 20850 1000 Hilltop Circle (application organization site) Baltimore, MD 21250 (contractual arrangement site) KEY PERSONNEL. See instructions. Use continuation pages as neededio provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Role on Project O'Leary, Timothy J. Armed Forces Institute of Pathology Principal Investigator Cunningham, Robert E. Armed Forces Institute of Pathology Research Associate Fabris, Daniele UNIVERSITY of Maryland, Bait. County Co-Investigator Mason, Jeffrey T. Armed Forces Institute of Pathology Co-Investigator Rait, Vladimir K. American Registry of Pathology Research Associate Sheng, Zongmei American Registry of Pathology Research Associate Disclosure Permission Statement. Applicable to SBIR/STTR Only. See instructions.' l~l Yes I No PHS 398 (Rev. 05/01) Page 2 Form Page 2 Principal Investigator/Program Director (Last, first, middle): O'Leary, Timothy Joseph [The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page.] RESEARCH GRANT
R21 CA120681-01 2006 PORTEUS, MATTHEW H UNIVERSITY OF TX SW MEDICAL CENTER-DALLAS Development of gene targeting in C. elegans and D. rerio using zinc finger
The goal of this research is to develop methods for the precise modification of specific target genes in two important genetic model organisms, the nematode Caenorhabditis elegans and the zebrafish Danio rerio. Both nematodes and fish are powerful experimental systems that combine elegant developmental biology with large scale genetics. Both systems have contributed to our understanding of fundamental problems in cancer biology, including programmed cell death, the control of organogenesis, the interaction of cancer susceptibility genes with the environment, and the genetics of melanoma. An important limitation of these model systems is that techniques for site-specific manipulation of the genome are not currently available in either nematodes or fish. Thus, in contrast to murine embryonic stem cells and the yeast S. cerevisiae, it is not possible to knock out specific genes or to precisely control the time and place of gene expression. In the last two years, a powerful new approach to gene-targeting has been developed and successfully used in flies and in mammalian somatic cells. This technique uses chimeric zinc finger nucleases to stimulate precise targeting of specific genes in their native genomic context. The aim of this proposal is to induce targeted, heritable genetic changes via zinc finger nuclease-mediated homologous recombination in C. elegans and D. rerio. Initially we will employ a well-characterized zinc finger nuclease that recognizes the green fluorescent protein (GFP) gene. We will introduce the nuclease into transgenic nematodes and zebrafish that express GFP. We expect the resulting double-strand DNA breaks to stimulate mutagenic non-homologous end joining (NHEJ), leading to the loss of GFP signal. In the second phase, we will simultaneously introduce the nuclease and a repair template that will allow us to create precise mutations in the target locus by homologous recombination. Based on the success of this work we will then target native genes in the worm and the fish by designing novel nucleases and testing them in vitro and in vivo for activity against the targeted gene. We expect that, if successful, this novel approach would be a practical, flexible, and powerful technique that would find wide application, significantly increasing the power of these systems to illuminate human cancer biology.
R21 CA123018-01 2006 SHI, HUIDONG UNIVERSITY OF MISSOURI-COLUMBIA Epigenetic Targeting in Non-Hodgkin's Lymphoma
Hypermethylation of promoter CpG islands plays a prominent role in cancer. In partnership with alterations in histone acetylation/methylation, this epigenetic event establishes a repressive chromatin structure that leads to silencing of key cancer-related genes. The occurrence of DNA methylation within the genome is not random, but rather patterns of methylation are generated that are gene and tumor type specific. How DNA methylation patterns are established is still poorly understood. Since various transcriptional factors or regulators are found in association with DNA methyltransferases (DNMTs) in vivo, we hypothesize that: 1) Oncogenic transcription factors can recruit DNMTs to target gene promoters and define a unique epigenetic signature in tumor cells; 2) Dissecting such complex epigenetic hierarchy will identify novel molecular targets for diagnosis, prognosis and therapeutic intervention. To test our hypothesis, we developed a high throughput technique for genome wide analysis of DNA methylation associated with specific proteins such as histones, transcription factors or any DNA binding proteins. The new approach named ChlP-Chop-DMH will combine both genome wide location analysis (also known as ChlP-on-Chip) and Differential Methylation Hybridization (DMH) analysis, two emerging technologies used in epigenetic research. The proposed method has distinct advantages over current protocols: first, this method directly examines the in vivo interaction of specific proteins with methylated DNA throughout the genome; second, this method may uncover novel biological properties of transcription factors; third, this method can be applied to discover novel epigenetic biomarkers relevant to tumorigenesis. In preliminary studies, we have verified the utility of this method with methylated histone H3 at lysine 9 and lysine 4 in human cancer cells. In the R21 phase, we will continue minor refinement of the method and pursue three aims: 1) Improve and optimize the ChlP-Chop-DMH method for analyzing genome wide association of DNA methylation with histone modification; 2) Utilize the proposed method to investigate the association of DNA methylation with chromatin remodeling factors: 3) Show proof-of-concept using the array to examine primary non-Hodgkin's lymphomas (NHLs). In this development phase we will focus on the sensitivity, reproducibility and accuracy of the proposed method. In the R33 phase, our goal is to utilize the technology to test biological hypotheses. We will fully implement the method and pursue these aims: 1) Discover epigenetic target genes associated with known oncogenic transcription factors c-Myc and BCL6: 2) Validate the identified epigenetic targets and investigate the regulatory role of the associated oncogenic transcription factors. This systematic approach will provide a powerful tool for future mechanistic studies as well as cancer diagnosis.
