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R21 CA157298-01A1 2012 AKSAN, ALPTEKIN UNIVERSITY OF MINNESOTA TWIN CITIES Development of Room-Temperature Storage Technique for Plasma/Serum Biospecimens
Currently, millions of serum biospecimens are being stored in biorepositories across the nation, while tens of thousands of new biospecimens are added to the pool daily. These biospecimens are stored for future research, mainly for proteinaceous biomarker discovery and verification (e.g. for diagnostic, therapeutic, and epidemiologic outcomes). The success of biomarker research not only depends upon the availability of the tools (proteomic, peptidomic, lipidomic and metabolomic technologies) to extract information from biospecimens, but also on the availability of ""high quality"" biospecimens. However, in most biorepositories, serum is stored by freezing without following any preservation protocol; the samples are directly placed in -20, -40 or -80oC freezers, in the absence of any cryoprotectant, where they experience very slow cooling (1-2o C/min). It is well known that these conditions impose very harsh chemical and physical stresses on macromolecules, altering their characteristics (structure and activity), often irreversibly. Recent evidence has shown that some of the most promising proteinaceous cancer biomarkers are indeed very susceptible to freeze/thaw and frozen state storage. Therefore, it is plausible that frozen state storage may cause most of the potential biomarker information in the stored sera biospecimens to be lost forever. Our long-term goal is to eliminate the requirement for frozen state storage and develop the techniques to store serum biospecimens at room temperature using isothermal vitrification technology. Isothermal vitrification is the process by which liquids doped with sugars are desiccated to a ""glass"" (a very viscous fluid). In this state, biochemical reactions are halted, degradation of the specimen is stopped, and macromolecules are stabilized in their native states. Isothermal vitrification will eliminate the exposure of the proteinaceous biomarkers to freeze/thaw stresses and to frozen state storage damage and thus will substantially increase the quality of the stored biospecimens. It will also present a more economical alternative to freezing, since storage of specimens in freezer-farms will no longer be needed. We will accomplish our goal by achieving the following four Aims: Aim #1. Develop a panel of lyoprotectant chemicals to be added to serum samples for isothermal vitrification: Demonstrate that the lyoprotectant cocktail enables rapid and uniform vitrification while stabilizing sera proteins. Aim #2. Determine the retention and elution efficiency of sera proteins following vitrification by adsorption in standard filter paper using the developed lyoprotectant cocktail. Aim #3. Determine whether specific sera proteins are altered following optimized vitrification and elution conditions from the optimal matrix; document protein depletion, aggregation and degradation. Aim #4. Develop and validate an electrospun sponge for streamlined vitrification of serum samples at sera collection sites. PUBLIC HEALTH RELEVANCE: The advent of genomics and proteomics for personalized medicine has placed biospecimen research on the forefront of NIH priorities, since research projects are only as good as the biospecimens used. We are developing methodology to stably store liquid biospecimens, such as sera, at room temperature. This technology will eliminate the need for long-term storage of serum samples in freezer farms, which are very costly to operate.
 
R21 CA173245-01 2012 CARON, MARC G. DUKE UNIVERSITY A Cancer Rainbow Mouse for the Simultaneous Assessment of Multiple Oncogenes
Understanding the genetic mechanisms which underlie tumor development will provide a foundation for developing new generations of better and more effective cancer therapies. To address this fundamental issue, new technologies are needed that are superior to those currently available. Current state of the art mouse models enable testing the stochastic activation of oncogenes in a cell type specific fashion and provide a mechanism for testing targeted therapies. Despite these desirable features, their limited accessibility and feasibility precludes their implementation in most laboratories. Moreover, as the list of oncogenes continues to grow, reliable and efficient technologies are needed that can assess several oncogenes simultaneously in order to facilitate rapid analysis of tumorigenicity and their responses to therapy. This application provides a solution to all these issues by developing an innovative transgenic mouse platform (Crainbow) to rapidly, efficiently, and cost effectively create mouse models of tumorigenesis. The Crainbow technology will enable multiple user selectable oncogenes to be incorporated into a single transgenic mouse whereby each oncogene will be identifiable on the basis of a unique epitope tag and a separately expressed spectrally resolvable fluorescent protein. Using a strategy of Cre induced stochastic activation, the Crainbow technology enables the contribution of each oncogene to be assessed coincidentally in the same tissue with single cell resolution. Additionally, clonally derived tumor cell populations can be studied in any tissue and cell type by breeding to the plethora of validated cell type specific Cre mouse lines. Underlying the flexibility of this technology is a novel plasmid construct for incorporating the oncogenes of interest. The Crainbow plasmid can be easily modified for oncogene targeting using the molecular biology skills available in most all biology laboratories. Crainbow transgene mice can then be produced rapidly on site or obtained from other investigators or repositories. The utility and versatility of this Crainbow platform technology is demonstrated through the following specific aims which: (1) Establish a flexible and rapid strategy for cancer rainbow transgene design and construction, (2) Generate and validate cancer rainbow transgenic mice, & (3) Generate tissue specific tumors using cancer rainbow transgenic mice. The Cancer rainbow technology addresses in a single mouse shortcomings of current technology by providing a comprehensive method to simultaneously monitor the activity of multiple genes, and a means to also rapidly screen novel targeted cancer therapies. Consequently, the Cancer rainbow technology will provide a paradigm shift for studying tumorigenesis. PUBLIC HEALTH RELEVANCE: This application will establish a powerful but easily employed technology that will supplant previous chimeric tumor mouse models. Therefore, cancer treatment strategies targeting these oncogenes can be easily and economically tested, resulting in more effective patient therapies and better clinical outcomes.
