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Click on any project title for a more detailed description of the project. For more information about any of these awards (e.g., PI contact information or associated publications), please use the corresponding project number to search for information at the NIH Reporter website.

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Project # Year
of Award
PI Name(s)
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Abstract Text (Official)
R21 CA154958 2011 ALEXANDRAKIS, GEORGIOS UNIVERSITY OF TEXAS ARLINGTON Scanning Correlation Microscopy Methods for Quantifying DNA Repair Kinetics
The kinetics of most fluorescently tagged DNA repair proteins subsequent to exposure to ionizing radiation, or radiomimetic chemical agents, cannot be quantified in the living cell without serious perturbation of the system under study. With the exception of only few proteins that attach in large numbers near DNA damage sites (e.g. 3-H2AX, 53BP1), most other proteins attach in fewer copies near the DNA damage sites and cannot be visualized by fluorescence microscopy. This because of the high background from freely moving, or immobile, fluorescent proteins that mask the weak aggregation of DNA repair proteins at damage sites. As a result, DNA damage is usually visualized by inducing clustered DNA damage by illumination with a laser beam. This illumination induces very high accumulation of repair proteins in one or more large spots in the nucleus. Nevertheless, the laser-induced damage is complicated in nature and may not be a good surrogate to ionizing radiation or radiomimetic agents for studying DNA repair in vivo. In this work we propose to develop quantitative microscopy methods that can overcome the current major limitation of not being able to quantify the kinetics of fluorescently tagged DNA repair proteins at sparse DNA damage sites. More specifically we will apply raster image correlation spectroscopy (RICS), a technique that analyzes the spatio-temporal fluorescence intensity fluctuations in image pixels, to quantify the repair kinetics of proteins with sparse accumulation in the cell nucleus. We will use RICS to quantify the repair kinetics of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), in its wild type and repair-deficient 7A forms after exposure to 3-rays and bleomycin that are both double strand break forming agents. We will also test hydrogen peroxide, a single strand break (DSB) forming agent, as a negative control. We will show that the DNA-PKCS kinetics after formation of DSBs can be quantified by RICS. Furthermore we propose to develop two specialized forms of RICS to further enhance the quantification of DNA repair kinetics of these proteins: (i) Photo-Activation RICS (PA-RICS) will offer control of the fluorescently tagged repair protein concentration, and (ii) Coherent Control RICS (CC-RICS) will optimize laser pulse characteristics to enhance fluorescence emission by up to an order of magnitude without increasing excitation power. PA-RICS and CC-RICS will enable improved quantification of the binding kinetic constants of DNA-PKCS variants at DNA damage sites. Importantly, the proposed RICS techniques can potentially be used to quantify the kinetics of a wide range of DNA damage sensing, signaling, and repair proteins with sparse accumulation patterns in the nucleus. Therefore, the proposed methods are very generally applicable to the DNA repair field and beyond. PUBLIC HEALTH RELEVANCE: We propose to develop fluorescence microscopy techniques that will enable researchers to monitor if and how fast cells can fix DNA after this is damaged by X-rays or chemical agents. By looking at how cancer cells respond to treatment-induced DNA damage when their repair proteins work properly, or when they don't due to a genetic mutation, we can understand the mechanisms of treatment resistance and design better therapies.
R21 CA140036 2011 BERMUDEZ, HARRY ; DECAPRIO, ANTHONY P (contact); ROTELLO, VINCENT M. FLORIDA INTERNATIONAL UNIVERSITY Platform for High-Throughput Analysis of Protein Adducts for Carcinogen Exposure
The long-term goal of this project is to develop a high-throughput technology that can characterize covalent protein and DNA modifications (""adducts"") induced by reactive carcinogens on a global basis with high sensitivity and specificity. Virtually all carcinogens, even the most inert, either contain an electrophilic center or undergo some level of oxidative metabolism to form reactive electrophiles. DNA and protein adducts are the products of the chemical reaction of such electrophiles with nucleophilic sites within these macromolecules. DNA adduction (including methylation and oxidative changes) has been widely implicated in mutation and carcinogenesis, while protein adducts (i.e., post-translational modifications) may have indirect roles in the mechanism of carcinogenesis if they involve DNA-related proteins or increase the rate of tumor promotion. In addition, such adducts are being explored as short- term, long-term, and/or cumulative markers of exposure to carcinogens. Current methods of adduct quantitation are expensive, highly chemical-specific, and labor-intensive. A technology for rapid and comprehensive profiling of macromolecular adduction in human subjects would be an important tool for carcinogen exposure and health risk assessment. This exploratory/developmental application proposes a proof-of-concept study to adapt three recently developed technologies, i.e., combinatorial chemical synthesis, antibody phage display, and microarray technology in a system to rapidly profile blood protein adducts of two important classes of carcinogens. Libraries of adducted peptides will be created to mimic structures formed in vivo in humans exposed to carcinogens. Adduct-specific scFv probes will be selected by screening with phage display libraries and validated with human blood specimens in an ELISA system. Results of ELISA-based adduct quantitation will be compared to those from conventional mass spectrometry-based analysis of blood protein adducts. If the proposed proof-of-concept studies are successful, expanded funding will be sought to develop a microarray-based global adduct detector for screening blood specimens from individual subjects. It is anticipated that the technology developed in this project will have wide applications for carcinogen exposure assessment, biomarker discovery, clinical diagnosis, and assessment of cancer treatment efficacies. PUBLIC HEALTH RELEVANCE: Many environmental carcinogens are able to chemically react with proteins and DNA in the body to form products called ""adducts"". These reaction products can be critical to the occurrence of cancer, or, alternatively, they can provide information on a person's exposure to these carcinogens. We propose to develop a rapid, reliable system to detect and measure these adducts in human specimens on a global basis, a technology that would be an important tool for carcinogen exposure assessment, clinical diagnosis, and assessment of cancer treatment efficacies.
R21 CA155568 2011 BIEBERICH, CHARLES J. UNIVERSITY OF MARYLAND BALTIMORE Application of an Innovative Technology to Develop Low Toxicity Kinase Inhibitors
Kinase inhibition is a viable treatment approach for multiple diseases including cancer, diabetes, and Alzheimer's. Although small molecule inhibitors have occupied center stage in the kinase inhibition arena, challenges with toxicity and emergence of resistance have limited effectiveness and impact. The growing recognition of the need to combine agents to target multiple signaling pathways to treat cancers compounds these already significant challenges. It is imperative that every possible route to developing effective new kinase inhibitors be explored with alacrity. Peptides that interfere with kinase-substrate interactions have the potential to serve as low toxicity, high specificity agents to fill a critical unmet need in this area. We propose to combine multiple innovative technologies to develop and test, in vivo, novel peptide inhibitors of an oncogenic kinase. Using the reverse in-gel kinase assay in combination with novel high-resolution chromatofocusing-based protein separation, naturally occurring Protein Kinase CK2 substrates with the highest observed catalytic efficiency will be identified. Phosphoacceptor sites will be determined by Electron Transfer Dissociation-assisted mass spectrometry, and sites of protein-protein interaction will be probed by Chemical Shift Perturbation-mediated Nuclear Magnetic Resonance spectroscopy. Substrate- competitive pseudosubstrate-class peptide inhibitors, and docking site-class peptide inhibitors will be designed and analyzed for kinase inhibitory activity in vitro and in vivo. For in vivo analyses, kinase inhibitory activity will be monitored using a novel approach to determine the phophorylation state of CK2 and substrates in peripheral blood mononuclear cells after parenteral administration in mice. The successful completion of these aims will provide proof-of-principle that the innovative application of an existing IMAT-funded technology can facilitate the development of new peptide kinase inhibitors to treat cancers and other diseases where aberrant kinase activity underlies disease initiation or progression. PUBLIC HEALTH RELEVANCE: The successful completion of this project will fill a need for new technologies to support the development of peptide kinase inhibitors. It will provide proof-of- principle that the reverse in-gel kinase assay can identify candidate substrates from which inhibitory peptides with therapeutic potential can be derived.
R21 CA151140 2011 BLAIR, SARAH L UNIVERSITY OF CALIFORNIA AT SAN DIEGO Non-circulating microparticles for improved localization and resection cancer
Surgical resection for cancer is becoming more minimally invasive with smaller incisions and less patient morbidity. However, this process switches the burden to the surgeon to remove all the disease with suboptimal visualization. This project proposes to develop a technique to employ pre- operative injection of ultra-sound contrast enhancement stationary microbubble and microshells to enable intraoperative detection of tumors even in cases with small skin incisions to allow surgeons to better identify and completely resect tumors and decease patient morbidty. Aims: To determine the optimal dose and lifetime of stationary microbubbles in a rabbit model as an ultrasound contrast agent in order to localize small breast tumors for surgical resection. To study the localization ability of a novel gas filled silica microshells compared to microbubbles as an ultrasound contrast agent and localization method for occult breast cancers using a rabbit model. PUBLIC HEALTH RELEVANCE: Because surgical resection of small early cancers remains so important in local control and prevention of metastases, techniques to improve its efficacy in visualizing and removing tumors completely can have a major impact on breast and other cancer treatment. We believe that improving targeting of nonpalpable cancers is crucial. This fact is particularly important in this era in which tiny cancers are detected by imaging and more advanced techniques are needed for localizing tumors. This project may be able to decrease positive margins, local recurrence and possible cure of early stage cancers.
R21 CA140080 2011 BUCHANAN, JANICE PAIGE; STRAUSS, STEVEN H. (contact) COLORADO STATE UNIVERSITY-FORT COLLINS Nanocaged Metal Tags in Massively Multiplexed Leukemia Bioassay and Beyond
Characterization of an acute leukemia (AML) cell requires measurement of biomarkers such as proteins, genes and small molecules. An unambiguous biomarker signature cannot be obtained by determining only a few proteins. A quantitative, massively multiplexed bioassay (MMB) for a constellation of proteins, small molecules, and gene transcripts would be a significant medical breakthrough. ""Personalized health care"", unambiguous disease identification, and patient- specific diagnosis/prognosis will be enabled by the simultaneous quantitative determination of many biomarkers in a patient's sample. We will develop a new, robust, multimetallic tagging system based on functionalized polymer microbeads (PMBs) analyzed by the proven, high-sensitivity, time-resolved, and element-specific detection technique of flow-cytometry inductively- coupled-plasma mass spectrometry (FC-ICP-MS). Our interdisciplinary group of recognized experts will address this significant challenge. Prof. S. Stevenson (UNIVERSITY. of Southern Mississippi, USM) will optimize the synthesis of 7 different M3N@C80 endometallofullerenes (EMFs) with interference-free metals that are not endogenous in biomedical samples. Prof. S. H. Strauss and Dr. O. V. Boltalina (Colorado State UNIVERSITY., CSU) will optimize the synthesis of chemical derivatives of the EMFs, determine the optimum type and number of functional groups to achieve uniformity. Preferred derivatives will be sent to Prof. J. P. Phillips (USM), who will attach polymerizable groups to EMF derivatives, optimize preparation of narrow-size-distribution PMBs containing many specific mixtures of the seven metals (all of which will be permanently sequestered in their carbon nanocages and therefore cannot leach out over time). The UNIVERSITY. of Toronto (UofT) group, led by Prof. V. I. Baranov, will develop purpose-specific analytical methods that combine robotic sample introduction and FC-ICP-MS instrumentation to allow massively multiplexed detection and classification of thousands of multimetallic encoded beads with element-tagged reporter affinity molecules. They will estimate the tagging multiplexity and show the integrity and virtually infinite shelf-life of the tagged PMBs, and that massive multiplexing with thousands of differently tagged PMBs is possible when this technology is reduced to practice. Dr. O. I. Ornatsky (UofT) will perform analyte selection, testing and validation of the encoded beads, immunoassays, and oligonucleotide hybridization, and will covalently link a range of antibodies (Abs) to the surfaces of the tagged PMBs, which will be tested in sandwich assays with secondary Abs linked to a reporter tag. We will prove that our tagging system has the potential to be used for massively multiplexed bioassays in which thousands of antigens, gene transcripts, and small-molecule cell markers for many diseases and conditions are determined simultaneously in a single sample. We will test our massively multiplexed bioanalytical platform on acute myeloid leukemia (AML) cells. We will show the advantages of FC-ICP-MS analysis of multi-metal-tagged PMBs coated with capture Abs against cytokines, chemokines, growth factors, and soluble receptors present in human serum. PUBLIC HEALTH RELEVANCE: The goals of the proposed investigation are the development of an advanced metal encoding system for bead-based assays. This novel system uses combinations of trimetallic endometallofullerenes embedded in polymeric microbeads to encode hundreds of thousands of unique carriers which together with time-resolved multi- element detection by flow-cytometry inductively-coupled-plasma mass spectrometry (FC-ICP-MS) will enable quantitative gene expression analysis and massively multiplexed immunoassays. Unambiguous disease identification and patient-specific diagnosis and prognosis will all be enabled by the simultaneous, rapid, and quantitative determination of many biomarkers in a patient's sample.
R21 CA155572 2011 CHEN, JIN ; LI, DEYU (contact); WEBB, DONNA J VANDERBILT UNIVERSITY MEDICAL CENTER VEC3-Valve Enabled Cell Co-Culture Platforms for Cancer Biology Study
It is becoming increasingly clear that the tumor microenvironment plays a key role in tumor progression, pointing to a need to develop technologies to study tumor cell behavior in different microenvironments. Toward this goal, we have created Valve Enabled Cell Co-Culture (VEC3) platforms, which are a new class of microfluidic devices designed for analyzing interactions between tumor cells and others in the tumor microenvironment. The technology enables separate culture of distinct cell types and cell interactions through either soluble factors or physical contacts between spatially separated cell populations while maintaining fluidic control over their individual culture environment. In addition, through selective blockage of the exchange of specific ligands between distinct cell populations, the platform can be used to identify the functions of relevant ligands of interest. Traditional cell co-culture techniques and reported microfluidic cell co-culture platforms have limitations and cannot address all important cell co-culture needs. The proposed VEC3 cell co-culture platform, through the introduction of a simple, robust, and user-friendly pneumatically or hydraulically controlled valve to reversibly separate or connect adjacent cell culture chambers, not only allows for separate culture and treatment of individual cell types, but also permits real-time, live imaging of cellular interactions. To date, as a proof of principle, VEC3 has been applied to observe dynamically synapse formation between hippocampal neurons, analyze tumor-endothelial interactions in normoxic and hypoxic environments, study tumor-fibroblasts interactions in 3D matrices, and quantify tumor-endothelial cross migration mediated by various molecules. In the proposed research we will further develop VEC3 through quantitative characterizations of cellular microenvironments, including cell density and uniformity, glucose and oxygen concentration, and cell interaction rates. Through improved design and performance engineering, we will develop optimized platforms and operation protocols to increase the success rate of VEC3-based assays. In addition, we will implement new functions such as controlled cell interactions via blockage of the exchange of specific ligands between two cell populations. More importantly, we will apply VEC3 to study tumor- endothelial cross migration mediated by various ligands and receptors and identify the functions of specific ligands in cell interactions. Therefore, successful execution of the proposed research will lead to a new class of versatile, multifunctional VEC3 microfluidic platforms that are widely applicable to cancer biology. This device will be used to elucidate the molecular mechanisms underlying tumor angiogenesis, intravasation and metastasis, which could eventually lead to better cancer treatments. The overall quantitative milestone is to achieve 95% success rate in assays using the VEC3 platforms for tumor angiogenesis, intravsation and metastasis studies with quantitative parameters of cellular communication. PUBLIC HEALTH RELEVANCE: Cell migration is critical for many biological processes, and tumor-endothelial cross-migration is of fundamental importance to tumor angiogenesis. The goal of this research project is to develop novel microfluidic cell co- culture platforms that allow for the quantitative assessment and identification of the molecular mechanisms that regulate cell migration and tumor-endothelial interactions. This will lead to new therapeutic approaches for treating various diseases, such as cancer, that arise from aberrant cell migration.