R21 CA120691-01 2006 TOLLEY, LUKE SOUTHERN ILLINOIS UNIVERSITY CARBONDALE Development of Dynamic Isoelectric Focusing for Cancer Proteomics
Though great progress has been made in the area of DNA analysis for cancer, understanding the proteins encoded by DNA can provide more answers, but is also more challenging. As the study of cancer proteomics advances, it is clear that new analytical tools and technology are needed for the comprehensive profiling of the proteins in a cell so that our understanding of carcinogenesis and the differences between healthy and cancerous cells can progress. Further understanding of cancer proteomics will drive the discovery of new drug targets as molecular changes in the cell are observed without preconceived ideas about what changes would be the most valuable to monitor. Due to the very large number of proteins in a cell, comprehensive analyses require the use of separation methods that have high peak capacities. Capillary isoelectric focusing (cIEF) has shown great promise in this area with a peak capacity in excess of 1400. This greatly exceeds traditional separation methods, such as liquid chromatography (LC), capillary electrophoresis (CE), or mass spectrometry (MS), which often have peak capacities of less than 200. An increase in the total peak capacity of a system can be achieved when multiple separation techniques are combined, leading to the popularity and performance of tandem methods such as LC/LC or LC/MS. Though the superior performance of cIEF over CE and LC would seem to make it a preferred choice in a tandem system, it is not able to be efficiently interfaced with other methods. This is the primary reason it is not widely used. The proposed research will continue the development of dynamic isoelectric focusing, which is a new technology developed by the PI that will be able to provide the high peak capacity of cIEF while also efficiently coupling with other techniques. The combined systems made possible will easily outperform other tandem methods and will have a high impact on the molecular analysis of cancer because they will permit the acquisition of a more comprehensive profile of the proteins in cancerous cells than is currently possible. The capabilities of dynamic IEF will be demonstrated by interfacing it to MALDI-MS and using the system to analyze and observe differences in extracts from treated and untreated PC-3 prostate cancer cells. The cell treatment will be based on compounds currently researched by the Co-PI, such as bisdehydrodoisynolic acid, which is an estrogenic carboxylic acid shown to be effective at reducing the proliferation of prostate cancer.
R21 CA116102-01A1 2006 TSOURKAS, ANDREW UNIVERSITY OF PENNSYLVANIA Novel Probe for Quantitative Imaging of Gene Expression
We propose to develop a molecular imaging probe that will provide quantitative information on the expression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probe will be designed to form a stem-loop structure and will be labeled with a 'reporter' fluorophore at one end and a quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeled with a second optically distinct 'reference' dye/nanoparticle, which will be selected such that it is unquenched regardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will be tested for their suitability in serving as the reference dye. We hypothesize that beneficial features of this novel probe compared with conventional molecular beacons will include (1) the ability to monitor transfection efficiency due to the presence of the unquenched reference dye. This will reduce false-negatives by allowing for the differentiation between untransfected cells and cells with low levels of gene expression. (2) The ability to remove via ratiometric imaging (i.e. reporter fluorscence/reference fluorescence) the impact of instrumental and experimental variability. (3) The ability to quantitatively compare variations in gene expression levels between samples, between cells within individual samples, and even between subular compartments by using the reference dye as a point of reference (4) The ability to quantify gene expression with spatial and temporal resolution since the covalent linkage between the reporter and reference dye ensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use the quantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivo imaging. (6) The possibility of an improved signal-to-background due to quenching of the 'reporter' dye by both the quencher molecule and the 'reference' dye. To evaluate these features we will pursue two major aims during the proposed research: 1) We will design, synthesize and characterize the 'quantitative' molecular beacon (QMB) in terms of its signal-to-background and lower detection limit (in vitro and in vivo) and 2) we will evaluate the ability of the QMBs to quantify endogenous mRNA expression in breast cancer cells in real-time. It is envisioned that the approach proposed here will allow significant advancements in our understanding of human health and disease and could potentially prove to be a powerful diagnostic tool.