 
R21 CA173303-01 2012 CARTEGNI, LUCA SLOAN-KETTERING INSTITUTE FOR CANCER RES Controlled premature termination of translation to generate designated truncated
Many diseases, including several forms of cancer, depend on the inappropriate expression of pathological proteins, such as oncogenes. Frequently, the structure-function of these proteins belies a modular construction, with an essential functional domain in the C-terminal portion of the protein (for example the transactivation domain of a transcription factor). Mutants lacking the C-terminal domain can have altered and antagonistic functions, and in some cases they can behave as dominant- negative variants. Such variants are sometimes naturally generated by alternative splicing/polyadenylation. However, these are not always present or not easily activatable. A method to specifically induce these dominant negative variants would be highly desirable and have a significant therapeutic potential in a broad range of diseases, including cancer. In order to generate a pre-designed, desirable truncated variant instead of the pathological one we propose development of a novel UNIVERSITYersal methodology, based on a brand new design of antisense molecules that are able to redirect termination of translation from the normally translated full-length ORF to an upstream specified location. These Self-Wrapping Antisense Translation Termination (SWATT) compounds work by introducing an insurmountable steric block that cannot be resolved by the helicase activity of incoming translating ribosomes, which can normally unwind extensive secondary structures within the pre-mRNA. This roadblock would thus result in ribosome drop-off and expression of a shorter, C-terminally truncated variant. We will validate the viability of this novel strategy by assessing SWATT compounds on specifically designed reporter luciferase constructs and, eventually, by showing their biological relevance using Notch signaling as a proof-of-principle substrate in cancer cells. Induction of a C-Terminal dominant negative version of Notch, lacking the intracellular signaling domain, should inhibit Notch signaling by engaging Notch ligands in non-productive interactions, and thus prevent its oncogenic functions. SWATT compounds will provide an additional tool to dissect the structure-function properties of proteins in their physiological contexts, and they wil constitute the basis for a novel therapeutic approach in the treatment of cancer and other diseases. PUBLIC HEALTH RELEVANCE: We plan to develop a novel technology to re-direct the production of proteins that contribute to tumor progression to variants of the same proteins with opposite, anti- tumorigenic properties (Dominant-Negative variants). This will be obtained by introducing a physical 'road-block' into the path of ribosomes during translation at specific, pre-determined points, and therefore forcing them to interrupt protein synthesis there. If successful, this work might constitute the basis for a novel, alternative approach to cancer therapy, with potential application to a broad range of tumors.
 
R21 CA173092-01 2012 CRAMER, SCOTT D; DAVALOS, RAFAEL VIDAL (contact) VIRGINIA POLYTECHNIC INSTITUTE AND STATE UNIVERSITY Isolation of Tumor Initiating Cells (TICs) using Contactless Dielectrophoresis
Tumor initiating cells (TICs), also known as cancer stem cells, are a finite population of cancer cells that have the ability to transplant a new tumor from an existing one. They are also putatively responsible for the metastatic properties of tumors. Isolation of TICs is the first step towards understanding the role of these cells in the pathogenesis and progression of cancer and is critical towards the development of improved specific therapies for cancer. The TIC is the most relevant therapeutic target. However, current efforts in isolation and characterization of the TIC are hampered by the lack of suitable high throughput and rapid methods to isolate these cells. Contactless-dielectrophoresis (cDEP) is a new, non-invasive technique to detect and enrich rare cells suspended in a medium based on their physical and electrical properties, independent of their genotype. Dielectrophoresis (DEP) relies upon the motion of a particle due to its polarization induced by a non-uniform electric fiel. Conventional DEP microfluidic systems are susceptible to electrode fouling and require complicated fabrication procedures, because the electrodes are in direct contact with the sample, which limits their lifetime and cost effectiveness. cDEP alleviates these limitations by using external electrodes placed in secondary, highly conductive, channels that are separated from the cell sample by a thin insulating layer. Since the devices do not require complicated fabrication techniques, mass production is readily achievable and, therefore, this technique can be used by a large population of biologists and researchers for cell isolation. The application of DEP to separate target cells has been studied extensively in the last two decades, and the results indicate that rare cells can be separated from other cells in a cell mixture. Our central hypothesis is that cDEP is capable of discriminating between tumor imitating cells (TICs) and non-TICs. We will utilize a multifaceted approach involving experimentation and computational modeling to design new cDEP devices to assess and optimize the effectiveness of cDEP technology to physically separate and enrich tumor initiating cells from other cell populations found in a tumor. This multi-faceted approach will include 1) characterize the dielectrophoretic response of tumor initiating cells compared to typical tumor cells, 2) assess the performance of cDEP at isolating and selectively concentrating tumor initiating cells, 3) investigate possible subpopulation of TICs by sorting cells using cDEP microdevices, and 4) use primary human prostate tissue and repeat Aims 1, 2, and 3 with primary human prostate cells. Success in isolating TICs will lead to increased understanding of the most appropriate therapeutic strategies to ablate this cell population. PUBLIC HEALTH RELEVANCE: Contactless Dielectrophoresis (cDEP) is a new technique to isolate and enrich rare cells from a cell mixture or from biological fluids. The goal of this projet is to develop cDEP as a tool for physicians to isolate patient specific tumor initiating cells (TIC) from non-TICs. This will facilitate understanding of TICs role in the pathogenesis, progression of cancer, and will lead to increased understanding of the most appropriate therapeutic strategies to ablate this cell population.