R21 CA128692 2011 CLARY, BRYAN M DUKE UNIVERSITY In Vivo Selection of Tumor-Specific RNA Binding Motifs
It is the overall goal of this application to better define the differences between hepatic colorectal cancer metastases and the normal host tissues within which they reside and in the process of developing this understanding to develop reagents that specifically traffic to in vivo tumors. Recognizing and understanding these differences will help to create therapeutic strategies that are targeted to patients with metastatic colorectal cancer who as a group face an expected 5-year survival prognosis of less than 10% with current therapies. The specific aims of this study are to: 1.To create RNA binding motifs through a novel in vivo selection process that specifically bind human intrahepatic colorectal cancers residing in immunodeficient mice and that possess the ability to traffic in vivo to the sites of tumor deposits upon systemic administration. 2. To define the protein targets of these tumor-specific RNA binding motifs and ascertain whether they possess binding characteristics consistent with RNA aptamers. 3.To determine if the identified RNA aptamers possess inhibitory actions on their protein target and/or whether they are capable of escorting therapeutic moieties to intrahepatic tumors. 4. Determine whether these same targets are also present in a broader spectrum of human colorectal metastases harvested by the applicant (BC) at the time of hepatic resection. To accomplish these aims, RNA aptamers will be generated through a novel selection strategy whereby mice bearing hepatic colorectal metastases will be injected via tail vein with a random library of RNA oligonucleotides. After a brief period of circulation, tumors are harvested and RNA retrieved, reverse transcribed, amplified, and transcribed back to RNA for repeat injection. This cycle is repeated until the population of RNA binding motifs is heavily enriched at which point cloning and sequencing is then performed. RNA aptamers created through this mechanism will then undergo in vitro binding assays to identify those with specific binding for tumor tissue. This process will be performed utilizing xenotransplants harvested from multiple patients in an effort to retrieve aptamers relevant to a broad spectrum of patients. Selections will also be carried out whereby different xenotransplants are utilized in alternate rounds. The resulting RNA binding motifs will be explored in their ability to traffic to the site of intrahepatic tumors. Through a ligand-mediated approach, the target of these aptamers will be isolated and sequenced. The ability of the RNA aptamers to inhibit the function of their target proteins and the proliferation of in vitro and in vivo tumors will be determined. In addition, the ability of RNA aptamer:bacterial toxin conjugates to effect cytotoxicity in vitro and in vivo will be determined. The relevance of the aptamers to a broad spectrum of patients with mCRC will be explored via cDNA arrays and IHC of resected tumors and through in vivo trafficking studies in mice bearing xenotransplants. PUBLIC HEALTH RELEVANCE: It is the purpose of the proposed research to better define the differences between human tumors (metastatic colorectal cancer) and normal tissue. Colorectal cancer is one of the leading causes of cancer death in the United States and rarely curable when spread (metastases) are detected. Defining the differences between cancer and normal tissues will allow for the further development of targeted therapeutic agents that are capable of inhibiting metastatic colorectal cancer while minimizing toxicity to normal tissues.
R21 CA157383 2011 ESTROFF, LARA A; FISCHBACH, CLAUDIA (contact) CORNELL UNIVERSITY Mineralized 3-D tumor models to study breast cancer bone metastasis
Project summary Breast cancer frequently metastasizes to be where it leads to osteolysis and poor clinical prognosis; however, the role of hydroxyapatite nanocrystals (HA, the mineral component of bone) in this process remains unclear due, in part, to the lack of appropriate culture models. The overall goal of these studies is to design a mineralized 3-D tumor model that captures the intrinsic 3-D cell-microenvironment interactions within bone- metastatic niches and nanostructural alterations of HA that may occur due to disease and aging. Specifically, we will develop porous poly (lactide-co-glycolide) (PLG) scaffolds that incorporate HA nanoparticles of defined physicochemical characteristics and assess the applicability and relevance of this 3-D tumor model to test the functional relationships between HA and osteolytic bone metastasis. This work will be accomplished in three specific aims: In Aim 1, we will develop the 3-D matrices by synthesizing monodispersed nanoparticles of HA with controlled/variable aspect ratios, crystallinities, and lattice substitutions and incorporating them into porous PLG scaffolds. In Aim 2, we will utilize these scaffolds to analyze the proliferative and osteolytic capability of breast cancer cells in response to varying HA nanoparticle characteristics in vitro and evaluate possible molecular mechanisms that may be involved in these changes. In Aim 3, we will correlate the physicochemical properties of HA with tumor growth and osteolytic capability in vivo and validate the contribution of specific tumor cell secreted factors as identified in aim 2. Interleukin-8 (IL-8) will be the initial focus of the proposed cytokine signaling studies, as this factor modulates metastasis-related osteolysis and tumor progression. Additionally, we will use the mineralized tumor model to identify novel factors that are regulated by nanostructural changes of HA and that may explain the molecular signature of bone-metastatic tumor cells. The novel combination of cancer biology with engineering and materials science approaches will result in a highly reproducible and pathologically relevant culture platform that will allow us to deconvolute the complexity of bone metastasis and identify molecular targets for improved therapies. By elucidating the importance of materials-based mechanisms, the proposed technology has the potential to challenge the currently accepted paradigm of bone metastasis as a disease that is solely mediated by cellular and molecular changes. Lastly, the proposed 3-D culture systems will enable radically new approaches of investigation of other physiological and pathological situations (e.g., osteoporosis, tooth regeneration) and their dependence on 3-D interactions with bone mineral. PUBLIC HEALTH RELEVANCE: Project Narrative: Bone metastasis is the leading cause of breast cancer-related deaths among women worldwide; however, the role of the bone mineral hydroxyapatite in this process remains unclear. This research will develop a three-dimensional culture platform to systematically elucidate the functional relationship between the bone mineral matrix, mammary tumor cell behavior, and metastatic osteolysis. Our studies will combine materials science with engineering and cancer biology, and this interdisciplinary approach has the potential to not only revolutionize our understanding of bone metastasis, but also provide a widely applicable culture model to study hydroxyapatite-dependent physiological and pathological processes.
R21 CA155535 2011 EVANS, CONOR LEE MASSACHUSETTS GENERAL HOSPITAL Hyperspectral and Structural Microscopy Platform for Therapy of Resistant Cancer
Although much progress has been made in detecting and treating cancer, disseminated metastatic disease remains the leading cause of death in cancer patients. Frustratingly little is known regarding how these deadly lesions respond to and eventually resist treatment in vivo due to their widespread nature, often sub-clinical sizes, and highly heterogeneous microenvironments. The lack of appropriate imaging tools to understand and overcome treatment resistance on the microscale in metastatic cancer represents a major unmet need in both cancer research and therapeutics. To address this critical challenge, this application introduces an innovative microscopic technological paradigm for visualizing and understanding the microscale treatment response and resistance factors found in disseminated metastatic cancer. The goal of this work is to develop a high- throughput, live, molecular and structural optical microscopy platform capable of visualizing and monitoring therapeutic response in micrometastatic lesions in vitro at both the cellular and nodular levels. Such an imaging platform will enable a bottom-up approach for basic cancer and cancer therapeutics research by building a detailed spatiotemporal picture of cancer therapy from single cells all the way to the whole tumor. Based upon the complementary molecular and structural imaging technologies of hyperspectral microscopy and optical coherence tomography (OCT), the automated multiplexed therapy-imaging platform will enable long-term cellular and nodular-level studies that map the 3D distribution of molecular treatment factors within a structural context. The new real-time, tunable, near-IR optimized hyperspectral microscopy system will allow for 3D, multi-fluorophore, live imaging of treatment resistance factors deep in tumor nodules. For longitudinal microscale visualization of the complex structural changes caused by treatment, the non- perturbative and label-free time-lapse OCT (TL-OCT) modality will be integrated along with the hyperspectral microscope. Applicable to many metastatic cancers, this new imaging platform will be validated using a physiologically relevant, repeatable, and multiplexed in vitro 3D model of micrometastatic ovarian cancer for imaging-based screens. In the first application of this approach, the multimodal technological platform for cancer biology research will focus on visualizing and correlating several crucial treatment resistance factors, such as drug uptake and diffusion, hypoxia, and pH, with treatment response. This imaging-based approach to systematically and quantitatively characterize cancer therapy response and resistance represents a departure from traditional methods that miss crucial molecular and structural information. These technologies can be readily translated for imaging patient tissue samples ex vivo, or in situ microendoscopically, for image-guided therapeutic planning. By building our knowledge of treatment response and resistance at the microscale, this innovative imaging platform forms the foundation of a transformative, new investigator-led research program aiming to improve current cancer therapeutics and defeat treatment-resistant metastatic disease. PUBLIC HEALTH RELEVANCE: Cancer that becomes metastatic is too often fatal due to the spread of numerous microscopic lesions that grow to be treatment resistant. The goal of this application is to address the root causes of treatment resistance in cancer through an innovative microscale imaging approach capable of visualizing the crucial molecular and structural factors involved in therapeutic response. By building an understanding of how deadly lesions escape therapy from the cellular level to the whole tumor, and using this knowledge to defeat treatment-resistant disease, the technology developed in this application aims to significantly improve the lives of patients suffering from advanced metastatic cancer.
R21 CA155543 2011 GULLEY, MARGARET L. UNIVERSITY OF NORTH CAROLINA CHAPEL HILL Enhanced Formalin Fixation to Improve Tests on Solid Tissues
Standard pathology practice relies on automated processing of tissues fixed in 10% neutral buffered formalin followed by staining protocols that were optimized over the past century for microscopic visualization. In the last two decades, molecular assays are increasingly applied to formalin fixed, paraffin embedded tissues although this effort is hampered by lesser quantity and poorer quality of nucleic acid compared with that recovered from fresh or frozen tissue. Hypothesis to be tested: We propose that, in order to improve fixation technology that will be embraced by the pathology community, key steps of standard formalin fixation cannot be altered. On the other hand, addition of chemical stabilizers to standard reagents, and altering the temperature of the initial phase of formalin fixation, are realistic changes that could improve downstream molecular analysis without adversely impacting morphology and immunostain outcomes. Based on synthesis of a diverse literature, we present a two-part hypothesis to drive development of enhanced formalin fixation protocols: A). The irreversible damage to nucleic acid occurring during formalin fixation is mainly biochemical and can be largely prevented by inhibiting endogenous nuclease activity during formalin infusion. To address this, broad-spectrum nuclease inhibitors will be identified that are small enough to co-diffuse with formalin into tissue spaces, and these will be tested with or without refrigeration in an otherwise-standard, automated tissue processing protocol. B). Nucleic acid damage accrues after fixation, due mainly to slow, persistent, oxidation by reactive oxygen species (ROS) derived from atmospheric O2, trapped inside the tissue block. To address this, ROS scavengers will be identified that are water-soluble, inexpensive, and small enough to diffuse rapidly into tissue spaces during the first ""post-formalin"" dehydration step, yet are poorly soluble in alcohol or xylene so that, upon tissue transfer into water-free solvents, the scavengers are embedded in the dehydrated tissue block matrix where they stand ready to quench newly-formed ROS during storage in situ. Relation to a follow-on R33: When this R21 is completed, procedural improvements will have been made which preserve DNA & RNA during ordinary 10% buffered formalin fixation and subsequent storage as paraffin embedded tissue. In R33 work, these compounds will be subjected to pilot scale manufacture as beta test kits, to be validated on diverse human cancer tissues at multiple sites.
R21 CA148068 2011 HAGEDORN, CURT H. UNIVERSITY OF UTAH Sentinel Pol II RNAs for Measuring RNA Integrity in Biospecimens
The presence and quantity of specific RNA polymerase (Pol) II transcripts in biospecimens, that reflect gene expression patterns, are increasingly being used as biomarkers to make major patient care decisions (e.g., MammaPrintTM, array assay for 70 mRNAs). However, mRNA molecules in biospecimens are highly susceptible to degradation by RNases during sample collection, handling, and storage. Reliable data obtained by transcriptome analysis of biospecimens, with gene arrays or RNA-Sequencing, is highly dependent on the quality of the RNA analyzed. Current standard measures of RNA integrity focus on 28S and 18S ribosomal RNA. However, diagnostic and prognostic gene expression markers of cancer focus on changes in mRNAs which are RNA Pol II transcripts. The 5' ends of Pol II RNAs are unique in that they have a 5' m7GpppN cap. We will use new technologies to identify a panel of sentinel Pol II RNA transcripts in human colon, breast and liver biospecimens that mirror mRNA quality and use these RNAs to develop a 3'/5' PCR based assay that more accurately assesses the integrity of Pol II transcripts in biospecimens. We developed a new approach to identify and characterize all Pol II transcripts present in biospecimens by isolating 5' m7G capped RNAs and analyzing them with RNA-sequencing technologies (Illumina). This allows us to identify and quantitate both protein coding and non-coding regulatory RNAs in biospecimens. It also allows us to define the entire length of each RNA, define their 5'-3' pattern of degradation, and develop a qRT-PCR based assay of their 3' and 5' regions to measure their level of intactness. The degradation patterns of Pol II RNA transcripts will be assessed in freshly collected human colon, breast and liver biospecimens (normal and cancer) after increasing times at room temperature and commonly used handling procedures. This analysis will identify a panel of candidate sentinel RNAs that can be used to develop an assay for mRNA integrity in biospecimens. The Specific Aims are: 1) To identify candidate sentinel Pol II RNAs that mirror mRNA decay in specific biospecimens (see Example) and; 2) To develop a 5'/3' quantitative reverse transcription PCR (qRT-PCR) assay that measures the intactness of sentinel RNA transcripts in specific biospecimens to better measure mRNA integrity. This study stimulates technology innovation by combining a new RNA purification technology for RNA Pol II transcripts and next generation RNA-sequencing to identify sentinel RNAs and developing a practical assay for RNA integrity in biospecimens. Our studies will optimize the use of both currently stored and future collections of biospecimens in identifying gene expression biomarkers for the early diagnosis, prognosis, and response to therapy of cancer patients. The long-term goal of this technology is improving patient care and therapeutic outcomes by better determining the quality of RNA in biospecimens before conducting diagnostic or predictive gene expression tests. Two international experts in colon (Dr. Burt) and breast (Dr. Buys) cancer will provide expert advice during the development of this assay. PUBLIC HEALTH RELEVANCE: Thousands of clinical biospecimens are collected every year at cancer centers throughout the United States for the proper diagnosis and prognosis of breast, colon, and liver cancer. RNA molecules in these biospecimens are increasingly being used in diagnostic and prognostic testing and more recently in major therapeutic decisions. Unfortunately, the procedures for collection and storage of these biospecimens vary and many times not optimized to ensure sample integrity and quality. The purpose of this study is to develop an innovative technology to identify and assay a subset of mRNA molecules that are highly susceptible to degradation and can be used as molecular ""sentinels"" to better determine the quality of mRNA in clinical biospecimens before measuring panels of gene expression biomarkers. The long term goal of this study is improving patient care and therapeutic outcomes by better determining the quality of biospecimens before running multi gene expression diagnostic tests.