R21 CA123329-01 2006 WANG, BINGHE GEORGIA STATE UNIVERSITY MRI contrast agents targeting carbohydrate biomarkers
Malignant transformation is often associated with alteration of cell surface carbohydrates. The expression or over-expression of certain carbohydrates, such as sialyl Lewis X (sLex), sialyl Lewis a (sLea), Lewis X (Lex) and Lewis Y (Ley), has been correlated with the development of certain cancers. These cell surface carbohydrates can be used for cell-specific identification and targeting of carcinoma cells. Recently, we have developed boronic acid-based small molecule lectin mimics (named boronolectins) that can recognize certain carbohydrates with selectivity. The same or similar methods can be used for the preparation of lectin mimics for a wide variety of carbohydrates. The long-term goal of this project is the development of conjugates of boronolectin-MRI contrast agents as biomarker-directed cancer imaging agents. Specifically, such conjugates can be used for the delivery of MRI contrast agents based on cell-surface carbohydrate biomarkers. In the R21 phase of this application, we plan to study the feasibility of this approach by (1) synthesizing boronolectin-MRI contrast agent conjugates using a boronolectin which is known selectively bind to sialyl Lewis X, (2) studying their ability to bind to cells with the target carbohydrate biomarkers, and (3) examining their ability to image implanted tumors in both an ex vivo and in vivo models. If the R21 phase is successful, in the R33 phase we plan to expand our biological evaluation to include tumors implanted at different positions, and to search for other lectin mimics that can bind specifically for other important carbohydrate-based cancer biomarkers. In addition, we also plan to examine the cytotoxicity of the boronolectin-MRI contrast agent conjugates. These small molecule-based recognition/delivery systems may have the following advantages over antibody-based systems: (1) greater stability during storage and in vivo; (2) lower propensity to elicit undesirable immune responses, (3) easier conjugation chemistry, and (4) more desirable pharmaceutical properties.
R21 CA116214-01A1 2006 WELSH, JOHN T SIDNEY KIMMEL CANCER CENTER Cancer Vertical Arrays
One of the experimental challenges in cancer molecular biology is assessing the validity and generality of biomarkers. This has become a critical bottleneck in the development of biomarkers from differential gene expression revealed by microarray studies. In this proposal, we develop the concept of using vertical arrays for exploration of differential gene expression in cancer. Vertical arrays explore the expression of a gene in many biological samples simultaneously, whereas standard microarrays explore the expression of many genes in response to one biological variable at a time. Vertical arrays are like dot blots in this regard, but vertical arrays are printed on glass slides, giving them better signal-to-noise behavior, and, rather than spotting the entire complexity of the RNA population in each spot, the RNA population is divided up among multiple spots. These low complexity representations have superb signal-to-noise performance. The work in this proposal will focus on establishing the feasibility of making a vertical array for studying gene regulation in many cancer samples simultaneously. Potential throughput is very high, such that multiple regions from each tumor can be studied simultaneously. This approach will be useful in confirming that a gene is indeed differentially regulated, in determining the distribution of expression of the gene in the transformed and surrounding normal tissue, and in determining whether the gene behaves in a similar manner in different cases of the same type of cancer and in different kinds of cancer. The goals require extensive and efficient microdissection, and we have built a novel instrument, the "tissue mill," to achieve these ends. Relevance: Biomarkers are useful for diagnosis, prognosis, and as potential therapeutic targets for cancer. There are hundreds of potential biomarkers, but further validation is needed before they can be exploited.
R21 CA118595-01 2006 WOODS, VIRGIL L UNIVERSITY OF CALIFORNIA AT SAN DIEGO DXMS-Facilitated Membrane Protein Construct Design/Cancer
Many cancer-implicated proteins are integral membrane proteins (IMPs). There is a pressing need for improved methods for the production of IMP constructs for use in high-resolution structure determination efforts. Three years ago, we completed a fifteen- year effort to develop methods for the performance of peptide amide hydrogen/deuterium exchange- mass spectrometry (DXMS). In collaboration with the Joint Center for Structural Genomics (JCSG), we recently demonstrated that DXMS can provide precisely the information needed to guide the design of well-crystallizing constructs of otherwise poorly-crystallizing soluble proteins. The NCI IMAT program is now funding our efforts to optimize DXMS-guided construct design for soluble cancer-implicated proteins (R33 CA099835). Until recently, we thought it unlikely that successful DXMS analysis of membrane proteins would be possible, and this funded grant contains no reference to membrane proteins (IMPs), nor does it support work on them. However, insights and preliminary studies described in the present application now make it likely that, with intensive development work, we can devise highly modified methods that will allow the facile DXMS analysis of IMPs. Development of membrane protein DXMS will greatly impact the structural biology of cancerimplicated IMPs, which are particularly difficult to prepare in crystallizable form. Initial year 1 development efforts will focus on the integrin allbbS, with which I have had considerable experience. Integrins are widely implicated in cancer cell and cancer vasculature biology, and findings with the prototypic allbbS integrin have proven applicable to the understanding of all integrins. The resulting IMPDXMS methods will be further refined and validated in year 2 through study of additional cancer- relevant IMPs and daughter constructs provided by Dr. Raymond Stevens, P.I. of the newly NIH-funded JCSG Center for Innovative Membrane Protein Technologies (JCIMPT). Once IMP- DXMS has been fully developed and validated, it will be made available to investigators studying cancer-implicated IMPs, by integrating the methods with our soluble-protein DXMS resource now supported by the NCI IMAT program. Thus the NCI's investment in presently funded DXMS work will be greatly leveraged by the relatively modest support requested for the development of IMP-DXMS.
