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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. Consistent with NIH policy, abstracts are not available for projects receiving their first award within the past year, so descriptions provided below are from the NCI program director.

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Project # Year
of Award
PI Name(s)
Institution Title
Abstract Text (Official)
R21 CA174541-01 2013 BAI, MINGFENG UNIVERSITY OF PITTSBURGH AT PITTSBURGH A Novel Theranostic Platform for Targeted Cancer Therapy and Treatment Monitoring
Cancer treatment currently relies heavily upon administration of cytotoxic drugs that attack both cancerous and healthy cells due to limited selectivity of drugs. Therapeutic efficacy and systemic toxicity can be improved by employing a multifunctional drug delivery system that allows targeted drug delivery, controlled drug release and therapeutic effect monitoring. The integration of therapeutic and diagnostic treatments has created a new genre in patient care and personalized medicine termed theranostics. Dendrimers provide an ideal theranostic platform due to their precisely controlled size, shape, and surface chemistry. These unique properties allow dendrimers to be developed with high structural monodispersity, desired plasma circulation time and biodistribution properties, as well as control over drug release. In our pioneering approach, we aim to develop the first quaterrylene-based (QR) near-infrared (NIR) fluorescent theranostic dendrimer platform and seek to shift NIR theranostic dendrimers away from those with poor chemical stability, quantum yield and photostability to a highly chemically stable, fluorescent and photostable NIR theranostic platform. As a proof-of-principle study to demonstrate that the QR theranostic dendrimers can be applied in targeted cancer imaging, we will attach a conjugable translocator protein (TSPO) ligand to the selected dendrimers and image the targeted agents in TSPO over-expressing breast cancer cells and in an animal model. We hypothesize that a quaterrylene-based dendrimer will provide a highly photostable, fluorescent and chemically stable theranostic platform for targeted cancer therapy and efficacy monitoring. Such innovative design avoids the photobleaching and self-quenching issues of current technology, thus allowing NIR theranostic studies with longer imaging time, higher fluorescence signal and more accurate quantification. It will be possible to conjugate various targeting molecules, signaling moieties and drugs to this innovative platform and therefore, this platform has the potential to be widely applied in cancer treatment and may transform the way that cancer patients are treated and monitored.
R21 CA174583-01 2013 CASTRO, CARLOS E. OHIO STATE UNIVERSITY Nanoscale tools for functional studies of cancer-relevant chromatin modifications
Dynamic organization of the human genome into chromatin regulates transcription initiation and elongation. Defects in chromatin modifications, assembly, disassembly and remodeling result in misregulation of oncogenes, which are associated with numerous cancers including ovarian, bladder, prostate, and colorectal tumors. Prior research has identified the components involved in chromatin transcriptional regulation (CTR), including histone variants and post-translational modifications (PTMs), histone modification enzymes, and histone chaperone assembly factors. Remarkably, genetic, biochemical, structural, deep sequencing and single molecule studies have not fully revealed the mechanisms of CTR. Therefore, new technologies are required to probe currently inaccessible dynamics and structure of chromatin assemblies at the 10-100 nm length scale, which encompasses critical molecular events the regulate DNA processing. This research will address current technological gaps through the development of nanoscale tools that measure mesoscale (10- 100nm) structure and dynamics of chromatin at specific cancer-relevant modification and processing sites. Specifically, we will develop 1) DNA origami nanostructures with multiple antibodies that recognize distinct physiological and cancer-relevant combinations of chromatin marks (histone modifications/variants and genomic DNA processing sites) and 2) DNA origami displacement sensors to study site-specific mesoscale dynamics at gene regulation sites. The long-term goal of this work is to develop tools and methods to probe chromatin function and dynamics at cancer-relevant chromatin modifications and oncogene regulation sites in vivo. Within the scope of this exploratory research, we will focus on the devic development, in vitro proof-of- principle, and characterizations of chromatin assemblies. Future work will build on the tools and experimental framework established here to implement DNA origami devices to probe intracellular function of chromatin assemblies.