R21 CA155615 2011 HARISMENDY, OLIVIER UNIVERSITY OF CALIFORNIA AT SAN DIEGO Identification of Somatic Mutations in Rare Subclones of Solid Tumors
Cells within a tumor sample are known to be heterogeneous, due to the contamination from non-malignant tissue or the presence of multiple sub-clones, each carrying different somatic mutations. The majority of somatic mutations identified to date are clustered (mutational hotspots) in the functional sites of a few ""cancer genes"" with key roles in cell signaling pathways of proliferation and survival. Somatic mutations in cancer genes modify their oncogenic potential or affect sensitivity to therapy. Currently available assays that are able to detect rare somatic mutations are not comprehensive. They are usually focused on a few commonly mutated loci, and not implemented in clinical setting due to cost or technical reasons. Therefore, a current technological need exists for an assay that can reliably detect and accurately measure the prevalence of multiple somatic mutations present only in a fraction of the cells in a heterogeneous tumor. Such an assay would facilitate translational research to study the selection of tumor sub-clones during disease progression and treatment. Additionally the assay could be used by clinicians to improve tumor characterization and selection of therapy choices during clinical trials. We propose to leverage the emerging technology of targeted high-throughput sequencing to develop a cost- effective assay capable of detecting somatic mutations that are present in e1% of tumor cells. Specifically we will perform ultra-deep targeted sequencing (UDT-Seq) of ~100 kb in each tumor assaying 518 mutational hotspots located in 46 cancer genes. The selected mutational hotspots cover ~87% of all entries in the COSMIC database. We will develop a streamlined sample preparation in collaboration with RainDance Technologies to ensure a straightforward implementation in the clinic. This sample preparation integrates the targeting PCR and the library preparation in one step using chimeric PCR primers. The amplified targeted hotspots (200bp long) will be thus directly sequenced on the Illumina Genome Analyzer (GAII) at a very high coverage (~20,000x). We will then precisely model the sequencing error using calibration samples to filter true mutations from the sequencing noise. Our specific aims are: 1) To calibrate the UDT-Seq assay by analyzing both pooled DNA samples containing precise ratios of known SNPs and DNA samples spiked with low amounts of mutated DNA from cancer cells. For this, we will develop a statistical sequencing error model to detect rare mutations in deep sequence. 2) To evaluate the accuracy of the UDT- Seq assay to detect rare somatic mutations in both frozen and formalin fixed paraffin embedded solid tumors. If the quantitative milestones set for this pilot phase of the UDT-Seq assay development are met, we will apply for R33 funding to further develop and make this assay broadly available to clinical oncologists for their own translational research through a CLIA laboratory. PUBLIC HEALTH RELEVANCE: We propose to develop an assay to analyze somatic mutations in rare subclones of solid-tumors. This assay will feature microfluidic-based sample preparation method and high throughput DNA sequencing. It would be the most comprehensive assay to identify rare somatic mutational hotspots in a clinical setting and clinical oncologists will potentially use this assay to identify new biomarkers for disease progression or predictive of drug response, for a more personalized treatment of cancer.
R21 CA155478 2011 HRUDKA, BRIAN BIOSPECIMEN PROCUREMENT SOLUTIONS, INC. Development of a system (devices and protocols) to improve biospecimen preservati
Normal and disease tissue biospecimens likely contain all the molecular information required to define disease prognosis and guide treatment. This has been reduced to practice in the diagnosis and treatment of breast cancer, evidenced by the commercial success of the Mamaprint(R) genomic test (Agendia, Inc; based on estrogen and progesterone sensitivity biomarkers). However, it is recognized that power and utility of today's exquisitely sensitive analytical tools is limited by the quality of the biomarkers measured. Biomarker pre- analytical variability resulting from improper and inconsistent biospecimen acquisition, fixation and shipment can affect the results of molecular analysis and therefore cancer research and therapeutic decisions. Recognizing this, Core Prognostics, Inc. (CPX) developed a unique system (device and protocols) to greatly enhance the convenience, success rate, and reproducibility of biospecimen acquisition and shipping. This passively-cooled system is laboratory-validated and has a proven track record of performance in biomarker- based multi-center clinical trials including three sponsored by NCI cooperative groups. However, as experience has been gained with the system, opportunities for improvement have been defined. Specifically, the experience of CPX shows that a passively-cooled system is inherently unable to guarantee tissue sample quality when simultaneously shipping multiple temperature specimens under different environmental conditions and when operated by technicians with varying experience levels. The specific aim of this project is to create a reusable actively-cooled system to conveniently and reproducibly refrigerate multiple tissue samples to < -700C, 40C and 150C and precisely maintain temperatures during shipment. The research strategy is to expand existing CPX technology partnerships to access the expertise required to achieve the considerable thermodynamic, refrigeration, design, fabrication, hardware and software innovation and laboratory testing necessary to achieve the goals of this proposal. ""Off- the-shelf"" components will be utilized to the greatest extent possible, to take advantage of existing technology, reduce project time and expense as well as the anticipated cost of the final product. The practical benefits of a successful project to improve biospecimen cryopreservation and shipping from patient to analytical laboratory will be to: 1.) substantially increase the reliability and reproducibility of biospecimen preservation and shipping; 2.) create broader access to the collection of a wide range of tissue types; 3.) improve the convenience and flexibility of tissue acquisition; and, 4.) reduce the cost and environmental impact over current single-use solutions. In the long-term, these benefits support the mission of NCI OBBR: ""to guide, coordinate, and develop the Institute's biospecimen resources and capabilities and ensure that human biospecimens available for cancer research are of the highest quality""1 1. National Cancer Institute (NCI), Office of Biorepositories and Biospecimen Research (OBBR) website. PUBLIC HEALTH RELEVANCE: Key to cancer research today, and widespread implementation of ""personalized"" medical technologies in the future, is the need for significantly increased consistency and accessibility of biospecimen preservation and shipping from patient to analytical laboratory. Improvement of this important and under-investigated ""patient-to- platform"" link, for the benefit of cancer research, prevention and treatment efforts, is the subject of this proposal.
R21 CA143408 2011 HUANG, SONGPING D KENT STATE UNIVERSITY AT KENT Prussian Blue Nanoparticles as Cellular T1 MRI Contrast Agents
This proposal is aimed at developing Prussian blue nanoparticles (PBNPs) as a new generation of T1-weighted MRI contrast agents (CAs) with high relaxivity, long blood circulation times and ability to penetrate the cell membrane. Prussian blue (PB) is iron(III) hexacyanoferrate(II) with anidealized formula Fe4III[FeII(CN)6]3.nH2O (n=14-16) in which two different iron centers, Fe3+ (high-spin S=5/2) and Fe2+ (low-spin S=0) are bridged by the CN- groups. In the crystal structure of PB, a quarter (25%) of the FeII(CN)6 unit is absent from the crystal lattice, creating a large cavity inside the structure that is filled with water molecules. The missing FeII(CN)6 unit also causes the Fe3+ center to be coordinated by one water molecule and five CN- groups, thus giving rise to an active inner-sphere relaxation mechanism for enhancing the T1 relaxation. Due to the strong ligand-field effect and simultaneous coordination of the CN- group to both the Fe3+ and Fe2+ centers in the extended 3D network, the CN- ligand and the Fe3+/Fe2+ ions are completely locked in their lattice positions and cannot be released from the structure. As a result, PB has the lowest solubility product constant ever measured for any compound (Ksp=10-41). We have found that replacement of some of the Fe3+ ions with Mn2+ or/and Gd3+ ions in the crystal lattice can form the manganese- or gadolinium-incorporated nanoparticles, Mn@PBNPs and Gd@PBNPs with significantly increased r1 relaxivity. Besides, the structural rigidity and reduced tumbling rates of PBNPs in solution, as compared to the small molecular Gd3+-chelates, can contribute to additional T1-weighted MRI contrast enhancement in this new nanoplatform. Our goals are: (i) to explore methods for optimizing the r1 relaxivity by adjusting the nanoparticle size, level of Mn2+- or/and Gd3+-doping, and surface coating with small molecules or polymers; (ii) to systematically investigate the characteristics of cellular uptake and cellular imaging as well as potential for image-guided drug delivery applications; and (iii) to simultaneously incorporate Mn2+ or/and Gd3+ ions along with the radionuclide Ga-67 or Ga-68 for MRI-SPECT and MRI-PET bimodal imaging applications. We will endeavor to test the following four hypotheses: 1) Prussian blue nanoparticles, when properly tailored and engineered, will be effective in reducing the longitudinal relaxation time of protons from bulk water. Incorporation of Mn2+ or/and Gd3+ into this nanoplatform will significantly increase the r1 relaxivity; 2) Prussian blue nanoparticles will be internalized by cells, exhibit no toxicity and be effective in cellular imaging and in delivering small-molecular agents; 3) Prussian blue nanoparticles will be effective T1-weighted MRI contrast agents in vivo; and 4) Simultaneous incorporation of paramagnetic ions of manganese(II) or/and gadolinium(III) along with the radionuclide Ga-67 or Ga-68 into Prussian blue nanoparticles will produce effective bimodal contrast agents for in vitro and in vivo MRI-SPECT and MRI-PET imaging. Impact Our approach to exploring PBNPs as novel T1-weighted MRI is unprecedented and represents a paradigm shift in the design of new-generation CAs. The new paradigm that will emerge from this proposed research will prove to be revolutionary rather than evolutionary for increasing r1 relaxivity in a novel class of particulate T1-weighted MRI CAs, and thus will have high potential to produce a major breakthrough in MRI diagnostic imaging and may even completely change the landscape in this area of research. PUBLIC HEALTH RELEVANCE: Prussian Blue Nanoparticles as Cellular T1 MRI Contrast Agents We aim to develop novel Prussian blue nanoparticulate T1-weighted MRI contrast agents suitable for cellular imaging of cancer cells. Such agents will penetrate the cell membrane, exhibit no cytotoxicity, and can integrate imaging and delivering capabilities into a single platform for image- guided drug delivery. The design paradigm developed from this research will prove to be transformative and have potential to significantly impact the diagnostic MR imaging.
R21 CA155424 2011 KAY, BRIAN KENNETH; LAVIE, ARNON (contact) UNIVERSITY OF ILLINOIS AT CHICAGO Enzyme-delivery scaffold technology for targeted cancer killing.
Delivering a protein in its active state into a targeted cell is a major technological hurdle. However, the ability to do so would open up numerous applications in both clinical and basic research settings. We propose to develop cell-targeting systems that fulfill this task, composed of two components: the cell targeting module, and the protein cargo. The targeting modules will be based on the molecular scaffolds of single chain variable fragments (scFvs; ~25 kDa), designed ankyrin repeat proteins (DARPins; ~15 kDa), and affibodies (~ 9 kDa). Theses scaffolds will undergo optimization using phage display technology in order to acquire the needed attributes. These include tight and specific binding to a cell surface antigen, which is followed by efficient internalization and escape into the cytoplasmic compartment. These properties of the delivery scaffold will allow it to act as a transporter of proteins into cells. To test our technology, we will deliver an engineered version of human deoxycytidine kinase (dCKEN), which is a novel enzyme variant that has been endowed with thymidine kinase activity. We will test the efficiency and selectivity of the delivery scaffolds for their ability to ferry dCKEN into HER2 positive cells. The unique catalytic activity of this engineered enzyme will allow us to confine the activation of thymidine analogs only to cells that have internalized the enzyme. In this way, we would have developed a system that can be used to eradicate HER2 positive cells while not affecting other cells. The challenges to the delivery technology are to discover scaffolds that can be obtained at high yield in E. coli, that bind to the cell-surface marker with low nanomolar affinity, and that undergo efficient internalization and escape from endocytic vesicles into the cytoplasm. The novelty of this work stems from the type of delivery scaffolds used, which are much smaller than conventional monoclonal-based targeting systems. The advantages of using these smaller scaffolds include deeper penetration into solid tumors, reduced non-specific binding due to the absence of an Fc region present in monoclonal antibodies, and the ability for production in E. coli. Moreover, every aspect of the delivery scaffold, from binding affinity via internalization propensity, to escape from vesicles into the cytoplasm will be optimized by coupling phage display with the appropriate selection method. PUBLIC HEALTH RELEVANCE: A major technological hurdle confronting cancer therapeutics is how to take advantage of cancer markers to achieve targeted therapy. This application addresses this need by developing an enzyme delivery technology that transports a unique enzyme into the intracellular compartment of cancer cells. Subsequent administration of an otherwise non-toxic prodrug that is converted to its toxic form by the unique enzyme will result in the elimination of the targeted cancer cells. Importantly, this approach will spare healthy tissue.