R21 CA122673-01 2006 ZHANG, JIN JOHNS HOPKINS UNIVERSITY Fluorescent Activity Sensors in Analysis of Oncogenic P13K/Akt Signaling
Activation of phosphatidylinositol 3-kinase (PI3K) and the downstream serine/threonine kinase Akt (also known as protein kinase B) triggers a cascade of responses that are critical for tumorigenesis, from cell growth and proliferation to survival and mobility. Aberrations of components in the PI3K/Akt pathway have been shown to be present in a majority of tumors. We hypothesize that aberrant PI3K/Akt activation could be characterized by combined activity profiles and used as a diagnostic marker in cellular activity-profiling. To test this hypothesis, we propose the following specific aims: 1) To analyze the activities of PI3K and Akt in breast cancer cell lines and to further develop fluorescent activity sensors for various components in the PI3K/Akt pathway; 2) To develop cellular assay platforms for high throughput activity-profiling of oncogenic PI3K/Akt signaling. These studies will take advantage of a series of fluorescence resonance energy transfer (FRET)-based reporters we have recently developed for measuring the activities of Akt and PI3K in living mammalian cells. Fluorescent activity sensors and cellular assay platforms developed in this study can be used in systematic analysis of the critical components in PI3K/Akt pathway in various cancers to generate activity profiles. Correlation of genetic alterations with activity profiles and phenotypes should provide new insights into the molecular mechanisms of cancer development. On the other hand, molecular diagnostics based on such activity-profiling could identify the molecular defects and the malfunctioned key nodes in the signaling network for a given cancer, and guide appropriate molecular therapeutics as well as facilitate their development and evaluation.
R21/R33 CA123027-03 2006 COTE, RICHARD JAMES UNIVERSITY OF SOUTHERN CALIFORNIA Integrated Microdevice to Capture and Detect Circulating Tumor Cells
Metastasis is probably the most important event for determining outcome in cancer patients. The detection of occult metastases in the bone marrow, while known to be clinically important, has not become routine clinical practice. This is due to the technical difficulties and costs involved in the current methods for their collection and detection. Detection of circulating tumor cells (CTC) in the blood is less sensitive than in bone marrow and suffers from the same technical barriers as the detection of tumor cells in the bone marrow, but offers the distinct advantage of being less invasive and better for patient compliance. Therefore, sensitive detection of earliest metastatic spread of tumor in a minimally invasive and user-friendly manner will have a great impact on the clinical management of cancer patients. The currently available methodologies for CTC capture and identification face significant barriers including multiple procedural steps, substantial human intervention, extremely high cost, and importantly, lack of reliability and standardization for the detection methods. We have demonstrated the potential for sized-based tumor cell capture using a parylene-based micropore membrane. We propose to develop this into a microchip device for processing blood, and eventually bone marrow and other fluids like pleural effusions or ascites. This microdevice, coupled with microfluidics, has the potential to revolutionize the approach to tumor cell capture and identification. Further, we propose to develop methods for on-chip characterization of the captured cells. First, in R21 Phase, we will develop and optimize the capture device using a model system to isolate and molecularly characterize cultured cancer cells admixed in blood, followed by a pilot study to examine blood from 45 actual cancer patients with metastatic disease for breast, prostate or bladder cancer. In R33 Phase, we will extend the application of microdevice to assess about 310 patient samples from the same three malignancies, and we will also assess the molecular characteristics of the CTC using the Quantum Dots to understand the biological features of these otherwise rare cells (such as existence of putative stem cell sub-population which may be more malignant). At completion, studies in this project will develop a cost effective on-chip system for capture, identification, and characterization of CTC, easily usable in the clinical setting.
R21/R33 CA118631-02 2006 CREWS, CRAIG M YALE UNIVERSITY Analysis of Tumorigenic Signaling Pathways with PROTACS
A key part of determining the course of treatment for a specific cancer is the identification of the specific activated signaling pathways, which are causing the malignant growth. In fact the treatment for a given cancer can be dependent upon the activated signaling pathway; for example HER2/neu positive vs. negative breast cancers are treated differently. This personalized medicine approach is best exemplified by the development of the Abl tyrosine kinase inhibitor Gleevec, which has revolutionized the treatment of CML. As more drugs targeting specific signaling pathways are developed, it will be important to identify those oncogenic signaling pathways activated in a given tumor biopsy. Towards this end, our long-term goal is the development of a library of small molecules to be used as diagnostic tools for assessing primary cancerous tissue samples. We have recently developed a new technology known as PROteolysis TArgeting Chimera molecules (PROTACs) that can selectively knock down a specific protein in vivo. These cell permeable hetero- bifunctional molecules utilize the cells own ubiquitin/proteasome protein degradation pathway to selectively destroy a target protein of our choosing. We propose to adapt this technology so that proteins required for continued tumor growth are degraded only in those cells with a particular activated tyrosine kinase pathway. In this way, it will be possible to identify those signaling pathways upregulated in a particular tumor cell and which are required for its growth. Towards the goal of novel tumor diagnostic technology development, in the subsequent R33 application, we propose to develop a panel of PROTACs that can be used in identifying the activated cancerous cell signaling pathways. This panel will be tested for use as a diagnostic tool for determining the best course of drug treatment.