R21 CA174594-01A1 2013 CELEDON, ALFREDO ANDRES TWISTNOSTICS, LLC Single molecule microarrays for the detection of mutant DNA in body fluids
The formation of malignant tumors is associated with accumulation of genetic mutations that enhance cell proliferation. Detecting these mutations in body fluids can be used for cancer management and early cancer detection. However, current cancer diagnostic methods are not well suited for the detection of mutant DNA in body fluids which have prevented the development of effective, cost-efficient cancer screening strategies. Here, we propose the development of microarrays based on Twist-Biosensor technology to dramatically improve and simplify detection of biomarkers in body fluids for cancer screening. Twist-Biosensor is a novel microarray technique in which hybridization is detected with single molecule resolution. In addition, Twist-Biosensor applies disrupting torsional stress to DNA hybrids, a novel strategy uniquely designed to give microarrays high point mutation selectivity. Therefore, the technique is ideal for detection of mutated DNA at low concentration in the presence of a large proportion of wild-type DNA. The overall aim of this application is to demonstrate the capabilities of Twist-Biosensor microarrays for cancer screening. We will develop assays to detect mutations of the KRAS gene and test them with plasma and urine samples from pancreatic cancer patients. Each of the proposed aims of this application focuses on establishing two complementary properties of the microarrays: Detection of multiple cancer mutations (Aim 1) and ultra-sensitive detection of mutated DNA (Aim 2). In addition, both aims will demonstrate that Twist-Biosensor microarrays are faster and require fewer steps than alternative diagnostic techniques.
R21 CA182608-01 2013 CHENG, JI-XIN PURDUE UNIVERSITY WEST LAFAYETTE Quantitative Spectroscopic Imaging of Cancer Metabolites in Live Cells and Intact
While altered cell metabolism is an emerging hallmark of cancer, there is a crucial need of new tools for quantitation of metabolites. Though NMR spectroscopy, mass spectrometry, and Raman spectroscopy are widely used for molecular detection in tissue extracts or intact tissues, these tools do not tell the spatial locations of the analytes inside the cell. We address this unmet need via development of multiplex stimulated Raman scattering (SRS) microscopy to enable quantitative vibrational imaging of metabolites in live tumor cells and intact biopsies. The recently developed SRS microscopy allows high-speed, high-sensitivity imaging of single Raman bands in live cells. However, the single-frequency SRS imaging technique has limited capability because it cannot resolve molecular species that often have overlapped Raman bands. We propose to overcome this technical barrier via parallel detection of spectrally dispersed SRS signals enabled by a homebuilt tuned amplifier array. In a pilot study, we demonstrated multiplex SRS imaging of live pancreatic cancer cells with a pixel dwell time of 40 ¿s. In Aim 1, we will develop multiplex stimulated Raman loss (SRL) microscopy and multivariate analysis algorithm to enable quantitative vibrational imaging of lipid metabolites in live cells. In Aim 2, we will develop epi-detected multiplex SRL microscopy to enable high-speed, large-area spectroscopic imaging of tumor biopsies. By accomplishment of the two aims, we will generate a high- sensitivity, high-speed, spectral imaging platform for molecular analysis of live cells with sub-micron spatial resolution. This platform will permit label-free visualization of metabolic conversion in live cancr cells, which is not possible with proteomics tools. Such capability is critical for mechanistic understanding of cancer metabolism and precise evaluation of drugs targeting cancer metabolism. This platform will also permit large- area mapping of intact tumor biopsies and offer information about metabolic biomarkers (e.g. cholesteryl ester) that are indicative of cancer aggressiveness.