R21 CA160157 2011 KOPELMAN, RAOUL UNIVERSITY OF MICHIGAN AT ANN ARBOR Magnetorotation: a Rapid Assay for Single Cell Drug Sensitivity of Cancer Cells
The current state of the art of many high throughput cell-based assays, used for cancer research, drug development and determination of precision therapy, often requires growth on 2D surfaces and requires the time-consuming and labor-intensive culturing step. Furthermore, current assays use large cell populations, which can pose a challenge when working with the critically important, but small in number, cancer stem cells (CSCs). When investigating CSCs, differentiation can occur in as little as 1-2 cell divisions. Currently, no high throughput method exists to measure cytotoxic effects on suspended cancer stem cells. So it is desirable to have a high throughput assay that is sensitive to volume and shape changes in single suspended cells and small spheroids. In response to the limitation of current protocols, we are developing a cell magnetorotation method, based on asynchronous magnetic bead rotation (AMBR), that is sensitive to single changes, and that will allow for rapid growth and cytotoxicity analysis of individual suspended cells and spheroids. Due to its submicroscopic resolution, high throughput, and short observation time, we anticipate that this AMBR assay will drastically reduce the culture step time, the overall time to results, and the number of cells needed for such an assay. For the preliminary validation of this new approach we selected to investigate single cells, single stem cells and single spheroids relating to prostate cancer. We set up a collaboration using the nanotechnology and nanomedicine expertise of the Kopelman Lab and the prostate cancer and drug sensitivity expertise of the Pienta Lab. Based on preliminary single cancer cell results, we hypothesize that the volumetric changes, resulting from the response to chemotherapy agents by cancer cells and spheroids, can be accurately and sensitively monitored. Furthermore, due to the combined sensitivity, speed and simplicity of the proposed approach, the typically very small number of stem cells can be tested rapidly, before their turning into a heterogeneous population. This will enable rapid drug sensitivity tests on cancer stem cells and on spheroids containing cancer stem cells, leading to a fast and smart selection protocol for therapeutic agents that can be tailored to a specific patient. We plan to achieve growth and cytotoxicity demonstration measurements on individual prostate cancer and cancer stem cells. We also plan to demonstrate that the AMBR method can be used to measure the growth of prostate cancer spheroids and their response to the cytotoxic agent docetaxel. Our long-term objective is to develop an assay device that will detect target cells directly in liquid clinical specimens (circulating tumor cells from patient blood) and provide automated results, with no manual steps, for personalized identification and drug sensitivity testing, for any form of cancer. PUBLIC HEALTH RELEVANCE: This proposal is relevant to public health because determining the optimum treatment for the individual patient is of prime importance in medicine, and particularly when it comes to cancer cases. A new approach, based on cell magnetorotation, enables drug sensitivity tests with higher sensitivity, throughput, speed and fidelity. Therefore, this proposal is relevant to the part of NIH's mission for developing medical discoveries that improve human health.
R21 CA155472 2011 LAI, JONATHAN ALBERT EINSTEIN COLLEGE OF MEDICINE YESHIVA UNIVERSITY Methods to Identify High-Affinity Antibodies that Target Tumor-Associated Glycans
Monoclonal antibodies are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, the use of anticarbohydrate antibodies to target glycans for these purposes has been less successful. Glycans contain less hydrophobic functionality than do proteins or nucleic acids, thus individual glycan-antibody interactions are relatively weak. Information encoded by glycans often involves subtle variations of branched oligosaccharides that cannot be detected with conventional antibodies. It has therefore been difficult to obtain reagents that enable a complete understanding of biological glycan function. Changes in cell surface glycan composition are associated with cancer (and other disease states); therefore, general methods to identify specific and high- affinity glycan antibodies would greatly facilitate cancer research and provide new avenues for cancer therapies and diagnostics. We propose to develop methods to identify such reagents using novel antibody phage display technologies. It has recently become possible to select specific and high affinity antibodies for virtually any protein antigen from phage libraries that bear tailored diversity elements encoded by synthetic DNA ('synthetic antibodies'). This innovative technology platform obviates the requirement for animal immunization, thereby circumventing many limitations of traditional hybridoma methods. We will use the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120, as the template for our design. The two heavy chain variable domains of 2G12 IgG exchange positions to create an extended recognition surface containing four oligomannose binding sites per IgG molecule. We have developed a phage vector that allows the functional display of the 2G12 scaffold. We will prepare synthetic 2G12 antibody libraries to determine minimal physicochemical requirements for high affinity glycan recognition in this scaffold. Next, we will use the information gained from these studies to identify novel 2G12 variants with altered specificity profiles for tumor-associated glycan targets. This innovative strategy will result in novel cancer antibodies that accelerate cancer research and pave the way for development of new cancer therapies and diagnostics. PUBLIC HEALTH RELEVANCE: Cell-surface oligosaccharides ('glycans') play critical roles in cancer and many other disease states, but the biological functions of glycans are poorly understood. Our goal is to develop strategies to identify glycan-targeting antibodies for use as research reagents, diagnostics, or therapeutics. Our approach employs novel methods for antibody isolation that circumvent many limitations of conventional antibody technologies, and will therefore yield antibodies with enhanced properties for biomedical applications.
R21 CA160052 2011 LAWRENCE, DAVID A WADSWORTH CENTER Holistic diagnostics of host during development of cancer
Our microfluidic Multiplex Immunodiagnostic Tumor Technology (¨MITT), which is able to sort blood components and catch a multitude of analytes, will be used to assess early host changes due to presence of a cancer. The ability to quantify a wide range of constituents from multiple organ systems as well as the cancer, itself, with high sensitivity and specificity will provide a holistic approach to evaluation of the physiological profile of cancer development. We predict that this instrumentation will be a substantial new technology for rapid early diagnosis of circulating tumor cells (CTCs) and of the host response induced by breast cancer. This device will be able to simultaneously quantify soluble analytes in plasma, blood leukocyte subsets and their released products, and CTCs, the critical and key parameter for this proposal. The fabricated chip at the heart of the instrument is able to fractionate blood into plasma, RBC/platelets, leukocytes, and CTCs with use of a micro-pillared array (plasma:cell sorter chip, PCSC) that precedes a grating-coupled surface plasmon resonance (GCSPR) chip capable of GCSPR or GCSP-enhanced fluorescence (GCSPEF) imaging; GCSPEF increases sensitivity approximately two logs, but requires secondary antibodies. The plasma constituents are captured on regions of interest (ROIs) with select antibodies. The plasma constituents will be screened for the host's immune and neuroendocrine response and stress-related proteins as well as tumor-specific antigens. Aim 1 will validate GCSPR/GCSPEF analysis with Luminex analysis and cell quantification by PCSC with flow cytometry. Aim 2 will determine the efficiency by which this technology can detect cancer and unique host biomarker profiles defining health status prior to detection of any palpable tumor. Cancer stage will be assessed by histology after blood assessments; female MMTV-PyMT mice with rapid onset of cancer will be compared to males and MMTV-neu mice as well as FVB controls used to obtain baseline values for blood biomarkers. Captured cells can be further phenotyped for released products or intracellular constituents after lysis. The technology will be optimized for sorting and capturing analytes for the greatest diagnostic and prognostic potential. The quantified profiles will delineate unique biosignatures that determine the characteristics of the host:cancer interaction. This technology will be useful for early cancer diagnosis, and we foresee using the technology to monitor tumor cell dissemination after surgery, tumor reoccurrence, and longitudinal therapeutic interventions. PUBLIC HEALTH RELEVANCE: The objective of this project is to establish and validate an innovative technology with the ability to provide relatively rapid diagnosis of the health status of a patient with a developing cancer, a patient after cancer surgery, or therapy to eliminate the cancer. Our system is designed to quantify a more holistic picture of patient status by assessing both presence of CTCs as well as the host's immune and stress profile. Quantification of CTCs in the blood would not be new, but our novel technology is designed to eliminate factors that could interfere with capture of CTCs (soluble TSAs). Additionally, we will be able to quantify i) immune factors, which have been implicated in many different aspects of cancer; ii) number and activity of circulating lymphocytes; iii) biomarkers of stress, e.g., CRP and HMGB1; and iv) intracellular biomarkers after lysis of quantified, captured cells (both lymphocytes and CTCs).
R21 CA157366 2011 LEVY, MATTHEW ALBERT EINSTEIN COLLEGE OF MEDICINE YESHIVA UNIVERSITY Targeting Cancer Cells with Functionalized Nanoparticle Libraries
We propose to use a combination of two powerful techniques: aptamer selection (systematic evolution of ligands by exponential enrichment; SELEX) and in vitro compartmentalization (IVC), to develop a new method of generating libraries of functionalized nanoparticles and that can be screened directly for function. While the goal of this application is to develop a novel system for the direct identification of targeted nanoparticles which localize to human tumors, we will develop a platform technology which extends beyond the detection and treatment of cancer and other diseases and will result in the generation of a novel class of capture agents that will find use in a variety of applications and fields of research. PUBLIC HEALTH RELEVANCE: We are engineering a new platform technology that will be of great utility in a variety of different fields for diagnostic, therapeutic and research applications. We will use this technology to develop a new class of targeting agents that are specific for cancer cells.
R21 CA151164 2011 MAKRIGIORGOS, G. MIKE DANA-FARBER CANCER INSTITUTE High-throughput technology that enables sequencing depth for colorectal CA
With the advent of second generation sequencing (SGS), for the first time there is a truly viable possibility of sequencing a substantial portion of an individual cancer patient's genome within a short time period and at relatively low cost, thus detecting mutations that can have prognostic or predictive value, or can serve as a fingerprint for tumor follow-up in a particular patient. However, there is still a missing link in providing a truly reliable identification of mutation fingerprints in patient tumor samples, as clinically-relevant mutations in tumors with heterogeneity, stromal contamination or in bodily fluids is problematic due to admixture with wild type alleles and can still be missed. And yet, the clinical significance of identifying these low-level mutation fingerprints is major in several situations as frequently these are the mutations that confer resistance, offer prognostic and predictive information and that would be useful for treatment follow-up. Unfortunately the new sequencing technologies 'lose steam' when it comes to detecting low-level mutations, and for SGS currently it's either deep sequencing or high-throughput capability, but not both. Thus integration of SGS with clinical practice cannot be effectively exploited. We developed Co-amplification at Lower Denaturation temperature (COLD-PCR), a new form of PCR that amplifies preferentially the minority alleles from mixtures of wild type and mutation-containing sequences, irrespective of where the mutation lies, providing a strong enrichment of the mutated sequences during PCR. We propose to establish massively-parallel COLD-PCR to enrich mutant sequences prior to their screening via SGA (Illumina), thus enabling 'deep' sequencing while also retaining high-throughput capability. To enable massively-parallel COLD-PCR, a micro-fluidic device that dispenses DNA and PCR reagents within individual nano-droplets (RainDance"") prior to PCR will be adapted to perform COLD-PCR in millions of separate nano- reactions simultaneously. The novel combination of technologies will be used to identify mutational fingerprints in tumors from 20 colon cancer patients, including low-level mutations, and then follow these fingerprints in plasma in the course of radio-chemo-therapy, to provide a molecular surrogate to therapy response. The proposed use of Novel Transformative Emerging Technologies is also applicable to other types of cancer and provides a solution bridging the gap in technology and enabling SGS to be applied to clinical oncology practice. Therefore relevance to Public Health is high. PUBLIC HEALTH RELEVANCE: Screening of individual patients' tumors for genetic alterations over many genes in a rapid and cost-effective manner is a significant challenge that must be fulfilled in order to realize the promise of individualized cancer treatment. Although major advances have been made, there is still a significant gap in technology that prevents clinical integration of the most powerful platforms for molecular profiling and follow-up of individual patients treatment. We propose an answer to this challenge by combining 3 cutting edge technologies, COLD-PCR, RainDance nano-droplets and Next Generation Sequencing. The novel combination of these technologies bridges the existing technology gap and enables reliable mutation screening in multiple genes simultaneously, in surgical cancer samples or bodily fluids. In view of the fundamental role of mutations in causing cancer and modulating tumor response to drug treatment this project has significant implications for public health.
R21 CA155479 2011 MCDONALD, JOHN F GEORGIA INSTITUTE OF TECHNOLOGY Use of nanogels to target delivery of siRNA to cancer cells in mice
Ovarian cancer is the leading cause of death from gynecologic cancer. Despite high initial tumor response rates of 80% to surgical debulking and chemotherapy, most women with advanced ovarian cancer will eventually develop drug-resistant disease. Because second-line chemotherapeutic agents provide a response rate of only 15-25%, there is clearly a need to develop better therapeutic strategies. Small interfering RNAs (siRNAs) are a class of RNA molecules that previously have been demonstrated to be highly effective in inactivating cancer-causing genes (oncogenes) in cancer cells grown in culture. However, the development of siRNAs as clinically significant therapeutic agents has been hampered by the fact that siRNAs are extremely unstable at physiological conditions. In addition, there has been no effective way to target these potentially therapeutic molecules specifically to cancer cells. We have recently demonstrated that gelatinous-like nanoparticles called nanogels can be easily loaded with siRNAs and targeted to deliver their potentially therapeutic payload to ovarian cancer cells grown in culture. Nanogels protect the siRNA molecules from degradation until released into the cancer cells. The proposed studies seek to demonstrate that these non-toxic nanogels can be used to effectively target siRNAs to cancer cells in living organisms (experimental mice) as well. Such studies are pre-requisite to the potential clinical application of the technology for the treatment of cancer in humans. Specifically, siRNAs directed against the EGFR (epidermal growth factor receptor) gene will be loaded into nanogels engineered to specifically target proteins expressed on the surface of ovarian cancer cells. The EGFR gene is highly expressed in ovarian and most other types of cancer cells and is known to induce cells to rapidly divide and become resistant to most commonly used chemotherapy agents. Thus, the inactivation of EGFR in cancer cells has great therapeutic value. Demonstration that nanogels can be an effective vehicle for the delivery of therapeutic siRNAs to cancer cells in mice will open the door to the development of this exciting new technology of clinical application in humans. PUBLIC HEALTH RELEVANCE: The ability to deliver siRNA against oncogenes specifically targeted to cancer cells will be a major contribution to the treatment of cancer. Development of a mouse model in which to demonstrate and refine this targeted delivery nanotechnology is a key step in moving the technology into clinical practice.