 
R21 CA173124-01 2012 DAYTON, PAUL A; JANZEN, WILLIAM PERRY (contact) UNIVERSITY OF NORTH CAROLINA at CHAPEL HILL Cavitation Enhancement of Biospecimen processing for Improved DNA Fragmentation
Random, unbiased fragmentation of DNA is necessary for next-generation sequencing (NGS) and chromatin immunoprecipitation (ChIP). Since DNA fragmentation can be a very problematic step for both NGS and ChIP, any technology that increased the efficiency and consistency of this step will be highly desirable for both research laboratories and in clinical diagnostics. Also, a technology that could make this step easier with no new equipment and very little cost to the laboratory would be ideal. We propose to apply the use of lipid encapsulated microbubbles to the fragmentation of DNA from both purified genomic and formaldehyde crosslinked samples. We have recently explored the feasibility of this technology, and our results were impressive. Preliminary data indicate that microbubbles can greatly improve the consistency of acoustic DNA fragmentation. Additionally, these bubbles are added in microliter volumes to the DNA or cell suspension at a cost ranging from one to ten cents per well, and can be used with any standard acoustic sonicator, presenting substantial cost savings compared to other techniques to improve DNA shearing. Furthermore, the microbubble technique greatly reduces the time required to optimize shearing, potentially greatly improving the throughput of this technique. As a second aspect, we will also assess the potential of microbubble technology to enhance tissue processing of formalin-fixed paraffin embedded (FFPE), or microbiopsies. Our goal will be to determine conditions for optimal performance of this phenomenon. Variables tested will include buffer reagents, microbubble size, microbubble concentration, acoustic frequency, acoustic peak pressure, and sonication duration. The project will conclude with publication of Standard Operating Procedures to disseminate the utility of this new technology. PUBLIC HEALTH RELEVANCE: Next-generation sequencing is playing an increasingly important role in understanding genetic mutations associated with cancer. DNA fragmentation is a crucial, but problematic step in this technique. Our preliminary data suggests that we have a technology to improve the robustness of this technique, as well as reduce time and cost through the application of acoustically active microbubbles to the shearing suspension. We will explore and optimize aspects of this novel approach.
 
R21 CA160060-01A1 2012 EWING, ROBERT (contact); WANG, ZHENG CASE WESTERN RESERVE UNIVERSITY Developing novel technology for mapping dynamic oncoprotein interaction networks
Many oncoproteins and tumor suppressive proteins shuttle between the cytosol and nucleus and thus interact with changing sets of protein partners in different subular compartments. Furthermore, increasing evidence indicates that oncogenic mutations of these proteins can result in alteration of cellular localization and rewirin of oncoprotein interaction networks. Characterization of the dynamic interaction network of oncoproteins and tumor suppressive proteins is therefore crucial to understand their roles in tumorigenesis. Robust technologies for mapping dynamic protein interaction networks under physiological conditions within the cell are not yet available. We propose to develop a novel approach for mapping dynamic oncoprotein interaction networks using endogenously epitope-tagged proteins for affinity- purification and quantitative proteomics for accurate network identification. This application builds upon our successful development of an innovative method to introduce epitope tag- encoding DNA into endogenous loci by homologous recombination-mediated knock-in in human cancer cells and our intensive experience in quantitative proteomics analysis. The endogenously tagged proteins are expressed at physiological levels and provide physiologically-relevant environments and compartments for protein complex identification and are therefore ideal for mapping dynamic protein networks. The goals of this application are twofold. First, we propose a detailed quantitative comparison of our technique in terms of distinguishing mutant and wild-type oncoprotein complexes against two conventional approaches for studying protein complexes. Second we test the potential of our new approach for studying the dynamic interactions that occur in response to cell signaling. Successful development of this technology will provide a platform for understanding the reconfigurations to protein interaction networks that result from oncogenic related translocation or mutation. As large-scale projects such as The Cancer Genome Atlas (TCGA) proceed, many more novel oncogenes and tumor suppressor genes will be discovered; our technology provides an important pipeline for understanding the function of these genes at the interaction network level by mapping their dynamic interaction networks. PUBLIC HEALTH RELEVANCE: Cancer development is driven by mutations in oncogenes and tumor suppressor genes. We aim to develop an innovative approach to facilitate studies of how these gene products function in cancer cells. Such studies will provide the fundamental knowledge required for the development of novel cancer therapeutic approaches.