R21/R33 CA122890-02 2006 HAAB, BRIAN B. VAN ANDEL RESEARCH INSTITUTE Defining Secreted Glycan Alterations in Pancreatic Cancer
The development of methods to accurately detect early pancreatic cancer and to better differentiate benign from malignant disease could greatly improve the outcomes for pancreatic cancer patients. It is known that malignant transformation of epithelial cells of the pancreas results in alterations in the carbohydrate chains of certain proteins secreted or released by these cells. Glycosylated proteins form the basis for current biomarkers for detecting pancreatic cancer and other adenocarcinomas, and refinement of these tests are predicted to enable detection of early pancreatic cancer. Our preliminary data has shown that a novel antibody-microarray technology allows the efficient detection of glycans on distinct proteins and the identification of specific glycan structures associated with pancreatic cancer. The method uses antibody microarrays to capture specific proteins from serum samples, followed by the incubation of a glycan-binding protein (such as a lectin) to quantify specific glycans on the captured proteins. Two classes of glycoproteins, mucins and carcinoembryonic-antigen-related proteins, are particularly associated with cancer, both in altered expression patterns and in altered glycan structures on the proteins. In the R21 phase, we will determine the levels of multiple specific glycans on members of those protein classes to test the hypothesis that the measurement of specific cancer-associated glycans on specific proteins, as opposed to measuring just protein or just glycan levels, will yield improved sensitivities and specificities for cancer detection. The R33 phase of the project will expand and thoroughly test the approach. The sensitivity and specificity of detecting pancreatic cancer using measurements of glycans on mucins, CEA proteins, and proteins identified in the R33 phase will be characterized in a large set of serum samples from subjects with pancreatic cancer, benign pancreatic disease, other cancers, and no disease. We expect to characterize the value of these measurements for disease diagnostics and to gain insights into the generality and frequency of specific glycan alterations on secreted proteins. Relevance to public health: The ability to more accurately diagnose cancers at earlier stages could lead to improved outcomes for many patients. This research could lead to significantly improved blood tests for the detection of cancer, as well as a powerful, generally- applicable platform for studying carbohydrate alterations on multiple proteins.
R21/R33 CA120732-02 2006 MEYER, TOBIAS STANFORD UNIVERSITY Dissection of the Modular Structure of Cancer Signaling Systems
The development of human cancer is a multistep process in which future cancer cells acquire mutant alleles of proto-oncogenes, tumor-suppressor genes, and other regulatory genes. Many or most of these genes are signaling related proteins and we are focusing here on the design principles of signaling networks that control the cancer related processes of proliferation, migration and endocytosis. We will test the key questions of 1) whether these cancer related signaling networks have a modular structure and 2) whether cancer cells have missing or added signaling modules that cannot be observed in normal cells. We have made significant advances to answer these questions by developing a method to create 2304 in vitro Dicer generated siRNAs against a core set of human signaling proteins. Using these siRNAs, we have already discovered the function of STIM1, a Ca2+ sensor in the ER lumen that controls Ca2+ influx into cells, and which also acts as a tumor suppressor. We have also developed quantitative microscopy- based measurement tools to track signaling processes and cell functions. Phase 1 of the proposal will demonstrate the overall feasibility of using a microscopy-based siRNA strategy to investigate multiple cancer-related cell functions. Phase 2 will address the questions posed above using an expanded set of 6000 siRNAs and a focus on six cell-types, 3 non-transformed and three breast cancer epithelial cell lines. We will screen to identify signaling siRNAs that alter proliferation, cell migration or endocytosis and then utilize follow-up studies with live cell biosensors that we developed to measure the duration of different cell cycle phases, as well as migration velocity and other kinetic parameters. We will then link genes that alter these cell functions to a subset of cancer-relevant signaling pathways using secondary siRNA screens. Based on these functional and signaling datasets, we will create a modular map of signaling systems using clustering methods. We will experimentally test the predictive power of modular maps using perturbations with pairs of effective siRNAs. We will show if and how modularity in a signaling system can be used to predict how cell functions can be manipulated using combinations of siRNAs and learn if and what distinguishing features exist that define modularity of signaling systems in cancer versus non-cancer cells. This will likely lead to the identification of new cancer drug targets and new therapeutic strategies.