R21 CA143362 2011 MESSMER, BRADLEY T UNIVERSITY OF CALIFORNIA AT SAN DIEGO Molecular Evolution of Multifunctional DNA Nanoparticles
We have developed a DNA nanoparticle library technology for the selection of cell binding DNA nanoparticles. Rolling circle amplification (RCA) of circular oligonucleotide templates containing randomized nucleotides produces libraries of single stranded DNA nanoparticles that can be screened for cell binding properties. The main goal of this project is to create multimodal DNA nanoparticles that specifically bind to cancer cells. The particles will be ""bred"" by a novel iterative selection and re-assortment method to create modular DNA nanoparticles that contain multiple distinct recognition elements within a single particle. This project addresses a significant challenge in many areas of cancer research and treatment, mainly the lack of cancer cell specific binding agents. Our DNA nanoparticles differ from other affinity reagents in that there is intrinsic multivalent display of the modules, allowing avidity to compensate for low monovalent affinity. The modular nature of the particle template construction allows multiple distinct recognition elements to be assembled into a single molecular entity. Furthermore, the combinatorial selection method allows the optimal particle to be evolved in the same molecular context in which it will be used. Collectively, the unique features of our DNA nanoparticle libraries represent a novel paradigm for cell affinity reagents that replaces high affinity binding to one or two defined molecular targets with a diverse landscape of high avidity interactions. The specific aims for this application are: Aim 1. Validate and optimize combinatorial selection methodology for multi- module particles. We have identified single module particles that bind to a mouse pancreatic cancer cell line. We will optimize the multi-module selection strategy with this line and confirm on two human pancreatic lines (MiaPaCa-2 and Panc-1) as well as a leukemia line (K-562) to demonstrate the feasibility against both solid and liquid tumor types. Aim 2. Demonstrate cancer specific cell binding of selected particles. Three applications will be addressed: histology, flow cytometry, and cell capture. Fluorescently labeled particles will used on tissue arrays for fluorescent microscopy and on suspension cells for flow cytometry. Particles tagged with biotin or iron oxide will used for magnetic cell separation. Aim 3. Identify the molecular targets of the cancer cell specific DNA nanoparticles. Two approaches will be used. In the first, we will perform co- precipitation experiments after crosslinking biotinylated DNA nanoparticles to the cell surface molecules. In the second, we will use ""click"" chemistry between azide or alkyne tagged particles to specifically react with cells that are either indiscriminately labeled with the partner click chemistry or with cells that contain the partner chemistry in specific protein modifications (e.g., farnesylated proteins). PUBLIC HEALTH RELEVANCE: The main goal of this project is to create a new type of particle that can bind to cancer cells but no to normal cells. These particles, made out of DNA, can help understand the differences between cancer cells and normal. They can also be used to capture and observe cancer cells in clinical applications.
R21 CA138333 2011 MO, YIN-YUAN SOUTHERN ILLINOIS UNIVERSITY SCH OF MED Methods of systematic microRNA target validation and identification
It is well known that microRNAs could play a fundamental role in regulation of diverse cellular functions. As key gene regulators, microRNAs work through a posttranscriptional repression mechanism. Increasing evidence indicates that deregulation of microRNA expression could lead to a variety of disorders including human cancer. Although significant progress has been made in the past years in discovery of microRNAs and their biogenesis, and their role in many cellular phenotypes, it is not fully understood how microRNAs exert their cellular functions because a single microRNA can have hundreds of targets. Hence, identification of microRNA targets is a critical step toward understanding of molecular mechanisms of microRNA-mediated gene expression in normal and disease processes. Currently, this largely relies on computer-aided algorithms, which unfortunately are still unable to provide a precise picture of microRNA regulatory networks, and thus the predicted targets need further experimental validations. It is evident that target validation is a bottle neck in our effort to dissect microRNA pathways. In this application, we propose to develop a novel selection method for microRNA target validation and identification through two complementary approaches. The first approach is to determine microRNA/mRNA interactions using our pre-microRNA collection against a specific target cloned in our selection plasmid; the second approach is to determine microRNA/mRNA interactions using a 3'-UTR (untranslated region) library against a specific microRNA. We believe that our selection method is innovative, simple and powerful. An additional benefit of this method will allow us to determine whether there are any new features, besides the seed sequence homology, which could contribute to the specificity of microRNA targeting. Accordingly, this study will greatly enhance our understanding of microRNA targeting and gene regulation by providing a valuable research tool. PUBLIC HEALTH RELEVANCE: MicroRNAs are master gene regulators that work through a posttranscriptional repression mechanism. A single microRNA could target hundreds of targets. However, we do not have a full picture of microRNA/mRNA interactions yet. This study will develop a selection system to systematically determine microRNA/mRNA interactions. The success of this study will benefit microRNA research fields by providing a simple but powerful tool for microRNA target validation and identification.
R21 CA157395 2011 PARKER, LAURIE L. PURDUE UNIVERSITY WEST LAFAYETTE Label-free, real-time detection of kinase activity in vitro and in single cells u
Our long-term goal is to develop a sensitive, multiplexed detection platform for real-time single monitoring of prognostic kinase activity in tumor samples. The objective of this application is to develop the first steps towards a multiplex quantification of kinase activity in a breast cancer model system using surface-enhanced Raman spectroscopy (SERS) and peptide-functionalized nanoparticle (NP)-based biosensors. Recent unpublished work in our laboratory suggests that exogenously-added peptide substrates and SERS will allow for sensitive, direct monitoring of kinase activity in biological environments such as cells. In this multidisciplinary application, we combine the kinase biosensor expertise of Parker lab and the extensive SERS experience of Irudayaraj lab to develop a quantitative SERS platform to monitor the activity of Akt, Erk, Src, and c-Abl, kinases associated with drug resistance and clinical outcome of breast cancer patients. Towards demonstrating this proof-of-concept, we propose the following specific aims: Specific Aim 1. Standardize a SERS platform for the quantification of Akt, Erk, Src and Abl kinase activity. Specific Aim 2. Develop single cell mapping schemes to monitor differential kinase activity in response to various stimuli and inhibitors. Our approach represents a novel use of SERS microscopy and kinase substrate NP biosensor design. This technology is non-destructive (leaving cells viable and intact after analysis) and is adaptable to single molecule (and thus single cell) microscopy formats, providing exquisite spatial resolution and allowing us to monitor localized signaling in living cells. It is also tunable for different kinase substrates to allow simultaneous monitoring of more than one kinase activity, in other words 'multiplexing' the analysis, so we will be able to visualize kinases as complexes and systems rather than in isolation. This project has the potential to transform personalized medicine and therapy selection by facilitating single cell monitoring of therapeutic response. Using handheld SERS devices in the clinic, this technology could potentially benefit thousands of patients by giving pathologists (and thus clinicians) nearly 'real-time' mechanistic information about an individual's disease and therapeutic response. This work will also advance the field of signal transduction biology as a whole by enabling the analysis of multiple kinase activities (as opposed to just detecting the phosphorylation state of known endogenous substrates) in single cells in a microscopic platform. Upon demonstrating proof-of- concept with this pilot project, we will have the tools and experience in hand to undertake further technology and instrumentation development to facilitate real-time, live cell imaging using our technique, as well as implementation of a point-of-care, handheld SERS readout using commercially-available devices (goals for a future R33 application). Maturation of the detection platform to that stage would allow biologists for the first time ever to detect signal transduction in real-time in live cells without needing to develop and express e.g. a fluorescent genetic construct, potentially transforming cancer research and drug discovery. PUBLIC HEALTH RELEVANCE: This project has the potential to transform personalized medicine and therapy selection by facilitating single cell monitoring of cancer therapeutic response. Upon further development, this technology could benefit thousands of patients by giving pathologists (and thus clinicians) nearly 'real-time' mechanistic information about an individual's disease and therapeutic response.
R21 CA160129 2011 PARKER, LAURIE L. PURDUE UNIVERSITY WEST LAFAYETTE Biosensor technology to monitor leukemia-related kinase activity in patient cells
Even with the recent development of blockbuster kinase inhibitor drugs like Gleevec, leukemias are still high on the list of deadly cancers. One reason for this is that patient response to inhibitor drugs is monitored by in- direct means (hematological remission, cytogenetic response, mRNA expression levels). We are combining two emerging methods: our peptide-based targeted intracellular kinase sensors and Multiple Reaction Monitoring (MRM) mass spectrometry to develop a kinase assay technology that will provide direct information about kinase inhibition from patient material. The strategy will also be compatible with simultaneous analysis of more than one kinase at a time, so the systems-level biology of phosphorylation signaling can be addressed. In this pilot study, we will examine various parameters to determine the optimal sensor concentration, cell number, analytical protocols and statistical analysis to obtain meaningful kinase activity data from patient material. We will also optimize the sample handling protocols for collecting, processing and storing the patient material prior to the kinase assay. Upon successful demonstration and optimization of this technique, we will be poised to expand this project through additional technological development using the R33 mechanism, and/or further study of signaling biology in leukemia through a hypothesis-driven mechanism. We anticipate that our technology will provide unprecedented levels of sensitivity and detail for examining kinase activity in primary patient cells, and may someday become a tool for monitoring inhibitor drug effects in relatively 'real-time,' giving physicians the opportunity to adjust dosage to maximize the positive outcomes of kinase inhibitor treatment for their patients. PUBLIC HEALTH RELEVANCE: We are developing technologies to monitor the inhibition of kinases by drugs in leukemia. Our assay combines two emerging technologies to potentially provide information about drug response in patients that physicians could use to adjust dosages and improve treatment.
R21 CA138353 2011 RAO, JIANGHONG STANFORD UNIVERSITY Nanotechnology for Multiplex Detection of Enzymes
Current detection platforms are essentially designed to assay the protein expression level in the biological samples, but for enzymes, not just the expression level but the catalytic activity are also important to their functional role in the pathologic process. We have recently developed a quantum dot-bioluminescence resonance energy transfer (QD-BRET) nanosystem. When a mutant of Renilla luciferase (Luc8) and the QDs are in close proximity, the biochemical energy generated in the oxidation of its substrate, coelenterazine, by Luc8 can be transferred to the QDs through BRET, generating light emission from the QDs. Since this QD-BRET process is distance-dependent, it offers a unique detection platform to assay the enzyme activity. This QD-BRET nanosystem offers many advantages over existing platforms for bioimaging and biodetection. First, by eliminating the need for the external light input, QD-BRET avoids problems associated with fluorescence detection such as background fluorescence, direct acceptor excitation, and photobleaching. Second, it offers great sensitivity for detection. Third, existing detection technologies are subject to the interference from the hemoglobin absorbance, and require serum rather than whole blood analysis. QD-BRET is compatible with analysis of whole blood, thus circumventing the need for serum preparation. Fourth, the QD-BRET platform is amenable to multiplexing and will enable analysis of several targets in a single test. In addition, by avoiding external illumination sources, this technology is much easier to be miniaturized and to be developed into portable diagnostic devices. Fifth, in comparison to the traditional FRET protocols using QDs, QD-BRET uses QD as the emitter which utilizes their outstanding absorption cross-section and broad absorption spectrum. This R21 application seeks to further advance the QD-BRET technology by developing competitive QD- BRET (cQD-BRET) sensors for highly sensitive multiplex detection of enzyme targets in biological samples with three specific aims: 1) Establish the cQD-BRET sensor design for multiplex detection of proteases in a homogenous assay with serum, whole blood and tissue samples. 2) Multiplexed detection of protease activity on a microplate reader platform with immobilized antibodies. 3) Comparison of detection sensitivity with commercial ELISA assay. PUBLIC HEALTH RELEVANCE: The proposed research aims to develop a novel nanotechnology to detect critical enzymes that are implicated in tumor formation, migration and metastasis. This new nanotechnology will enable highly sensitive detection of these enzyme molecules in biological samples to help early detection of cancers and to advance our understanding of the differences of enzyme activity between tumor and normal tissues during cancer formation and metastasis.
R21 CA147922 2011 SANTANGELO, PHILIP J GEORGIA INSTITUTE OF TECHNOLOGY Characterizing gene regulation with single molecule sensitive probes
Deregulation of gene expression contributes to aberrant phenotypes and behaviors of cancer cells. Acquiring a new profile of expressed proteins and their subsequent activation enable cancer cells to re-enter the cell cycle, or give them survival and migratory advantages over those of the ""normal cells"". Alterations in cis-acting sequences, RNA binding proteins, or in upstream signaling pathways affect the stability and/or translational efficiency of mRNAs encoding proto-oncogenes, cytokines, cell cycle regulators and other regulatory proteins to promote tumorigenesis and cancer progression, especially during cellular stress induced by hypoxia or reactive oxygen species (ROS). In addition, these environments can also induce stress granule formation, a consequence of translational suppression and eIF21 phosphorylation, which can inhibit apoptosis. In this application we will focus on developing technology to characterize the deregulation of gene expression by: 1) evaluating the transcriptional activity of cancer cells by assessing mRNAs already transcribed, and 2) by characterizing mRNA/RNA binding protein interactions in the cellular/tissue milieu. To do this we will further develop probes to image low copy number native mRNAs in both fixed and living cells, and develop a novel method for detecting RNA-protein interactions with single interaction sensitivity. Recently we have developed a new probe consisting of four high-affinity, nuclease resistant, linear nucleic acids, labeled with multiple, high quantum-yield fluorophores linked together by streptavidin, via the biotin-streptavidin linkage. This design results in a multivalent, single RNA sensitive and versatile imaging probe. Because these probes contain a protein at their core, streptavidin, it is our contention that when combined with antibody-based proximity ligation assays (PLAs) and multicolor fluorescence microscopy, single RNA-protein interactions can be detected in fixed tissue or post live hybridization, fixed cell samples. Proximity ligation assays utilize oligonucleotide-linked antibodies, ligation, rolling circle amplification (RCA) and fluorescence detection to achieve single interaction sensitivity. Last, once these interactions are identified, fluorescent proteins will be developed for the relevant RNA binding proteins to allow for the monitoring of these interactions in live cells during cancer relevant processes. Thus, through the development of this technology, we will be able to characterize these events in cellular models of prostate tumor progression, both fixed and live, and in clinical tumor samples. From this characterization we hope to identify new targets for therapeutic development and possibly gain insight into mechanisms that may make tumor cells refractory to chemo and radiation therapy.
R21 CA160080 2011 SHENDURE, JAY ASHOK UNIVERSITY OF WASHINGTON Ultrasensitive identification and precise quantitation of low frequency somatic m
The ultrasensitive detection of clinically relevant somatic alterations in cancer genomes has great potential for impacting patient care, e.g. for early detection, establishing diagnoses, refining prognoses, guiding treatment, and monitoring recurrence. However, current technologies are poorly suited to the robust detection of somatic mutations present at very low frequencies. Massively parallel sequencing represents one path forward, but its sensitivity to detect very rare events is fundamentally constrained by the sequencing error rate. Our goal is to develop a new experimental paradigm that overcomes this limitation. In our approach, each copy of a target sequence that is present in a sample is molecularly tagged during the first cycle of a multiplex capture reaction with a unique barcode sequence. After amplification, target amplicons and their corresponding barcodes are subjected to massively parallel sequencing. During analysis, the barcodes are used to associate sequence reads sharing a common origin. Through oversampling, barcode-associated reads error-correct one another to yield an independent haploid consensus for each progenitor molecule, i.e. ""molecular counting"". Furthermore, the collapsing of commonly derived reads inherently corrects for any allele-specific bias during amplification, such that estimates of mutant allele frequency can be accompanied by precise confidence bounds. In our first aim, we will develop experimental methods and analytical tools that enable the robust detection of targeted somatic mutations via molecular counting to frequencies as low as 1 mutated copy in a background of 100,000 unmutated copies. In our second aim, we will develop three ultrasensitive, multiplex molecular counting assays that are specifically targeted at panels of clinically relevant cancer mutations or genes, and rigorously evaluate these for reproducibility. The availability of robust, cost-effective, generically applicable tools for the ultrasensitive, multiplex detection of rare somatic events will be a transformative step forward for the translation of discoveries in cancer genetics to a clinical setting. PUBLIC HEALTH RELEVANCE: As we enter an era of ""personalized medicine"", DNA sequencing technology will be increasingly important to public health, contributing towards the unraveling of the genetic basis of human disease, as well as for clinical diagnostics. This proposal aims to develop ultrasensitive methods for detecting cancer-relevant mutations in tumor samples. These technologies have the potential to directly enable the translation of discoveries made in cancer genetics to clinical applications such as the early detection of cancer and the monitoring of patients for cancer recurrence.