 
R21 CA173347-01 2012 GRZYBOWSKI, BARTOSZ ANDRZEJ NORTHWESTERN UNIVERSITY Microsystems for targeting Levy walks in metastatic cancer cells
Cell migration is a hallmark of metastasis, a most common cause of death from cancer. Research has established that metastatic cancer cells differ from the non-metastatic ones in terms of their genetics, molecular composition, and increased motility. Despite these advances, there are currently no approved drugs available that target motility of metastatic cancer cells. This is in large part due to our limited understanding f what processes underlie the increased motility of the metastatic cells. We have recently shown that non- metastatic and metastatic cells migrate in fundamentally different ways. While non-metastatic cells execute simple, diffusive random walks, their metastatic variants move not only superdiffusively, but also perform so-called L¨vy walks in which step-times are drawn from probability distributions with heavy power-law tails. L¨vy walk path structure is characterized by clusters of small steps separated by occasional but long ""flights"". The importance of this finding is that it is known from the theory of stochastic processes that L¨vy walks represent an optimal search strategy - one that is often employed by animal predators looking for scarce prey. In this context, metastatic cells can be viewed as ""cellular predators"" navigating human body in a manner that maximizes their chances of finding suitable loci for seeding metastases. As we have showed, these L¨vy walks can be reverted to the purely diffusive walks by synergistic inhibition of Rho and Rac pathways - this finding paves the way to rationally controlling and ultimately limiting the ""predatory"" walks of metastatic cells. Our current application aims to develop conceptually novel technological platform for quantitative analysis of L¨vy walk motility of metastatic cancer cells. Specifically, micro- and nanofabrication and surface functionalization schemes will be combined to develop high-throughput cell migration assay in which linear 1D microtracks will be integrated with 96-well format. Software modules will be developed to automate the microscopy and image acquisition/analysis and will be interfaced with data analyses and processing based on statistical-physical models. A set of 50 genes known or predicted to be involved in cell migration - by regulating filamentous actin (F-actin) polymerization, formation of F-actin contractile bundles or microtubule-F- actin crosstalk - will b targeted in a focused short interfering (si) RNA screen. The ability of siRNAs to affect L¨vy walks will be quantified over large populations of cells using the fully automated 1D microtrack assay. These studies will result in the identification of novel regulators and/or regulator combinations whose inhibition abrogates metastatic cell L¨vy walks. The material systems developed in the context of this application will constitute a versatile technological platform extendable to large scale RNA interference and chemical library screens for the discovery of new drug targets and anti-motility/ anti-metastasis drugs. PUBLIC HEALTH RELEVANCE: We have recently discovered that as cancer cells metastasize, they develop the so-called L¨vy walking navigation strategy typically used by animal predators searching for scarce resources. This skillful mode of cell motility can help the metastatic cells to seed new tumors. The research we propose aims at the development of technology with which to (i) study this ""predatory"" mode of cell motility in analytical precision; and (ii) facilitate the discovery of proteins whose inhibition would revert the metastatic L¨vy walkers into ""benign,"" diffusive cells.