R21 CA177393-01 2013 DOYLE, PATRICK S; FAN, RONG(contact) YALE UNIVERSITY High-throughput, Multiplexed Detection of miRNA Biomarkers in Single Cancer Cells
MicroRNAs (miRNAs) are a class of small non-coding RNAs recently discovered as negative gene regulators. A large faction of miRNAs are located within the fragile regions of chromosomes, which are areas of the genome more tightly associated with human cancers, indicating the possible role of miRNAs in tumor development. It has been shown that miRNA expression profiles correlate with various cancers and the miRNA signatures are potentially unique cancer biomarkers to accurately diagnose and classify human cancers. However, human cancers are highly heterogeneous due in part to the high degree of intratumoral heterogeneity at the single-cell level, requiring the evaluation of a large number of single tumor cells, each of which the miRNA expression profile can be measured. Here we propose to combine a single-cell barcode chip platform and a novel ligation-based miRNA detection scheme to develop an innovative technology that for the first time offers both high-throughput and multiplexing for rapid miRNA detection in single cells without amplification and at low cost. More specifically, we will: (1) Design and validate a DNA barcode microarray for multiplexed detection of miRNA biomarkers without amplification, and (2) Integrate this barcode array with a nanoliter microfluidics chip to perform multiplexed detection of 12 miRNA biomarkers from a large number of single cancer cells. Our technology is a versatile platform to detect miRNA biomarkers implicated in a variety of human cancers. It can be further modified to detect other non-encoding RNAs and messenger RNA biomarkers from single cells. Moreover, the proposed microchip is inexpensive, scalable and easy to manufacture. It may find immediate applications in basic research and clinical stratification of human cancers.
R21 CA177519-01 2013 HARISMENDY, OLIVIER(contact); HOWELL, STEPHEN B UNIVERSITY OF CALIFORNIA SAN DIEGO In vivo detection and genome-wide location analysis of DNA-adducts
DNA adducts are the hallmark and most common form of DNA damage in the cell. They result from environmental carcinogen exposure (such as UV) or during chemotherapy using DNA modifying agents like cisplatin (cDDP) or alkylators such as chlorambucil (CLB). While mechanisms underlying sensitivity, agent homeostasis, detoxification, DNA repair and apoptosis, have been well investigated, the central molecular event, the formation of adducts, is not well understood in vivo. Evidence suggests that the epigenetic landscape and the structure of the chromatin influences the formation of adducts and mediates drug sensitivity. Therefore, there is a need to better identify DNA adducts and understand the association between the epigenetic marks in the cell. Currently there is no method to determine the exact location of DNA adducts in vivo nor at a high-resolution across the genome. In order to address this, we propose to develop a method, TdT-Seq, that will identify these adducts genome-wide at the single base pair resolution. The expertise of the investigators include knowledge in cancer biology and platinum drug pharmacology (Drs. Howell and Abada) as well as experience in high-throughput genomic assays and computational analysis (Dr. Harismendy); expertise that will be needed to successfully develop the assay. The TdT-Seq assay relies on adduct-mediated inhibition of the DNA polymerase in vitro. The resulting single strand DNA will be captured by a specific TdT mediated ligation, enriched, then sequenced in high throughput. We propose to establish the technical validity of the assay by determining 1) sensitivity at various cDDP concentrations and read depth, 2) specificity by the development of a locus specific method (Strand Specific Adduct Detection) and independent analysis of 50 adduct loci, and 3) quantativity using increasing cDDP concentrations and known spike-in controls. We will also perform specific experiments to establish TdT-Seq's use for clinical cancer research. In particular, we will optimize the protocol for the identification of UVor chlorambucil (CLB) induced adducts to broaden its applicability. We will also develop the protocol for low amounts of DNA originating from mouse tissues or heterogeneous tissue specimens. Finally, we will analyze the ability of TdT-Seq to measure the kinetics of DNA repair using genetically modified cell lines. TdT-Seq's development will therefore lead to a robust and innovative assay, with demonstrated performance and utility for cancer research. TdT-Seq will generate an entirely new type of data, which can be used in combination of other whole genome datasets from the ENCODE or TCGA consortium to provide a more precise and comprehensive description of the mechanism of DNA damage and repair in vivo in various cell types and cancers. The long-term benefits of such research include the prediction of drug sensitivity or the study of epigenetic modifying compounds to rationalize combinations for optimal drug efficacy.