R21 CA155536 2011 YAO, XUDONG UNIVERSITY OF CONNECTICUT STORRS Ultrathroughput Multiple Reaction Monitoring Mass Spectrometry for Large-Scale Ca
Protein biomarkers have enormous potential in the diagnosis, prognosis and treatment of cancer. Realization of the high therapeutic and financial potential of cancer biomarkers demands new technologies for speedy and cost-effective production of high-quality biomarkers and facile adjustments for measuring newly-produced biomarkers. Liquid chromatography- stable isotope dilution-multiple reaction monitoring mass spectrometry (LC-SID-MRM MS) of signature peptides of candidate biomarkers is an emerging technology well suited to this field. This application will establish the feasibility of transforming this mass spectrometry method to break through the sample-throughput bottleneck. The new technology is termed Ultrathroughput Multiple Reaction Monitoring (UMRM) MS and is an integration of LC-SID-MRM MS and peptide derivatization. Two specific aims will be pursued: (1) identification of suitable peptide derivatizations for UMRM measurements, and (2) validation of derivatizations for UMRM analysis of prostate-specific antigen in 108 serum samples in a single experiment. The fundamental novelty of the new technology rests on the novel transformation of LC-SID-MRM MS to produce unprecedented sample throughput. It has three unique advantages: the throughput advantage, the flexibility and immediate-impact advantage, and the economic advantage. The new technology can be implemented with inexpensive reagents and commercial mass spectrometers for focused quantitation of cancer biomarker candidates in many phases of a biomarker pipeline. The drastically increased sample throughput of the new technology will in particular impact the verification and validation of biomarker candidates, which require targeted quantitation of hundreds to thousands of patient-control samples. Thus, the UMRM technology will significantly accelerate new cancer biomarker generation. The new technology also has potential in advancing quantitative biology of cancer. PUBLIC HEALTH RELEVANCE: This project examines the feasibility of transforming he multiplexing potential to the sample- throughput potential of the emerging peptide multiple reaction monitoring mass spectrometry for cancer biomarker development. The new technology, capable of one-experiment quantitation of large numbers of samples, will significantly impact the large-scale validation of cancer biomarkers which uses hundreds and thousands of patient-control samples.
R21 CA157417 2011 ZEICHNER, STEVEN L CHILDREN'S RESEARCH INSTITUTE Development of an in vivo screening technology for cancer vaccine immunogens
This application addresses the mandates of the RFA by proposing to develop a highly novel and previously untested technology to identify, assess, and compare immunogens for cancer vaccines, employing an in vivo immune system in a high throughput screening mode. The development of the new technology will bring together multiple investigators experienced in a wide variety of complementary techniques, from different institutions and disciplines. If successful, the new technology will have great impact on oncology by providing investigators with an important new technology to evaluate and compare immunogens for cancer vaccines. We will develop and then employ this new technology in a test case to identify immunogens that elicit immune responses to the survivin tumor-associated antigen, an attractive and widely studied tumor vaccine candidate useful for this developmental proof-of-principle study due to its demonstrated activity in cancer vaccine models and relatively small size. The technology exploits an intact GI mucosal immune system as a massively parallel in vivo screening device. To develop this new screening technology we will: 1) Create a library of bacteria transformed with DNA bar-coded plasmids that include expression cassettes that place large amounts of chimeric protein, which will include sequences encoding an exhaustive, overlapping library of peptides derived from survivin, on the Gram-negative bacterial surface, 2) Feed the library to mice, 3) Use PhyloChip microarray technologies (or high throughput sequencing) to identify, via the barcodes, members of the library showing decreased relative abundance in the mouse feces over time, which we would take as evidence of the induction of a mucosal immune response against the chimeric proteins encoded by plasmids showing decreased abundance. We will rescreen the clones to confirm their ability to induce an immune response and evaluate sera and cells from animals inoculated with bacteria expressing the proteins and animals immunized with the proteins identified in the screen to determine if they induce anti-survivin humoral and cellular immune responses. Clones and the peptides encoded by the clones able to induce an anti-survivin response will be studied in an in vivo tumor vaccine model and compared to peptides already known to induce an anti-tumor immune response in the model. The study proposes to develop an innovative, rapid, high-throughput approach to the identification of potential immunogens useful as cancer vaccine employing the survivin tumor associated antigen as a model. If successfully developed, the technology also would be useful in the search for other immunogens for cancer vaccines and for comparing the immunogenicity of different candidate proteins/peptides. Since the proteins are expressed in Gram-negative bacteria, including vaccine strains of Salmonella, the technology could be used to rapidly and cheaply produce a candidate vaccine. Although the project is risky, the project could therefore potentially have a very large impact on cancer immunotherapy. PUBLIC HEALTH RELEVANCE: Cancer immunotherapy or vaccines for the therapeutic treatment of cancers hold much promise, but to date have not proven effective. We propose to develop a new screening system that uses the gastrointestinal immune system to identify proteins or smaller pieces of proteins that may be good candidates for new vaccines. We then propose to use this new screening system to identify a protein or a piece of a protein, which we will instruct bacteria to produce using recombinant DNA technology, that can be developed into a vaccine to treat cancer.
R33 CA160344 2011 ALARID, ELAINE T; BEEBE, DAVID J (contact) UNIVERSITY OF WISCONSIN MADISON Integrated Micro Scale transcriptional profiling of cell communication networks
In cancer, differences in risk factors and genetics generate diverse clinical behaviors (disease progression, response to therapy) at the patient level. Yet, despite critical implications for therapeutic outcome, few tools are available to study patient samples at the cellular level in a rigorous way with sufficient replicates to enable clinically meaningful assessments. This challenge is particularly acute in the context of cancer metastasis, where micro-tumors establish themselves in ectopic environments which are significantly different in cellular make-up than primary tumor. But few tools allow one to probe intercell network communication (e.g. paracrine signals between different cell types). A sample preparation bottleneck is particularly acute for the study of heterogeneity requiring large numbers of samples (e.g. each patient sample is different, each requiring nucleic acid purification). Thus, there is a need to perform culture (including co-culture), treatment, and analyte purification from a large number of samples (multiple patients, treatments, and repeats). Our goal is to apply simple micro scale technology to profile intercell regulation on heterogeneous patient samples. The cornerstones of our approach are an immiscible oil barrier, termed the ""Phase-Gate,"" that separates the upstream ""dirty"" side (culture, treatment, lysis) of sample processing from the downstream ""clean"" side (analyte purification and detection) and micro chamber co-culture with increased sensitivity to paracrine signaling. Our preliminary data predicts that neighboring stroma play a critical role in regulating key therapeutic targets and that advances in experimental capabilities (throughput, sensitivity, compartmentalization) will provide new tools and biological insights that will advance basic and translational cancer research. Here, we will apply a co-culture and transcriptional analysis platform to dissecting cell network communication - specifically how intercell regulation contributes to nuclear receptor regulation and ultimately to tumor progression and therapeutic resistance. The validation and advanced development of this technology directed towards an important area in cancer biology (nuclear receptor response) will accelerate its use enable studies not feasible previously. PUBLIC HEALTH RELEVANCE: We anticipate that the research proposed in this grant will not only help to elucidate how intercell regulation contributes to nuclear receptor regulation, but ultimately to tumor progression and therapeutic resistance. More broadly these studies will provide new tools and biological insights that will advance basic and translational cancer research.
R33 CA147988 2011 COMINS, DANIEL L; HAWKRIDGE, ADAM M; MUDDIMAN, DAVID C. (contact); PETITTE, JAMES N NORTH CAROLINA STATE UNIVERSITY RALEIGH Development and Application of Novel Glycan-Specific Reagents to Facilitate Early
Mass spectrometry is an extraordinarily powerful bio-analytical technique that has had a profound impact on our molecular understanding of human health and disease. Major advances in mass analyzer technology, dissociation techniques, and ionization methods are largely attributed to the central role that mass spectrometry plays in the field of systems biology. While mass spectrometry has evolved over the last century into a highly effective analytical tool, there are still opportunities for new advances to be made allowing an even more diverse array of biological questions to be addressed. This proposal is centered on the development and characterization of novel glycan- specific tags for biological mass spectrometry that facilitate the relative quantification of glycans cleaved from plasma proteins with increased ion abundance. The short-term objective of this proposal is to develop these novel tags and then test them using the avian model of spontaneous epithelial ovarian cancer. These results will provide a solid bio-analytical technology foundation from which our newly developed chemical tags can be applied to achieve the long-term objective of effectively elucidating glycan biomarkers for the early detection of epithelial ovarian cancer in women. Public Description of Proposed Research Mass spectrometry (MS), the science related to the ""weighing of molecules"", has had a profound impact on the study of human health and disease including cancer, heart disease, neural development, and auto-immune diseases. However, front-end chemistries for MS-based glycomics will be exploited to dramatically improve the ability of MS to detect and quantify glycans cleaved from plasma proteins. This will allow a more diverse array of contemporary biomedical questions to be addressed including the quantification of diagnostic and prognostic biomarkers. This proposal is squarely centered on the elucidation of a glycan-specific biomarker(s) for the early detection of epithelial ovarian cancer. PUBLIC HEALTH RELEVANCE: This proposal seeks support to develop and apply novel tags that will facilitate both global and targeted quantitative mass spectrometric analysis of N-linked glycans with significantly improved limits-of-detection. Once synthesized, the novel tags and ancillary methods will concurrently be applied to the experimental chicken model of spontaneous epithelial ovarian cancer for comparative analysis (i.e., biomarker discovery) and fully disseminated to the glycomics research community. The long-term application of these novel tags, as developed in our laboratory for this study, will be their use to elucidate glycan biomarkers for the early detection of epithelial ovarian cancer.
R33 CA160138 2011 DORSEY, KATHLEEN CONWAY ; THOMAS, NANCY E (contact) UNIVERSITY OF NORTH CAROLINA at CHAPEL HILL High-Throughput DNA-Methylation Profiling from Fixed Melanocytic Tissues
Project Summary/Abstract Melanoma has the capacity to metastasize early and its course is rarely impacted by medical intervention. Because of the pronounced difference in survival between localized and metastatic disease, it is imperative to diagnose melanoma in its earliest form; however, even expert pathologists can have difficulty distinguishing melanomas from benign nevi (moles), especially atypical nevi. DNA methylation holds promise as a tool, in conjunction with histopathology, for enhancing melanoma diagnosis by providing molecular information. High- throughput DNA-methylation array profiling has the potential for discovery of candidate DNA-methylation sites useful for diagnosis but must be valid on formalin-fixed paraffin-embedded (FFPE) tissue, which is typically the only diagnostic material available for melanocytic lesions. In an R21 phase, we demonstrated technical feasibility of DNA-methylation profiling using FFPE tissues and identified a ""proof-of-principle"" signature for discriminating melanomas from nevi. However, many challenges remain in the application of this technology to FFPE tissues, including the ability of the assay to handle small specimens and methods for verifying percent tumor. Our long-term goal is to develop a practical clinical assay for molecular diagnosis of melanoma at the earliest possible stage, while avoiding false-positives and minimizing the overall cost of diagnosis. The objectives in this application are to identify internal markers to assess percent tumor, determine valid conditions for high-throughput DNA-methylation profiling of small FFPE tissues; and confirm DNA-methylation differences using additional assays. The central hypothesis of our proposal is that high-throughput DNA- methylation array profiling of FFPE tissues can be further developed to handle small samples and internal standards can be identified to assess percent tumor. The rationale for the proposed work is that allowing the majority of samples to be profiled will decrease bias in selection of candidate DNA-methylation differences, while the internal standards will exclude samples that will not give valid results. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify candidate DNA-methylation differences that distinguish melanocytic (nevus or melanoma) vs. non-melanocytic (surrounding skin or lymphocytic infiltrate) cells for use as internal quality control standards to quantify sample percent tumor; 2) Identify valid conditions for high-throughput DNA-methylation array profiling of small-sized FFPE melanocytic tissues; and 3) Confirm candidate DNA-methylation differences from high-throughput profiling using more quantitative assays. The approach is innovative because it couples emerging high-throughput DNA- methylation technology to a biospecimen repository and FFPE melanocytic specimens, as they are typically prepared in hospital and community-based dermatologic practices. The proposed research is significant because valid high-throughput DNA-methylation profiling of the majority of FFPE specimens along with internal quality control measures will open new diagnostic opportunities for melanoma and other malignancies. PUBLIC HEALTH RELEVANCE: Project Narrative Melanoma has a predilection to metastasize when only a few millimeters in depth; however, early detection and diagnosis are difficult due to the overlap in clinical and histologic appearances of melanomas with highly prevalent benign moles. High-throughput DNA-methylation array technology holds the promise of discovery of candidate DNA-methylation markers useful for improving melanoma diagnosis but must be valid on small formalin-fixed tissues embedded in paraffin blocks, which is typically the only diagnostic tissue available for primary melanomas and moles. The goal for this R33 is validation and advanced development of methodology to overcome the challenges when this novel technology is directly implemented for analysis of small formalin- fixed paraffin-embedded specimens, along with identification of criteria for quality assessment.