 
R21 CA173164-01 2012 STEINMAN, RICHARD A UNIVERSITY OF PITTSBURGH AT PITTSBURGH Exosomal Recombinase-a tool to dissect metastasis and the cancer microenvironment
We propose a tool to engineer cancer or host cells to irreversibly mark nearby cells by means of novel ""exosomal Cre recombinase"" constructs. By creating cancer cells that transport Cre, the proximal microenvironment in Cre-responsive animals can be analyzed or manipulated to an extent well beyond our current capabilities. These sorts of manipulations would enable the detailed dissection of molecular mechanisms that shape the directionality of cancer spread, sustain dormancy or control cancer-immune cell interactions. Exosomes are nanovesicles that are robustly produced by cancer cells. Specific tags directing Cre to cancer exosomes will generate a visual (fluorescent) map of the trajectory that the cancer cells used during metastasis in fluorescent reporter animals. This will also provide a means to isolate each cell that contacted the cancer so that the transcriptome of those cells can be compared with that of similar cells that were na¨ve to the cancer. This strategy is expected to offer an unprecedented tool for the dissection of the lung cancer microenvironment that is engaged during the metastatic process. Specific Aims of this application are to: 1. Create and evaluate tools that package fluorescent Cre-recombinase into exosomes for delivery to adjacent cells. This aim will establish the optimal construct for Cre transfer between cancer and surrounding normal cells. We will construct fusion genes that combine Cre, a red fluorescent marker, and specific trafficking domains of exosomal proteins. Exosome-specific Cre activity and transfer will be confirmed. Fluorescent conversion of GFP-reporter cells along the path of migrating e-C cancer cells will be visualized through live cell imaging and quantitatively evaluated. Aim 2. Enable and evaluate niche-specific activity of Cre-transfer by hypoxic cancer cells in vivo. This aim will demonstrate microenvironment-specific transfer of Cre from cancer to bystander cells. Luciferase-expressing Lewis lung carcinoma cells (LL2/luc- M38) will be stably transfected with red fluorescent exosomal Cre under control of the VEGF promoter 6, 7. Metastases following intravenous inoculation of syngeneic Cre-reporter mice will be visualized using whole animal imaging. Ex vivo multiphoton confocal microscopy will then measure host cell conversion to green fluorescence at candidate hypoxic regions and along metastatic tracks. Pimonidazole injection prior to sacrifice will be used to validate hypoxia in candidate regions. The ability to capture and analyze Cre-targeted peritumoral cells will be tested. Impact: This tool will allow robust collection of imaging and cellular data that unambiguously delineate cancer/bystander cell interactions that occurred in vivo in desired microenvironments. It is expected to create an unprecedented historical record of each cell that touched a cancer cell. This record could illuminate functional changes that occur in the microenvironment that impact metastases, and how these changes are associated with alteration in the route of spread of cancer. PUBLIC HEALTH RELEVANCE: Cancer cells require the collaboration of nearby normal cells in order to survive. Some details are known about how normal cells collaborate to support tumors when they first appear, however, little is known about whether or how bystander cells regulate the trajectory of cancer during the metastatic process. This is partly because the exact route that a cancer cell takes during metastasis cannot be visualized. One of the aims of this project is to establish a new tool so that every cell that cancer touches during its transit throug the body will glow irreversibly. This will create a visual (fluorescent) map of the trajectory that the cancer cells used during metastasis. Moreover, it provides a means to isolate each cell that contacted the cancer so that the behavior of those cells can be compared with that of similar cells that did not contact lung cancer cells. This strategy can uncover the cellular actors involve in dialogue with metastasizing cancer so that the pathological dialogue can be dissected and targeted.
 
R21 CA173205-01 2012 TAO, NONGJIAN ARIZONA STATE UNIVERSITY-TEMPE CAMPUS Charge sensitive optical detection for high throughput study of small molecules
High-throughput detection of small molecules and post translational protein modifications that involve small molecule biochemical reactions are critical for understanding the mechanisms underlying cancer initiation and progression, for discovering proper biomarkers that enable early cancer detection, and for screening drug candidates for effective cancer treatment. The most widely used technology uses fluorescence labels, which is problematic for small molecules whose sizes are comparable to those of the fluorescent dyes. Various label- free techniques have been developed, but their sensitivity diminishes with the size of the molecules, making it extremely challenging to detect small molecules. To address the need, a charge sensitive optical detection will be developed in this project. The technology is particularly suitable for the detection of small molecules, and biochemical interactions that involve small mass changes. The detection method uses optical probes that compatible with the standard microplate technology, making it attractive for high-throughput detection. The success of the project will lead to a new detection technology to measure small molecule interactions, such as drug-target interactions, membrane protein signaling, protein phosphorylation and other post translational modifications, oxidative and reduction reactions that involves charge transfer. Many of these processes are highly important for the research, diagnostic and treatment of cancer. PUBLIC HEALTH RELEVANCE: A new charge sensitive optical detection technology will be developed for label-free measurement of small molecule interactions. The success of the project will lead to a new high-throughput screening tool for research, diagnostic and treatment of diseases including cancer.