R21 CA177447-01 2013 JEFFREY, STEFANIE S STANFORD UNIVERSITY A Droplet-Based System for Capture, Manipulation, and Biochemical Profiling of Ra
We are motivated by the view that development of a reliable and robust technology for efficient detection, characterization, and direct assay of minimal residual disease (MRD), including circulating tumor cells (CTCs) from blood and disseminated tumor cells (DTCs) from bone marrow, will make a major contribution toward elucidating the biology of the metastatic process and developing new methods for the management and treatment of cancer. These cells are important actors in how cancer may spread and kill people. Cancer is a dismal disease - within five years, almost 30% of cancer patients die, not from the primary tumor, but from the metastases or spread of the cancer to other organs in the body. It is thought that some cells can slough off the primary or secondary tumor and circulate in the bloodstream or find safe harbor in the relatively hypoxic bone marrow, and that some of these rare tumor cells (CTCs and DTCs) can lead to metastatic growth, especially if they acquire stem-cell like characteristics. The end goal is to demonstrate a transformative ability to capture and especially to manipulate rare cells on the same platform used to capture them, and to individually process captured cells in droplets for molecular characterization on the same chip. The novel idea embodied in this research is to use many small droplets containing immunomagnetic beads that probe for rare cells as the capture beads containing antibodies are sequentially incubated and washed with fluid droplets, in both a parallel and pipelined fashion. Gently probing small volumes with suspensions of magnetic beads should be much more efficient at capturing rare cells than probing a large volume, at the expense of requiring many repeated (but rapid) steps to eventually sample the full volume. Moreover, multiple antibodies may be used to probe one sample. Our aims are 1) to demonstrate that sub-populations of rare cells can be captured in a pipeline of separate, individual droplets; and 2) to show that droplets containing rare cell types can be manipulated and transported to a site for biochemical profiling, by implementing assays designed towards exploiting the full metabolic potential of these cells.
R21 CA174581-01 2013 LAI, JAMES UNIVERSITY OF WASHINGTON Biospecimen preparation technologies to enable high throughput and highly sensitive targeted proteomics
Many diseases result in specific and characteristic changes in the molecular profiles of biological fluids (e.g., urine) and tissues. For cancer, thes changes can be utilized for screening, or as indicators of disease progression or of treatment-associated changes, which can significantly improve the medical outcome. Mass spectrometry (MS), a highly sensitive analytical technology with multiplex analysis capability, has been utilized for detecting changes in protein expression. The sensitivity of MS is inherently dependent on the available analyte concentration and the level of background interference, so specimens such as urine require extensive preparation (i.e., HPLC) prior to the analytical phase. These preparatory processes can result in significant loss of the analyte and are cumbersome, time consuming, not amenable to automation, and more susceptible to contaminations, which significantly impact the overall assay performance such as limit of detection (LOD). To address the need, this project aims to develop a novel biospecimen preparation technology that can process entire available urine samples rapidly (d 30 minutes) by concentrating multiple target analytes (e 102- fold) to enable high throughput multiplex targeted cancer proteomics with significantly lower LOD. The proposed sample preparation utilizes the stimuli-responsive binary reagent system for effective and rapid chromatography without solid phase. The key innovation lies in the combination of stimuli-responsive reagents, including magnetic nanoparticles (mNPs) and polymer-antibody conjugates to maximize the analyte binding efficiency while maintaining effective magnetic separation. This binary reagent system can achieve significantly higher analyte binding then the existing magnetic microparticle by simply applying a larger amount of the antibody conjugates without increasing the mass of magnetic nanoparticles because the antibodies are not attached to the particle surfaces. When a stimulus (e.g., heating) is applied to the solution, both stimuli- responsive reagents, including mNPs and polymer-antibody conjugates, form large aggregates that can be separated using a modest magnetic field to concentrate the bound analytes. We will develop a temperature- responsive separation module for isolating multiple analytes simultaneously in urine. The separation module will be optimized for processing the entire available urine (ca. 200 ml) and interfacing with MS for target protein quantification. Additionally, we will utilize clinical urine samples to evaluate the multiplex capability of the proposed specimen preparation process in conjunction with the MS assay improvement. The success of the project will result in a biospecimen preparation technology that can rapidly (d 30 minutes) process the entire available urine sample to produce multiple concentrated target analytes (e 102-fold) to enable high throughput multiplex target proteomics via MS with significantly lower assay LOD. The technology is applicable to different specimens (e.g., serum) for different analyte panels, which will significantly accelerate cancer proteomics and facilitate high quality cancer diagnosis in the future.