R33 CA155554 2011 GARRAWAY, LEVI ALEXANDER (contact); MACCONAILL, LAURA E DANA-FARBER CANCER INSTITUTE High-Throughput Tumor Genomic Profiling by Massively Parallel Sequencing
Cancer diagnosis and treatment decisions have historically been based on anatomic sites of origin and spread; however, the emerging paradigm incorporates key genetic attributes of a given tumor to predict clinical behavior and specify the optimal use of targeted therapeutics. Ultimately, the delivery of personalized cancer medicine will require systematic characterization of all therapeutically informative tumor genomic alterations in the clinical and translational arena. Previously, we developed and deployed OncoMap, a mass spectrometric genotyping-based platform that enables high-throughput profiling of hundreds of known mutations across dozens of cancer genes. This platform performs well and has launched a robust translational oncology effort. However, the mass spectrometric genotyping technology remains limited in scope, assay sensitivity, and the type of genomic alteration that can be identified. Recently, it has become possible to render multi-faceted tumor characterization both technologically feasible and economically accessible through massively parallel sequencing (MPS) technology. Thus, the goal of this application is to migrate the OncoMap approach to an MPS platform (Illumina), empowered by innovations such as solution-phase exon capture and sample barcoding. Together with our colleagues at the Dana-Farber Cancer Institute and Broad Institute, we have generated preliminary data showing the feasibility and promise of each of these components, thereby raising the possibility of comprehensive tumor sequencing at a low per-sample cost. Accordingly, in this R33 application we will implement a transformative platform for systematic tumor genomic profiling, which we call ""MPS-OncoMap."" To accomplish this we will optimize the methodology for sample barcoding technology, solution-phase exon capture, and single-molecule sequencing to enable robust mutation profiling (base mutations, amplifications, and deletions) across ~150 cancer genes in at least 12 tumor samples simultaneously. We will test the performance of MPS-OncoMap using DNA from cancer cell lines, frozen tumors, and formalin-fixed, paraffin-embedded tumor tissue for which the ""ground truth"" is known for multiple genetic alterations. Finally, we will implement MPS-OncoMap at production scale to enable systematic analyses for many translational oncology applications. Achieving these Aims may inform a definitive path to comprehensive tumor genomic profiling with far-reaching impact in the translational and clinical oncology arena. PUBLIC HEALTH RELEVANCE: High-Throughput Cancer Gene Mutation Profiling y Massively Parallel Sequencing PIs: Levi A. Garraway, M.D., Ph.D. and Laura E. MacConaill, Ph.D. Many cancers are caused by mutations in the DNA that give rise to altered proteins or cellular processes. The use of anticancer drugs targeting such proteins can lead to significant clinical benefit, but it remains impractical to profile the tumor of each cancer patient for the presence of such alterations. This application seeks to adapt a powerful new sequencing technology together with other innovations to make it possible to detect many informative cancer gene mutations in clinical tumor specimens. Once implemented, this approach could speed the advent of personalized cancer medicine.
R33 CA151210 2011 GREIS, KENNETH DONALD UNIVERSITY OF CINCINNATI Validation of MALDI-MS-based inhibitor screening technologies for cancer targets
Validation of transformative MALDI-MS-based inhibitor screening technologies for cancer targets. Mass spectrometry (MS) has a long history as a transformative technology. Two specific examples include quantitative MS to measure the fate of drug compounds in biological systems and the development of proteomics techniques for protein identification and characterization. Recently MS-based applications have been demonstrated to be highly effective as the readout for high throughput screening (HTS) assays. The major advantage reported over the commonly used fluorescence and chemi-luminescence readout is the paucity of false positive or false negative readout. Other added benefits include a greatly reduced (>80%) reagent cost by using label-free substrates and the ability to multiplex assays such that multiple therapeutic targets can be screened for inhibitor hits with one pass through the compound repository thus saving millions of dollars in reagent and personnel costs. We have developed MALDI-MS based readout methods for measuring enzyme activity and inhibition for a variety of enzyme classes. However, the limited use of these methods with small test libraries has been insufficient to validate the overall utility of this readout for large screening campaigns. Thus the primary goal of this proposal is to systematically validate the reliability of the MALDI-MS readout compared to a traditional method of HTS using a library of 50,000 compounds. The measurements will include hit rates from the primary screen, the validation rate of the hits in secondary screening and other standard quality assessments common for HTS including signal to background, coefficient of variance (CVs) and Z' values. The target enzymes for these comparative assays will include two related kinases, PKC-zeta and PKC-iota, which appear to regulate two directly opposite effects on cancer initiation and development. Thus as a secondary aim of this proposal, the MALDI-MS readout will be assessed for its effectiveness to distinguish inhibitors from the compound repository that have selectivity for PKC-iota over PKC-zeta. We have shown that MALDI-MS readout is amenable to enzyme assays and inhibitor screening on a small scale with major advantages over existing readout methods. If, the MALDI-MS readout can be scaled to true HTS levels while maintaining all the advantages seen in the proof-of-concept studies, then this MS-based technology would likely transform the way we approach HTS, in much the same way as MS-based technologies have changed bioanalytical and proteomics applications over the past 15-20 years. Furthermore, by targeting kinases (a key class of regulatory enzymes whose dysregulation is often associated with cancer development) to validate the MALDI-MS readout approach, we can be assured that with the success of these studies, it will be clear that this technology can be readily reapplied to other cancer relevant kinases as well as expanded into other enzyme classes and disease categories. PUBLIC HEALTH RELEVANCE: Dysregulation of cellular function is a hallmark of cancer development and progression and can often be traced to the actions of one or a few cellular enzymes, thus these enzymes may act as primary targets to halt cancer development and progression. As such, rapid and accurate methods to evaluated inhibitors of these target enzymes represent an initial phase in the new drug discovery and development process. In this proposal, we investigate a new readout technology, based on mass spectrometry, to rapidly screen for inhibitors of a class of enzymes known to be involved in cancer development and progression.
R33 CA147966 2011 JU, JINGFANG STATE UNIVERSITY NEW YORK STONY BROOK Identification of post-transcriptionally regulated targets by TrIP-Chip/Seq
Resistance to chemotherapy is one of the major reasons for the failure of cancer treatment. Post- transcriptional and translational control plays an important role in chemoresistance. Currently there is no available high throughput approach to investigate post-transcriptional and translational control mediated by RNA binding proteins or non-coding microRNAs from a small number of cells. Our primary objective in the proposed project is to apply our newly developed Translational Immunoprecipitation-Microarray Analysis (TrIP-Chip) (1) to discover post-transcriptionally regulated mRNA targets mediated by miR-215 from a small number of colon cancer stem cells. Our recent studies revealed that miR-215 is involved in regulating some key anticancer targets, e.g. thymidylate synthase and dihydrofolate reductase. Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to form dTMP and dihydrofolate. Because the TS-catalyzed enzymatic reaction provides the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis, TS has been an important target for cancer chemotherapy for over 50 years. The enzyme dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. This reaction provides the key intermediate in one-carbon transfer reactions. DHFR plays a critical role in folate homeostasis, and is required for the de novo synthesis of purines, thymidylate and certain amino acids. Therefore, DHFR has served as a critical target in cancer chemotherapy. The mRNAs regulated post-transcriptionally by miR-215 will be validated at the protein level by a sensitive, high throughput shotgun proteomic analysis based on multi-dimensional protein identification technology (MudPIT). We will further develop a TrIP-Seq approach to increase both the coverage and sensitivity to detecting rare transcripts potentially at the single cell level. Three specific aims are proposed: Specific Aim 1. Identify post-transcriptionally and translationally regulated mRNA targets of miR-215 using TrIP-Chip approach. Specific Aim 2. Validate miR-215 mediated targets by high throughput proteomic analysis. Specific Aim 3. We will further develop the TrIP-Chip approach by integrating next generation sequencing based expression analysis (TrIP-Seq) to increase coverage and detection sensitivity from a small number of cells. PUBLIC HEALTH RELEVANCE: Currently, there is a lack of high throughput approach to study post- transcriptional and translational control with a small number of cells. We have developed a novel Translational Immunoprecipitation-Array Gene expression analysis (TrIP-Chip) technology that allow us to investigating post-transcriptional and translational regulated genes from as few as 500 cells. We believe with the improvement of the dynamic range of the next generation deep sequencing platform (e.g. Illumina Instrument), we can potentially analyze translational control from a single cell. The proposed project will develop and apply this new approach to discover miR-215 mediated targets and functional significance in colon cancer and colon cancer stem like cells. The technology will have a broad application not only in cancer but other human diseases as well.
R33 CA157403 2011 LIOTTA, LANCE ALLEN GEORGE MASON UNIVERSITY Implementation of phosphoprotein preservation technology for cancer biospecimens
An urgent clinical goal is to identify molecular networks associated with subpopulations of cancer patients who may respond individually to molecular targeted inhibitors. Current molecular targeted therapeutics is directed at protein kinases and/or their phosphorylated substrates. Therefore, measurement of this new class of phosphoprotein signal pathway epitopes in tumor biopsy samples is crucial for individualizing molecular targeted therapies. Phosphoprotein antigen epitopes are not adequately preserved by formalin fixation and paraffin embedding, and freezing of tissue is very expensive and compromises diagnostic accuracy. We propose the advanced development and clinical validation of an innovative and transformative technology for preserving tissue phosphoproteins and diagnostic histomorphology for clinical cancer molecular profiling. Applying knowledge gained under an NIH R21 funded study, we created a novel tissue preservation chemistry that stabilizes all classes of phosphoproteins, is compatible with paraffin embedding, while maintaining complete diagnostic histomorphology, and fully preserving critical diagnostic immunohistology (IHC) antigens including Estrogen Receptor, Progesterone Receptor, HER2, and Ki-67. These IHC antigens are not preserved by special research fixatives used for tissue RNA preservation. Our new non-formalin tissue preservative, termed Biomarker and Histology Preservative (BHP) can be seamlessly introduced into the current community hospital clinical diagnostic workflow with no additional steps or equipment. At the time of procurement, tissue can be immersed directly in the new fixative and processed into a paraffin block for routine diagnosis, obviating the need for costly freezing during shipping or storage. BHP offers the potential for substantial improvements over conventional formalin fixation. In the present application we propose the blinded clinical validation of our novel preservation chemistry in community hospital settings, utilizing a team of international pathologists for validation. The goal of the project is one-step paraffin block stabilization of all classes of cellular phosphoproteins, diagnostic histomorphology, and diagnostic immunohistochemistry antigens, while at the same time maintaining full diagnostic morphology equivalent or superior to standard formalin fixation. We will collect fresh surgical tissue, under informed consent, covering a broad variety of organs and cancer histology to develop an archive of 150 cases of matched paraffin and frozen specimens. We will measure 100 validated phosphoprotein epitopes spanning membrane, cytoplasmic and nuclear compartments from extracted paraffin sections using Reverse Phase Protein Microarray (RPMA) and Laser Capture Microdissection (LCM) technology. Following objective independent validation by diagnostic pathologists, this transformative technology will be ready for widespread clinical and research use. Adoption of the technology would mean that only one diagnostic paraffin block could be used for all classes of molecular profiling rather than the current requirement for multiple blocks. This would increase diagnostic accuracy while substantially reducing costs. PUBLIC HEALTH RELEVANCE: Our technology for preserving proteins, at the time of collection, will enable analysis of molecular targeted kinase inhibitors for individualized therapy, while considerably reducing healthcare costs, and increasing diagnostic accuracy. Our new non-formalin tissue preservative can be seamlessly introduced into the current community hospital clinical diagnostic workflow with no additional steps or equipment.
R33 CA155586 2011 MULVIHILL, SEAN J; PORTER, MARC D (contact) UNIVERSITY OF UTAH Advanced Development of a Multiplexed SERS-based Biomarker Detection Platform: A
This proposed research focuses on the advanced development and validation of an innovative, extensible diagnostics platform to markedly improve early cancer detection and cancer risk assessment through the ultrasensitive readout of biomarker panels. The creation of a surface-enhanced Raman scattering (SERS) immunoassay diagnostic platform, realized by coupling sensitive SERS detection with nanoparticle labels, enhanced analyte delivery, and immunoassay architectures, will result in a simple platform capable of multiplexed biomarker detection. This intelligent use of a panel of biomarkers will serve as the cornerstone in making subclinical cancer detection a reality. Integral to this work is the use of pancreatic adenocarcinoma (PA) as a disease model to validate the platform. PA exhibits traits common to most diseases that progress asymptomatically, including the unavailability of one ideal tumor marker with high clinical sensitivity and specificity. Since development of PA arises from a range of causative mutations in individuals, a panel of multiple biomarkers with overlapping detection capabilities is likely to provide improved accuracy. Assembly of such a panel from markers that have demonstrated limited correlative value individually provides a vehicle to assess the figures of merit and multiplexing ability of the SERS platform. Currently, our ability to identify health risk, disease susceptibility, and response to therapy remains unreachable due in part to our technical inability to easily screen a single sample for the large number of biomarkers that potentially make up a ""disease map"" and to do so at costs that enable routine testing for everyone. The SERS platform is designed to remove this hurdle. Once validated, the platform could also be used for marker and marker panel discovery and is uniquely suited for rapid deployment as a cost-effective, portable, and robust system for multiplexed diagnostic assays in a clinical setting or to form an essential part of the personalized medicine infrastructure. PUBLIC HEALTH RELEVANCE: This proposal seeks to make early cancer detection and the associated promise of improved outcomes accessible to the general public. Current technical limitations have hindered the ability to rapidly screen large numbers of biomarkers from individual patients, a method purported to have improved diagnostic, prognostic, and therapeutic efficacy over a single biomarker. The project proposes developing a platform that uses a rapid, cost-effective optical detection technology to simultaneously measure hundreds of biomarkers in a single drop of blood.
R33 CA155618 2011 SUPERFINE, RICHARD UNIVERSITY OF NORTH CAROLINA CHAPEL HILL Array Microscope Assay for Cancer Cell Mechanics
Array Microscope Assay for Cancer Cell Mechanics Abstract As cells become cancerous, characteristic changes take place in their behavior that affect cell division as well as the ability of the cell to migrate or metastasize. Metastatic behavior, including cell migration, motility and adhesion, is one of the most damaging hallmarks of cancer. Current assays of cell metastases involve the observation of the lateral mobility of cells in a ""scratch"" assay, or the translation of cells through porous membranes. These assays usually take several hours to days of cell tracking. Metastatic potential has recently been associated with protrusive ability and cell body mechanical properties. We propose to replace the migration assay with one that measures the cell stiffness and cell mechanical response. This involves performing a calibrated tug on the cell with the measurement of the probe displacement. This measurement takes only seconds. This would allow the replacement of a five to forty eight hour assay with a one minute assay. More important than the simple benefit of a faster measurement on a single specimen, we propose an assay system that will allow high throughput methodologies to be applied to elucidating the time course of the biochemical pathways at the heart of the mechanical, and hence, metastatic propensity. We currently have a prototype multiwell assay system demonstrated on cancer cell mechanics. Our next steps are to move from a 16 well prototype to a 96 well assay, and to validate our system on cell lines and on ex-vivo tumor cells. Our development of high throughput force assays will be applied to relate tumorigenicity to the regulated expression of TGF-2 superfamily receptors and subsequent TGF-2 superfamily signaling. TGF-2 and the related TGF-2 superfamily ligands, the bone morphogenetic proteins (BMPs) and inhibin, are potent regulators of normal epithelial cell proliferation, differentiation, survival and migration, with frequent disruption in these homeostatic mechanisms resulting in human cancers and driving human cancer progression, including the metastatic process. We will assess dynamic changes in biomechanical properties during epithelial- mesenchymal transition (EMT), and investigate the migratory, invasive and metastatic potential of these cell models both in vitro (cell lines) and ex vivo and correlate these results with the biomechanical measurements. These measurements will validate our high throughput force system for a wide variety of cancer cell biology studies, enabling the elucidation of the biochemical and genetic determinants of metastatic behavior. PUBLIC HEALTH RELEVANCE: Array Microscope Assay for Cancer Cell Mechanics Narrative We will develop a high throughput force assay system validated on cancer cell lines and on ex-vivo tumor specimens. This powerful system will be ready to be used for discovery of biochemical and genetic determinants of cancer cell metastatic properties to better understand the basic science, diagnosis and treatment of cancer.