 
R21 CA173390-01 2012 WANG, TZA-HUEI JOHNS HOPKINS UNIVERSITY PCR-free Multiplexed Detection of Circulating miRNA in Blood
Micro RNAs (miRNAs) are short nucleotides (~ 20 nt) that act as regulators of gene expression in nearly all cellular processes including differentiatio, proliferation, and apoptosis. In tumors, miRNAs have been shown to play key roles in cancer processes such as metastasis and tumorigenesis. Since a single miRNA can regulate many mRNAs, dysregulation of one miRNA can have far-reaching biological consequences and that a small panel of miRNAs may suffice for diagnostic purposes. Recent discovery of the existence of circulating miRNAs in the blood stream further raises the potential of miRNAs as noninvasive biomarkers for remote cancer detection. In addition, due to their small size and protection within an exosomal shell, miRNAs robustly resist RNA degradation in tissue and blood. These features make miRNAs exciting targets for cancer diagnosis and prognosis. Since cell-free, circulating miRNAs exist at very low physiological concentrations, current methods to detect these targets predominantly rely on highly sensitive RT-qPCR. However, RT-qPCR is generally limited to single-plex analyses while clinical assessment of miRNAs requires that a panel (e.g.10-100) miRNAs to be quantified in a rapid and inexpensive manner. Employing a large number of separate PCRs for each sample is costly and requires large amounts of miRNA, which is difficult to obtain from blood samples. On the other hand, existing multiplexed technologies such as miRNA microarrays are woefully lacking in the requisite sensitivity to detect these circulating miRNA panels in body fluids. In this project, we propose to develop a single molecule length coding platform to address the unmet clinical need for highly sensitive and multiplexed detection of circulating miRNA. The platform employs a ligation- based molecular length coding scheme to generate miRNA-specific length-encoded strands that are deciphered by size separation to facilitate multiplexed detection. It utilizes cylindrical illumination confocal spectroscopy to quantify low concentration targets through single molecule counting, achieving high sensitivity and quantitative accuracy. In addition, a microfluidic device will be developed to simultaneously concentrate multiple microliter-sized samples into picoliter-sized plugs for arrayed separation in sub-micron channels to enhance both the resolution of separation and the throughput of analysis. Finally, we will validate the proposed platform by determining the analytical sensitivity and specificity using control serum samples spiked with synthetic miRNA sequences. We aim to achieve PCR-equivalent sensitivity of < 10-22 mole, specificity of >1000:1 for unrelated miRNA and > 100:1 for related miRNA. Validation with clinical samples will be performed by analyzing a panel of 20 miRNAs in the serum of patients with advanced esophageal adenocarcinoma (20 samples) and healthy controls (20 samples) using d 200 mL of serum in a single reaction. The result will be compared to that obtained by RT-qPCR using 4 mL of serum split into 20 separate single-plex reactions. PUBLIC HEALTH RELEVANCE: The goal of the proposed project is to develop a single-molecule length-coding platform capable of highly sensitive and multiplexed analysis of panels of miRNAs in the blood stream, addressing the unmet clinical need of using circulating miRNAs as cancer biomarkers.
 
R33 CA122900-01A1 2012 DOVICHI, NORMAN J UNIVERSITY OF WASHINGTON Chemical cytometry for neoplasia prognosis-Barretts esophagus
This application is in response to RFA-CA-06-003, Applications of emerging technologies for Cancer Research. This RFA:"" invites applications for research projects to evaluate the usefulness of emerging molecular technologies that are ready for initial application to clinical or biological questions in cancer research. Projects should be designed to demonstrate that the technology is robust and yields reproducible measurements. Projects should also be designed to gather preliminary data to support the use of the technology in a future project(s) with a clinical or biological focus."" The application combines the efforts of researchers in the Departments of Chemistry and Pathology at the UNIVERSITY of Washington and at the Fred Hutchinson Cancer Research Center. These researchers will perform a pilot study to evaluate new technology for cancer research, chemical cytometry. This technology monitors protein and biogenic amine expression in single cells, and will be evaluated for the study of cells isolated from biopsies obtained from patients in the Seattle Barrett's Esophagus Cohort. We will systematically determine the variance in cellular composition within Barrett's esophagus biopsies We will dissect 20 cells from a single crypt isolated from a biopsy and perform chemical cytometry on those cells. This study will provide understanding of the variance in protein expression along a crypt and during development of a stem cell into its differentiated form. We will perform chemical cytometry on cells isolated from three crypts obtained from a single biopsy. These crypts will be closely related and the study will provide information on the intraclonal variation. We will perform chemical cytometry on cells isolated from crypts obtained from three biopsies from the same patient. This study will provide information on the interclonal variation. We will generate fingerprints from 5 patients, which will provide information on the between-patient variance. As a result of these studies, we will have identified the number of cells or crypts necessary to be assayed to characterize the neoplasia. Based on these results, we will propose a follow-up grant for the systematic validation of chemical cytometry for prognosis of the progression to adenocarcinoma, and for the development of a platform appropriate for large-scale clinical analysis.