R21 CA182330-01 2013 LEVY, MATTHEW ALBERT EINSTEIN COLLEGE OF MEDICINE Lectimers: Glycan-anchored scaffold libraries for targeting carbohydrate-binding
We propose to develop a novel platform technology that will produce high-affinity and high-specificity ligands to target carbohydrate binding protein (CBPs), a diverse class of proteins which possess a wide range of biological functions, playing roles cancer tumorigeneisis, immune modulation and viral infection. Our method builds upon advances in sequencing technologies and leverages the natural, low affinity, interactions of monovalent sugars for CBPs combined with the ease of synthesis of nucleic acids. Our glycan-anchored libraries will thus possess many of the attributes of nucleic acids - in particular structural rigidity, ease of synthesis and the ease with which they can be amplified and characterized - but with the enhanced chemical functionality observed in peptides, proteins and small molecule ligands. Because these reagents are based on nucleic acids, the resulting affinity agents can be readily synthesized at relatively low cost and can be easily modified with a variety of fluorophores or chemical moieties for diagnostic, therapeutic and research purposes.
R21 CA177535-01 2013 LIOTTA, LANCE ALLEN GEORGE MASON UNIVERSITY Protein Painting reveals hidden protein-protein interaction domains
We propose the creation of a wholly novel technology ""protein painting"", for the rapid, direct isolation and sequencing of hidden native protein-protein interaction domains. Protein-protein interactions are the basis of virtually all functional moleculr events driving cancer cell signaling and gene regulation. Currently no technology exists to directly isolate and sequence unknown interaction domains, or to directly detect whether known protein-protein binding domains are in contact in a cellular or tissue sample of native proteins. Tracking the interface domains between interacting proteins, or within misfolded proteins, is the basis for the next generation of therapies that block the molecular interactions driving cancer. We will create protein paint chemistries, a novel panel of synthetic organic small molecules that bind to protein molecules with high affinity and mask all the protease cleavage sites of the protein. We will use our new protein painting chemistry to isolate and sequence protein-protein interaction domains of cancer related proteins. Interacting native proteins in solution are painted with a palette of paint molecules that coat the exposed surfaces of the proteins but do not have access to the internal protein-protein contact domains. Thus if two native proteins are bound together, the interface domains will remain non-painted. The protein paints coat with a high resolution (< 3 amino acids). Following painting, the interacting proteins are dissociated, to reveal and expose the non-painted interaction domains that were inaccessible to the paint molecules when the proteins were interacting in their native state. Painted regions are masked from proteinase cleavage or antibody recognition, even after dissociation. The dissociated painted proteins can then be subjected to proteinase cleavage (e.g. trypsin) and ms sequencing, or antibody probing. Since the paint blocks the proteinase cleavage sites that are not in contact, proteinase fragments for ms sequencing will only be generated from the non-painted areas exclusively comprising the interaction domains. The Aims follow our discovery of the masking of trypsin cleavage sites (carboxyl side of arginine & lysine) by sulfonated anthracene organic dye molecule ""paints"" such as disodium 1-amino-9,10- dioxo-4- [3- (2- sulfonatooxyethylsulfonyl) anilino] anthracene -2 -sulfonate, which achieve a high degree of trypsin cleavage site coverage. We verified protein painting capabilities by direct ms sequencing of the hidden interaction domain of interleukin-1¿ bound to its receptor. In the past this domain could only be predicted by X- Ray crystallography. Our transformative technology addresses a broad and critical unmet need in cancer biology and cancer therapeutics. We envision that the technology can be used in the future to decipher interaction domains from tumor biopsy samples or cell cultures treated with a ligand or a therapy. Antibody binding assays can be used to quantify the number/type of occupied binding sites in a cell lysate. We will create a panel of 12 protein paints, and apply the paints to 3 model cancer related protein-protein interactions:1) receptor-ligand, 2) phosphorylation mediated association, and 3) transcription factor complexes.