R33 CA155252 2011 TANG, KEQI BATTELLE PACIFIC NORTHWEST LABORATORIES Validation and Advanced Development of Emerging Technologies
The primary goal of the project is to develop mass spectrometry (MS) based assays for high throughput and quantitative biomarker validation with sensitivity and specificity comparable to or better than antibody based immunoassays. The analytical platform will be primarily based on triple quadrupole mass spectrometry with its sensitivity, dynamic range, measurement specificity and sample throughput significantly enhanced by integrating the latest high speed liquid chromatography (LC), high efficiency electrospray ionization (ESI) source and unique MS interface technologies developed at Pacific Northwest National Laboratory (PNNL) and extending the instrument capability from performing traditional multiple reaction monitoring (MRM) measurements into MRM2 MS analysis. The expected limit of quantitation and dynamic range for the new instrument based assays will be expected to reach low pg/mL level and span eight orders of magnitude in sample concentration allowing quantitative measurements of low abundance protein biomarkers in complex biofluids, such as human blood plasma. The new instrument based assays will be applicable to a broad spectrum of clinical cancer biomarker validation applications and capable of validating hundreds to thousands biomarkers in a single experiment. Their performance will be rigorously optimized and tested using clinical samples. PUBLIC HEALTH RELEVANCE: The development of mass spectrometry (MS) based assays for high throughput and quantitative biomarker validation with sensitivity and specificity comparable to or better than antibody based immunoassays.
R33 CA157396 2011 TSENG, HSIAN-RONG UNIVERSITY OF CALIFORNIA LOS ANGELES Advanced Development of An Integrated CTC Enrichment Technology
Project Summary The long-term objective of this research proposal is to perform advanced development and analytical validation of a technology for enrichment of circulating tumor cells (CTCs). The goal is to further develop our highly efficient and specific CTC capture technology to pave the way not only for CTC enumeration to serve as a biomarker for PC to better predict clinical outcomes, but also as a source of clinical material (i.e., CTCs as a ""liquid biopsy"") for sequential molecular analyses that can be used to direct appropriate therapies for individual patients (i.e., ""the right treatment for the right patient""). Our joint team has demonstrated a unique, relatively inexpensive cell affinity assay, which is capable of identification, enumeration and capture of viable (preservative-free) CTCs in whole-blood samples collected from PC patients. Initially, we pioneered the concept of applying anti-EpCAM (epithelial cell adhesion molecule)-coated nanostructured surfaces as a high-affinity substrate for enrichment of CTCs. By integrating the high-affinity substrate with a microfluidic component capable of generating chaotic turbulence, further improved CTC capture efficiency (up to 99%) has recently been achieved as a result of the enhanced collisions between CTCs and the substrate. Side-by-side analytical validation was conducted to compare our nanostructure substrates with CellSearchTM assay using blood spiked with PC cell lines as well as 33 blood samples isolated from in PC patients at predefined stages. CTCs are cancer cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. Enumeration of CTCs has established clinical utility in patients with metastatic, castration-resistant (CR; i.e. hormone refractory) PC, in whom CTCs are an independent predictor for response to chemotherapy, disease free survival and overall survival. It is conceivable that the molecular and functional characterization of CTCs could provide much valuable information for predicting patient prognosis and monitoring therapeutic interventions and outcomes. Herein, we will first develop a new-generation integrated CTC chip capable of highly efficient and specific enrichment of CTCs with improved blood handling capacity, followed by comprehensive analytical validation using blood samples collected from PC patients at predefined stages (e.g., CRPC, PSA recurrence). We will then purify and isolate individual CTCs for quantification of 16 genes using a commercial real-time qPCR System. We propose to quantify expression of 16 genes, which we have chosen as markers of differentiation state, epithelial-mesenchymal transition, and the AR signaling axis. The molecular signatures imparted by these genes not only depict various cellular phenotypes but also offer the promise of predicting response/ resistance to anti-cancer therapeutics. PUBLIC HEALTH RELEVANCE: Relevance to Public Health The long-term objective of this research proposal is to perform advanced development and analytical validation of a technology for highly efficient enrichment of circulating tumor cells (CTCs). This new CTC-based diagnostic platform will pave the way not only for CTC enumeration to serve as a biomarker for PC to better predict clinical outcomes, but also as a source of clinical material (i.e. CTCs as a ""liquid biopsy"") for sequential molecular analyses that can be used to direct appropriate therapies for individual patients.
R43/44 CA161965 2011 Lim,Mark Ambergen, Inc Global Proteomic Screening By Maldi Spectrometric Imaging Of Protein-Bead Arrays
Sequencing of the human genome has led to a new and even more ambitious goal - characterization of the human proteome. Such an endeavor involves not only understanding the function of hundreds of thousands of different proteins expressed in human cells but also characterizing the millions of potential interactions that can occur with other cellular and extracellular molecules including proteins, nucleic acids, lipids and small molecules. The ability to rapidly perform such massive global proteomic screens would be a powerful tool in many areas of cancer research such as biomarker discovery, mapping cellular networks and drug development. Although high-density DNA microarrays introduced almost 20 years ago have had a major impact in facilitating the genomic revolution, high-density protein microarrays have not yet exerted a similar impact. Current limitations in protein microarray technology include low array density, poor reproducibility, high cost, poor assay kinetics and difficulty in detecting a diversity of bait-prey interactions as well as enzyme-induced protein modifications. In contrast, mass spectrometry used in conventional proteomics does provide many of these capabilities including label-free identification of small drug compounds, identification of protein modifications and protein identification. However, the separation methods used in conjunction with conventional mass spectrometry based proteomics such as two-dimensional gel electrophoresis and liquid chromatography are slow and not nearly as robust as the physical arraying/sorting of proteins inherent in a microarray. During Phase I we will evaluate a new approach developed by AmberGen for proteomics termed Bead-based Global Proteomic Screening (Bead-GPSTM) which combines the advantages of MALDI mass spectrometry imaging (MALDI-MSI) and microarray technology. This approach utilizes photocleavable mass-tags (PC-Mass-Tags) to encode a protein-bead library (bait library) as well as interacting prey molecules such as other proteins, all displayed on individual beads randomly arrayed at high-density (1,000,000 wells) in a Pico-well plate. Because we have shown in preliminary experiments that MALDI-MSI of high density protein-bead arrays has the potential to rapidly identify millions of different mass-tag combinations, with high sensitivity and spatial resolution, it is possible to perform highly multiplexed screening of bait-prey interactions far beyond the capabilities of conventional fluorescence microarrays. However, fluorescence imaging can still be used with Bead-GPS"" to pre- identify and quantitate positive interactions which are then decoded by MALDI-MSI. In addition, the power of Bead-GPSTM is further extended by the ability of MALDI-MSI to perform on-bead label-free detection of i) interacting prey molecules such as small drug compounds, ii) other proteins (protein fragmentation fingerprinting) and iii) protein modifications (e.g. serine or tyrosine phosphorylation). During Phase I we will fabricate a 100-member prototype protein-bead library using cell-free protein translation techniques in order to evaluate key features of Bead-GPSTM including PC-Mass-Tag coding (for both bait and prey molecules), protein-protein interaction analysis both with PC-Mass-Tags and by label-free means, detection of label-free protein-drug interactions, detection of protein modifications and serum profiling for cancer biomarker discovery. During Phase II, a full proteome-wide Bead-GPS"" platform will be constructed and tested. In order to accelerate commercialization of the products resulting from this project we will work closely during Phase I and II with Bruker Daltonics (Billerica, MA), a world-leading provider of MALDI-MS instrumentation, to develop a user- friendly, fully integrated instrument (and software) which will serve as a platform for the Bead-GPS"" technology.
R43 CA163403 2011 Ruan,Biao Potomac Affinity Proteins, Llc Protease Chain Reactions For Molecular Analysis Of Cancer Markers
Our objective is to develop a protease chain reaction technology (ProCR) which will enable ultra- sensitive molecular detection. A protease chain reaction has four basic components: 1) a protease conjugated to a binding molecule, 2) an unconjugated protease, 3) an inhibitor protein which contains a proteolytic cleavage site and 4) a protease substrate which generates a signal upon its cleavage. Fundamentally, a protein chain reaction is a powerful analogue computer with two key characteristics which greatly facilitate the detection of target molecules. 1) It can convert the concentration of a specific target molecule into a time signature. 2) It can create enormous signal amplification, analogous to the amplification of DNA by a polymerase chain reaction (PCR). Thus detection is enabled because the final observable signal produced by a target molecule can be very large and the time lag until onset of the signal is precisely correlated with the concentration of target molecule. The critical elements for controlling the protease chain reaction (and ultimately determining the sensitivity of assays linked to it) are very tight inhibition of the protease by the intact inhibitor and cleavage of the inhibitor by free protease. Accordingly, the three experimental Aims are: 1) Engineering the subtilisin prodomain to maximize inhibition; 2) Design and construct prodomain inhibitors with cleavable loops; 3) Construct and characterize a prototype chain reaction useful for detection. The Phase I goal is to demonstrate proof of principle for protease chain reactions and their applicability for molecular detection. The three milestones are: 1) Develop tight inhibitors with KI d 100pM; 2) Engineer cleavable loops into the inhibitors of Aim 1 which are cleaved a rate > 1000 M-1s-1; 3) Detect 1 femtomole of protease- conjugated antibody by ProCR in a microtiter dish assay. The long term goal is to develop protease- inhibitor complexes as enzymatic nano-processors which can be combined to detect multiple signals and to control output with multiple logic gates.
R43 CA163405 2011 Ozers, Mary Szatkowski Proteovista, Llc High Throughput Method To Assess Snp Functionality In Prostate Cancer
A critical unmet need in implementing personalized medicine is the ability to sort through the millions of single nucleotide polymorphisms (SNPs) present in the human genome and to pinpoint which of these DNA variations are causative in disease. A key under-studied function of SNPs is their ability to generate or disrupt genomic binding sites for transcription factors involved in cancer. Toward this goal, we are inventing the SNP-SNAP (Specificity and Affinity for Proteins) microarray as a prototype high throughput device to evaluate SNP function. The SNP-SNAP arrays will be used to display a quarter-million prostate cancer- related SNPs as double-stranded DNA molecules and to assay transcription factors (i.e., drug targets) for their binding to these SNP DNA sequences. The resulting data will be correlated with prostate cancer incidence. The million-plus data points from the SNP-SNAP arrays will be analyzed using SNP-Sequence Specificity Landscapes, creating a prostate cancer ""molecular signature"" that relates transcription factor binding, SNP preferences, and chromosomal position of the nearest genes. Our findings will also relate prostate cancer-associated SNP function with cancer stage and aggressiveness. Understanding SNP function will have a major impact on personalized medicine, by providing individualized disease risk assessment, identifying new personalized therapeutic targets, and predicting efficacy and potential off- target side effects of common therapeutics. The goals of this Phase I project are to: 1. Design and synthesize a customized SNP-SNAP DNA microarray to tile across a quarter-million SNPs that are associated with prostate cancer. 2. Examine the DNA binding specificity and affinity of 5 prostate cancer-related transcription factors, as purified proteins and from cell lysates, on the SNP-SNAP array and annotate the human genome with the transcription factor binding differences due to SNPs. Verify results with chromatin immunoprecipitation in prostate cancer cells. 3. Obtain SNP data from patients with prostate cancer and determine if there is a statistically significant association of functional SNPs, which yielded differential binding of prostate cancer specific transcription factors on the SNP-SNAP array, with prostate cancer incidence. This technology can assay millions of SNPs and multiple transcription factors simultaneously, thus representing one of the first methods to evaluate SNP functionality in a high throughput manner. Our SNP- SNAP technology, by virtue of the array custom design and ability to examine millions of DNA permutations, is also broadly applicable to any cancer type and disease model.
R43 CA163436 2011 Morachis,Jose Nanocellect Biomedical, Inc. Ading Color To Cancer, Adenovirus And Flow Cytometry To Identify And Capture Ctcs
We propose a novel technique to identify and capture circulating tumor cells (CTCs) using engineered adenoviruses and sophisticated flow cytometry. Current techniques for detection of CTCs include reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, fluorescence in situ hybridization, and, more recently, microfluidics. Unfortunately, RT-PCR does not distinguish between viable metastatic CTC versus nucleic acids or cellular fragments originating from the primary tumor. ! Antibody-based techniques cannot be used for detection of all cancers, but only those cancers that express the most common and well- characterized markers. As such, there is a desperate need to develop new diagnostic agents and tools that not only detect and capture CTCs but also quantify their malignant potential and identify 'up-front' the therapies that are most effective in ablating an individual patient's tumor. Despite the complexity and variability of cancers at a genome scale, a unifying theme is their growth deregulation phenotypes, the so-called hallmarks of cancer, which are conferred by mutations in a relatively small number of key pathways. Rather than focus on detecting individual genetic lesions that are numerous and highly variable between tumors, we propose to create diagnostic viruses that incorporate multiple transcriptional and molecular modules in their genomes to infect and detect a patient's tumor, report its molecular 'hallmarks' and its response to different therapies 'up- front'. Using these agents, the molecular lesions and malignant characteristics of any given tumor will be rapidly discerned (within 24 hours) and scored via a standardized automated platform. Furthermore, these agents could also be used as reporters to determine rapidly and directly if a patient's tumor is likely to respond to a particular therapy. Our goal is to develop a standardized automated platform that provides point-of-care diagnostics to inform clinical decisions at a level of molecular sophistication and prognostic power that is not possible with any other detection system, biomarkers or correlative gene expression signatures. To achieve this, we will combine transformative new technological platforms developed at the Salk, UCSD, and NanoSort that label tumor cells in different colors based on their acquisition of molecular lesions that dictate malignant progression and response to therapy, facilitating their detection, quantification and isolation using an integrated 'lab-on a chip' flow cytometer.