 
R33 CA160132-01A1 2012 LAM, KIT S UNIVERSITY OF CALIFORNIA DAVIS Discovery of Death Ligands Against Cancers
Recently, we have modified the one-bead-one-compound (OBOC) combinatorial library method by adding a known cell adhesion (or cell capturing) ligand to the surface of every bead in the OBOC library. When live cells are incubated with such novel one-bead-two-compound (OB2C) libraries, the cell membranes of the captured cells facing the bead surface are exposed to the library compounds displayed on each bead. With an appropriate reporter system, one should be able to rapidly detect beads that can elicit a specific biochemical or cellular response (agonists). Similarly, if the cells are stimulatd by an exogenous agonist, molecules that suppress specific biochemical or cellular response (antagonists) can also be discovered with this approach. In this R33 proposal, we plan to focus our effort on using the novel OB2C technology to discover pro- apoptotic cell surface acting molecules against both hematologic and solid malignancies. Specific aims of this proposed project are as follows: Aim 1: To design and synthesize OB2C combinatorial libraries for the discovery of synthetic and cell surface acting pro-apoptotic molecules or death ligands. Aim 2: To screen OB2C combinatorial libraries for the discovery of synthetic and cell surface acting death ligands against lymphoid cancer, acute myeloid leukemia and solid tumor cells. The lead compounds will be further optimized with focused OB2C combinatorial libraries. Aim 3: To resynthesize the lead compounds and evaluate their pro-apoptotic functions by themselves or after conjugation to the cancer cell surface targeting ligands. The mechanisms of action of these pro-apoptotic agents will be determined. Impact: The ultra-high throughput OB2C library method is highly efficient and economical. Once optimized, it can be readily applied by many academic investigators to their research. The death ligands to be developed in this proposed research can be developed into novel effective but less toxic cancer therapeutics. These ligands may also be used as biologically active probes for basic cancer biology and systems biology research, particularly after their mechanisms of action have been elucidated. PUBLIC HEALTH RELEVANCE: In this R33 proposal, we plan to focus our effort on using the novel OB2C technology to discover pro-apoptotic cell surface acting molecules against both hematologic and solid malignancies. These death ligands can be developed into novel effective but less toxic cancer therapeutics. They may also be used as biologically active probes for basic cancer biology and systems biology research, particularly after their mechanisms of action have been elucidated.
 
R33 CA173359-01 2012 LIOTTA, LANCE ALLEN GEORGE MASON UNIVERSITY Nanotrap technology for one step preservation and amplification of cancer biomark
We propose advanced development and validation of novel technology that will maximize the quality and utility of biologic fluid specimens, and permit the measurement of previously invisible low abundance biomarkers. The technology is transformative and paradigm-shifting because it immediately and economically solves fundamental roadblocks paralyzing the body fluid cancer biomarker field. In a single step, in minutes, our technology overcomes the severe problems of biomarker preservation, low abundance, and masking by unwanted proteins. The technology is novel porous, buoyant, core-shell hydrogel nanoparticles containing high affinity reactive chemical baits that harvests biomarkers in body fluids. Our nanoparticles can be pre loaded into Vacutainer(R) blood collection tubes, or other body fluid collection vessels. Upon contact with the sample, the suspended nanoparticles immediately affinity-sequester target biomarkers inside the particles, exclude albumin, fully protect the biomarkers from degradation (even at elevated temperatures), and massively concentrate the sequestered biomarkers into a small volume. The technology can dramatically (demonstrated up to 10,000 fold) improve the lower limits of detection and the precision of: a) mass spectrometry (MS) biomarker discovery, b) quantitation by multiple reaction monitoring (MRM), or c) quantification by any clinical grade immunoassay. All of the Aims and Milestones of the predicate IMAT NCI R21 CA137706 grant have been exceeded. We have discovered more than a dozen novel chemical baits with preferential high affinities (KD < 10-11 M) for specific low abundance protein analytes. We discovered a novel shell chemistry that selectively prevented unwanted entry of all size albumin-derived peptides without hindering the penetration of non-albumin small proteins and peptides. Labile body fluid biomarkers, that would otherwise rapidly degrade, are completely preserved at the time of collection, thus obviating the need for costly freezing or proteinase inhibitors. Low abundant proteins previously invisible to discovery by MS, and previously not measureable by MRM, or clinical immunoassays, can now be quantified with high precision within the linear range of the assay. Our technology is completely innovative, as documented by 3 allowed (2 issued) patents that have been commercially licensed, and 14 publications. No existing technology can solve all of the aforementioned roadblocks, and attain a capture/elution efficiency near 100%. The technology has recently permitted the MS identification of new serum and plasma proteins not listed in the international HUPO database. Under the proposed Aims we will scale up the technology to conduct blind validation of its performance precision, accuracy, improved detection sensitivity, and preservation capacity, in two large (n = 400 sera and n = 74 plasma) well-controlled clinical sera/plasma sample sets (64 analytes). We will extend the technology to urine and sweat to open up these biofluids as a new category for biomarker research, using innovative approaches to solve fundamental problems of volume, perishability, and low protein concentration for these biofluids. PUBLIC HEALTH RELEVANCE: We propose advanced development and validation of novel nanotechnology that will maximize the quality and utility of biologic fluid specimens, and permit the measurement of previously invisible low abundance biomarkers. The nanotechnology is transformative and paradigm-shifting because it immediately and economically solves fundamental roadblocks paralyzing the body fluid cancer biomarker field, permitting the measurement of biomarkers emanating from small (< 2 mm) early stage cancers with high precision and accuracy.