R21/R33 CA114304-02 2006 SCHMITTGEN, THOMAS D OHIO STATE UNIVERSITY Real-time PCR expression profiling of microRNA
microRNA is a newly discovered class of endogenous, small interfering RNA. MicroRNA binds to messenger RNA and translationally represses protein levels. While over 300 microRNAs have been discovered in humans alone, their biological function, targets, expression levels and role in disease remain largely unknown. A role between microRNA expression and carcinogenesis has been proposed. There is a lack of sensitive, high-throughput methodologies to monitor the expression of microRNAs. microRNA are challenging molecules to quantify because the microRNA precursors consists as a stable hairpin and the mature microRNA is only 22 nucleotides in length. We propose to evaluate sensitive and specific real-time PCR assays to quantify the expression of the mature and microRNA precursors. The microRNA expression will be analyzed in a number of important biological conditions relating to human cancer. The microRNA expression will be determined in specific sections of cancer and normal tissue isolated by laser-capture microdissection. The expression of mature and precursor microRNAs will be compared to using real-time PCR and a cDNA micro array. microRNA expression will be studied in clinical samples of human pancreatic cancer. The unparallel sensitivity and specificity of real-time PCR as applied to this new and exciting class of regulatory RNAs should propel the field into new directions not only in cancer but in other areas of human health.
R33 CA114306-01A1 2006 BRENT, ROGER VTT/MSI MOLECULAR SCIENCES INSTITUTE Tadpole Assays for the Molecular Assessment of Cancer
Biologists have long known that cancer cells sometimes announce their presence by shedding certain molecules into the blood. More recently, many have come to believe that some cancers might be detectable by "signatures", patterns of sets of molecules, perhaps normally present in the blood, but, in for certain cancers, present in higher or lower amounts than normal. Recently, we learned to make new kinds of molecules, which we call "tadpoles". We have demonstrated that we can use them to detect and count small numbers of molecules. These assays are relatively simple and relatively inexpensive, and they should be applicable to both kinds of cancer detection. During the next 3 years, we seek funding to develop and "harden" these assays to the point that they can be tested in clinical cancer diagnosis.
R33 CA118591-01 2006 CLAWSON, GARY A. PENNSYLVANIA STATE UNIVERSITY An RNA Sensor for Detection of Circulating Tumor Cells
This proposal seeks to develop an RNA Sensor to be employed for detection of circulating tumor cells. RNA detection is based upon an hybridization "sandwich". Two target RNAs have been chosen for clinically important cancers (prostate, breast, and melanoma), and library selection protocols will be utilized to identify/optimize accessible sites for antisense oligonucleotide (ASO) binding. Silicon nanowires will then be covalently derivatized with ASO to a library-selected site (ASO-,) in the target RNA. The ASOi nanowires will then be deposited by fluidic deposition onto chips, and integrated into the underlying CMOS circuitry. Target RNA will be purified from cellular preparations, and will then be hybridized to the ASd-nanowires. An ASO2, targeted to a 2nd library-selected site, will be covalently attached to 12 nm gold particles (ASO2-nanoprobe). Binding of the ASO2-nanoprobe to the target RNA-ASOi-nanowire complexes will induce a resonance frequency shift in the nanowires, which is greatly amplified by the mass of the gold particle. This resonance frequency shift (R??) will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (R??) will initially be detected optically). We have successfully measured R? of 300 nm silicon nanowires (with high Quality-Factors) under ambient conditions. Theoretical calculations predict very good Quality-Factors for silicon nanowires in H20, and detection of single binding events should be achievable. Preliminary data related to all aspects of RNA Sensor development have been obtained. These include: library selection of target sites in prostatic DD3 RNA, sandwich hybridization specificity "off-chip" synthesis and derivatization of nanowires, R?. measurements with nanowires, and fluidic deposition of nanowires on chips. After basic developmental steps are completed, experiments will include quantitative determination of target RNAs using the detection device compared to QPCR amplification. The Specific Aims for this funding period are designed to develop an RNA Sensor appropriate for subsequent use in clinical validation studies for circulating tumor cells. Successful development of this RNA Sensor would provide a major advantage over PCR-based assays, and could form the basis for high-throughput screening tests for simultaneous detection of many different circulating tumor cell types.
R33 CA118696-01 2006 CRAVATT, BENJAMIN F SCRIPPS RESEARCH INSTITUTE Microarray Platform for Profiling Cancer Proteases
Proteases are suspected to play major roles in cancer, including the activation/inactivation of growth factors and the degradation of extracellular matrix components to promote cancer cell migration and invasion. Consistent with this premise, transcript and protein levels for many proteases are upregulated in cancer cell lines and primary tumors. However, whether these changes in protease expression correlate with changes in protease activity remains a critical, but largely unanswered question. Indeed, proteases are regulated by a complex series of posttranslational events, meaning that their expression levels, as measured by conventional genomic and proteomic methods, may fail to accurately report on the activity of these enzymes. To address this important problem, we have introduced a chemical proteomics technology referred to as activity-based protein profiling (ABPP) that utilizes active site-directed probes to determine the functional state of large numbers of proteases directly in whole cell, tissue, and fluid samples. We have applied ABPP to identify several protease activities upregulated in human cancer cells and primary tumors. Recently, we have created an advanced antibody-based microarray platform for ABPP that enables profiling of protease activities with an unprecedented combination of sensitivity, resolution, and throughput, while requiring only minute quantities of proteome. The goal of this R33 application is to extend these studies to create the first ABPP microarray for the parallel analysis of key cancer-associated protease activities in any proteomic sample. These studies will deliver valuable new reagents and methods for the functional characterization of proteases that will be made freely available to the cancer research community. We envision that the general implementation of these innovative technologies will greatly accelerate the discovery of proteases with altered activity in human cancer. These proteases may in turn represent valuable new markers and targets for the diagnosis and treatment of cancer.
R33 CA112061-01A2 2006 ELENITOBA-JOHNSON, KOJO SEYS JOHN UNIVERSITY OF UTAH Quantitative proteomic analysis of lymphoma transformation
Non-Hodgkin's lymphoma accounts for approximately 50,000 new cases of cancer annually. This figure represents an increase beyond that seen for most other forms of cancer. Among the non-Hodgkin's lymphomas, follicular lymphoma represents the most common subtype of low-grade B lymphoma in adults, and typically pursues an indolent clinical course. In a significant proportion of cases there is histologic transformation from a low-grade neoplasm to an aggressive diffuse large B lymphoma with significantly decreased median survival. The recent advent of sophisticated mass spectrometry technology coupled with software algorithms that permit instantaneous protein identification, makes it feasible to study the pattern of protein deregulation that distinguish two biologic states. We propose to employ a combination of chromatographic techniques and tandem mass spectrometry in the identification of the alterations in protein expression that accompany histologic transformation. We shall be analyzing a cohort of matched pairs of follicular lymphoma and their transformed diffuse large B lymphoma counterparts occurring in the same individual. Relevance: Comprehensive identification of the qualitative and quantitative changes in protein expression that are involved in follicular lymphoma transformation will permit the delineation of deregulated pathways, identify distinct prognostic subgroups of transformed lymphoma, and facilitate the development of novel therapies that target susceptible elements in the deregulated pathways.
R33 CA112151-01A2 2006 ENGELWARD, BEVIN P. MASSACHUSETTS INSTITUTE OF TECHNOLOGY Applications of "Recombomice" for Cancer Research
Applications of "Recombomice" for Cancer Research Every time a cell divides, billions of base pairs of information must be accurately copied in the face of an onslaught of DNA damage. Homology directed repair (HDR) provides one of the most important mechanisms for coping with damaged DNA. If coding information is missing or corrupted, HDR can extract sequence information available elsewhere in the genome. Although HDR is generally beneficial, transfer of genetic information is risky, since misalignments can lead to tumorigenic rearrangements. To investigate the process of HDR in vivo, we have engineered the first mouse model in which HDR can be detected in somatic cells by the appearance of a fluorescent signal. In the fluorescent yellow direct repeat (FYDR) recombomice, recombination at an engineered substrate yields fluorescence. Recombination assays are simple and rapid, making it possible to do in days what used to take weeks. In addition, the FYDR mice overcome limitations of previous systems. For example, although APRT mice can be used to detect loss of heterozygosity, technically demanding assays are necessary to identify HDR events; in the pun mice, only embryonic recombination events can be detected. In contrast, FYDR mice yield a fluorescent signal that is specific to HDR events, and the recombination rate can be readily measured in cells from both embryonic and adult tissues. Furthermore, fluorescence makes it possible to capture in situ images of recombined cells, making it possible to discern independent lineages of recombinant cells in vivo. Our Specific Aims are to I) Evaluate the frequency of recombinant cells in multiple tissues; II) Develop methodology for quantification of recombinant pancreatic cells in situ and reveal the relative frequency of recombinant cells among two different cell types within a normal tissue for the first time; III) Measure the effects of environmental factors on recombination in vivo; and IV) Reveal how specific genes (Blm and p53) affect recombination susceptibility in vivo. The broad long term objectives of this work are to demonstrate the utility of this newly developed technology for studying recombination in mammals, to substantially expand the capabilities of the existing system, and to elucidate environmental and genetic factors that influence a person's susceptibility to spontaneous, environmentally-induced, and cancer therapy-induced DNA rearrangements.