 
R33 CA160011-01A1 2012 NOLLING, JORK N PRIMERADX, INC. A Highly Multiplexed PCR Platform for Gene Expression Profiling from FFPE Tissue
The overall goal of this application is to deliver a highly multiplexed, quantitative, and automated system for the rapid identification of diffuse large B lymphoma (DLBCL) subgroups using formalin-fixed paraffin-embedded (FFPE) material as the sample source; a technology that is not yet available to the research and clinical communities. DLBCL accounts for ~30% of all non-Hodgkin lymphomas and thus is the most common subtype of this cancer in the United States. While many patients respond well to treatment, there is a sizable subset that remains refractory or suffers relapse. Gene expression profiling has revealed that this difference in response is reflected in the biology of the tumor an two subgroups have been defined based on the origin of the tumor cell. Molecular signatures within a panel of 17 genes can differentiate these subgroups. Technologies to detect gene expression patterns currently center around two existing formats: microarrays and real-time PCR. The former, although able to detect hundreds to thousands of genes, suffer from their lack of sensitivity and quantitative capacity, while PCR, although quantitative and exceedingly sensitive, has extremely limited multiplexing capability. Therefore, technologies that combine attributes of multiplexing with sensitive, quantitative analysis are critically needed to advance the understanding of tumor biology toward translation into clinical diagnostic utility. Archives of FFPE tumors represent a valuable resource for translational cancer genomic research. However, utilizing FFPE tissue is challenging since it is often derived from small biopsies containing RNA that is fragmented during fixation and storage, thus rendering it less suitable for microarray analysis. As a result there is a lack of quantitative, multiplexed, sensitive, and robus assays that are able to utilize FFPE as a source of tissue for genetic analysis. In order to overcome these above obstacles we propose to develop a novel, rapid, and automated assay to detect, quantify, and classify the two main subtypes of DLBCL, thus facilitating timely diagnosis of the tumor type in order to inform clinical treatment options. Such a technology, that can detect and quantify multiple gene targets in a single-tube reaction using FFPE specimens as the source material, does not currently exist. The objective of this proposal will be met by completing three Specific Aims. These consist of (i) developing the technology, to be termed the 'ICEPlex Multiplex FFPE Assay', by adapting PrimeraDx's emerging STAR technology and ICEPlex instrument system for this purpose; (ii) validating the analytical performance characteristics of the resultant assay; and (iii) verifying performance of the assay by conducting a concordance study comparing results from the newly developed assay with a reference microarray technology currently used for DLBCL subtyping. PUBLIC HEALTH RELEVANCE: The past decade has seen a major shift in cancer diagnostics with the ascendance of gene expression profile analysis of tumors, which is leading to improvements in cancer detection, diagnosis, and treatment. However, to achieve the full potential of this approach, technology impediments involving the number of genes that can be detected in a single assay and the use of stored tissue samples that may be degraded need to be overcome. PrimeraDx will address these problems by providing an automated solution for a single-reaction, multi-sample analysis from stored tumor tissue material in order to classify types of a common human lymphoma, thereby facilitating rapid diagnosis, determination of treatment options, and analysis of response to therapy.
 
R33 CA173382-01 2012 ZU, YOULI METHODIST HOSPITAL RESEARCH INSTITUTE RFA-CA -12-003 - Activatable one-drop and one-step assay for circulating tumor ce
An ideal clinical assay for detecting circulating tumor cells should be high throughput (as simple as a one-step reaction) and highly sensitive (requiring as little as one drop of blood). The current assay to detect circulating tumor cells is time- and labor-consuming multi-step procedure, requiring isolation of tumor cells, staining with antibodies, and repeated washes to reduce background. To overcome these obstacles, we have developed a unique aptamer probe that carries a ""tumor cell-activatable"" reporting system. The aptamer probes specifically bind tumor biomarker(s) and are optically silent in the absence of target cells, thus giving no to minimal background. To translate these research findings to the clinic, the goal of this study is to develop a novel technology for circulating tumor cell detectio in a ""One-Drop (of blood)-One-Step Assay"" (ODOSA). The specific aims of this proposal are to: 1. Optimize sensitivity and specificity of the aptamer probes for the ODOSA technology; 2. Develop a multi-sample and high-throughput platform for the ODOSA technology; and 3. Validate the ODOSA technology in clinical specimens. Scientifically, this study presents an innovative concept that tumor cells can be specifically detected by activating the aptamer probes through a natural cellular process with exclusively intracellular signals and no background. Technically, the ODOSA is a technological breakthrough over the current antibody-mediated assays. Clinically, the ODOSA provides a high- throughput and multi-sample platform, and enables physicians to detect one single circulating tumor cell among millions of cells in real time using a minimal amount of blood. There is no alternative clinical assay available to date. PUBLIC HEALTH RELEVANCE: The proposed One-Drop (of blood)-One-Step Assay (ODOSA) presents transformative technological advance that would allow physicians to detect various circulating cancers in one drop of a patient's blood. The current antibody-mediated process is labor and time intensive, with multiple steps requiring large initial sample volumes. The proposed ODOSA is a technological breakthrough. Clinical implementation of the ODOSA would enable early detection and accurate monitoring of therapeutic response of cancer tumors.