R33 CA118479-01A1 2006 KNOWLES, DAVID W. UNIVERSITY OF CALIFORNIA-LAWRENCE BERKELEY LAB Novel Image-Based Screening of Mammary Tumors
Breast tumors are detected by self exam, clinical exam, and mammogram and suspicious lesions are biopsied. The ensuing histological classification plays a determining role in the treatment decision but the associated risk of malignancy, the appropriate treatment, and the risk of reoccurrence are difficult to determine. As a consequence, patient treatment is based on epidemiological findings rather than individual needs. Non- invasive imaging provides some information but early breast cancer detection by routine screening is not currently feasible. However, once a tumor has been detected and biopsied, there is urgent need for novel methods to aid in the detection and classification of sub-classes of lesions. One key epigenetic marker of cell phenotype is the organization of nuclear proteins which direct and reflect normal cell function. Based on the hypothesis that the redistribution of chromatin-related proteins reflects changes in gene expression that accompany alterations in cell phenotype, we have developed image analysis methodologies to quantify the nuclear distribution of specific chromatin-associated proteins from three-dimensional, high-resolution, fluorescently immunostained images. By applying these methods to culture models that mimic normal and malignant breast epithelial tissue, we have demonstrated that the distribution of nuclear mitotic apparatus protein (NuMA) and heterochromatin related protein histone-4 methylated on lysine-20 (H4-K20m) are biomarkers capable of clearly distinguishing non-neoplastic and malignant human mammary epithelial cells. The goal of this project is to quantify the distribution of specific nuclear proteins in culture models that mimic premalignant and malignant tissue to uncover epigenetic characteristics of premalignant disease. Our image-based methodologies will be expanded to produce a novel technique, the phenotype tissue-map, capable of resolving local tissue phenotype at cellular resolution and uncovering subtle differences in tissue morphology and behavior. The technology, which will work alongside the usual H&E staining and histological techniques, will be tested on needle-core biopsies of a variety of premalignant tumors with the aim of defining sub-classes of graded lesions. The results will be correlated with the histopathology of the initial needle-core and the follow-up surgical biopsy with the hope of predicting more aggressive phenotypes missed by the initial screen. The future public health benefit of this research is to aid the treatment decision process of breast cancer patients. By better understanding the organization of molecular components within the cell and how the organization of these components is altered during the progression to cancer, we can develop and provide pathologists with novel image analysis tools to aid and support the histological classification of biopsied breast tissue.
R33 CA116115-01A1 2006 LANDERS, JAMES P UNIVERSITY OF VIRGINIA Microdevice for Direct DNA Purification
With an ever-increasing interest in the molecular typing of cells from histologic tissue sections, the ability rapidly and efficiently extract DNA from the selected cells will be of paramount importance. The overall goal of this project is to develop a microchip-based sample preparation method for high efficiency, low-cost extraction of DNA from tissue samples - this microdevice will easily accommodate blood or other cells sources. The microdevices will be created using state-of-the-art microfabrication techniques coupled with fluidically controlled on-chip cell lysis and solid phase extraction chemistries. This project couples the industrial capabilities of HT Micro for facile fabrication of complex, high surface area microstructures, with surface modification chemistries developed at the UNIVERSITY of Virginia that enable efficient and high capacity DNA extraction. The microdevices will be tested using a variety of sample varying in type and quality, and extraction efficiency will be determined using real-time PCR. As a demonstration of integration with current laser-capture microdissection instruments, the microdevice will be fabricated to directly accept the cap from the Arcturus Pixcell IIe which will contain the selected cells bound to an ethylene vinyl acetate polymer membrane on its bottom surface. The tissue samples will be collected and laser microdissected by our surgical pathology collaborator here at UVa - samples of normal and malignant cells will be analyzed. The final device will offer the rapid analysis, high extraction efficiency, and high-throughput advantages of microdevices and, in addition, is expected to offer higher capacity, and lower cost-per-device than current conventional or microchip techniques.
R33 CA118602-01 2006 SCHNITZER, JAN EUGENIUSZ SIDNEY KIMMEL CANCER CENTER Technology/Map Endothelial Targets/Human Renal Tumors
The molecular complexity and in vivo inaccessibility of most tumor cells within solid tumors can greatly limit genomic- and proteomic-based discovery of useful targets for tumor-specific imaging and therapeutic agents in vivo. To overcome endothelial cell (EC) barriers and achieve more effective targeting and penetration into solid tumors, we shift analytical focus from the tumor cell to the vascular EC surface and its caveolae in direct contact with the circulating blood. To reduce data complexity to a meaningful subset of targetable proteins expressed on the EC surface, we will use tissue subular fractionation, novel multimodal mass spectrometric analysis, in silico subtraction, and bioinformatics interrogation of structure and function to unmask, from the >100,000 proteins in the tissue, those few intravenously accessible proteins differentially expressed on vascular endothelium in human renal tumors. This technology and overall approach has been validated in rodent solid tumors whereby new vascular targets have been uncovered permitting tumor-specific imaging, penetration, and effective radio immunotherapy (Nature, 429:629-35, 2004). But, currently very little is known about the expression of proteins in tumor neovascular endothelium, especially in human tissue. We now wish to apply our new technology to map comprehensively the proteome of luminal EC surfaces and caveolae in human renal tumors in vivo. It is likely that human tumors will express a different constellation of proteins on tumor neovasculature not yet uncovered or induced in animal models. To this end, we propose the following specific aims: 1) To use novel tissue sub fractionation and proteomic analytical approaches to map comprehensively vascular EC surfaces and caveolae in human renal tumors vs. matched normal renal tissue to unmask candidate tumor-induced/associated vascular proteins. 2) To create new antibodies to newly discovered human renal tumor EC targets and to use antibodies as probes to validate the expression of tumor-induced/associated proteins at the EC surface and its caveolae in human tissues and thereby to assess the degree of target specificity for the neovasculature of human solid tumors. Such mapping may also elucidate the effects of the tumor on the developing vascular endothelium and yield important tumor-specific vascular targets for improving noninvasive diagnostic imaging and therapy as well as yield new diagnostic and prognostic markers for the molecular classification of tumor biopsies