R21 CA174577-01 2013 LU, CHANG VIRGINIA POLYTECHNIC INSTITUTE AND STATE UNIVERSITY Sensitive and integrated microfluidic ChIP assays for studying transcriptional regulation
Dysregulated transcription is frequently involved in cancer development. Chromatin immunoprecipitation (ChIP) assay is the technique of choice for examining in vivo transcription factor-DNA interactions. The current ChIP technology is limited by the requirement of a large number of cells and the long assay time caused by extensive manual processing. These problems practically prevent its use on primary cells extracted from animals and patients. In this project, we will develop microfluidic ChIP assays for studies based on tiny amounts of primary samples from mice and humans. We will achieve a sensitivity of ~20-50 cells for ChIP-qPCR and ~1000 cells for ChIP-seq for transcription factor binding studies using primary cells by the end of this project. Taking advantage of the ultrahigh sensitivity, we will demonstrate two innovative applications of ChIP assays: 1. Study transcriptional regulation in a cellular subset from primary tumors, namely tumor-initiating cells (TICs); 2. Monitor temporal dynamics in transcriptional regulation by minimally-invasive examination of a single live mouse. These experiments cannot be conducted using current technology and will yield unique insights that improve the understanding of cancer development at the molecular level.
R21 CA182333-01 2013 LUKER, GARY D; TAVANA, HOSSEIN(contact) UNIVERSITY OF AKRON A Novel High Throughput Tumor Spheroid Microtechnology
Two-dimensional (2D) cultures of cancer cells are routinely used in drug discovery for screening and initial characterization of the efficacy of librry of potential drug compounds. Despite their simplicity and compatibility with high throughput screening instruments, 2D cell assays often fail to predict the efficacy of compounds in vivo, making drug development and discovery an extremely costly process. Disparity between 2D cultures and the complex 3D environment of cancer cells in vivo is the major shortcoming of monolayer culture systems. Development of novel chemotherapeutics requires compound screening against malignant tumor cells in a setting that resembles the 3D tumor environment. Cancer cell spheroids (CCS) are 3D clusters of malignant cells that are regarded as physiologic models of solid tumors; they possess similar metabolic and proliferative gradients to avascular tumors and exhibit the clinical expression profiles of solid tumors. Despite the inherent power of CCS to predict clinical efficacy of drugs, incorporation of CCS into the mainstream drug development process is severely hindered by complex and expensive methodological requirements for the formation and maintenance of CCS. We overcome the limitations of existing techniques by developing a technological platform to generate spheroids of consistent size in standard 384-microwell plates using an aqueous two- phase system (ATPS). A drop of the denser aqueous phase containing cancer cells is robotically dispensed into each well containing the second, immersion phase. The drop confines cells and remains immiscible from the immersion phase to facilitate aggregation of cells into a compact CCS of well-defined size. Importantly the overlay of culture media provides nutrients and minimizes the well-known problem of evaporation and changes in osmolality of media as in other assays. The entire process of generating 384 spheroids is done robotically and in a single step. The unprecedented ease of formation and maintenance of CCS and full compatibility with standard equipment in high throughput screening laboratories makes this microtechnology readily available to the researchers in academia and industry. We anticipate that this microtechnology will make drug testing and screening with 3D tumor models a routine laboratory technique prior to expensive and tedious in vivo analyses. In addition, it will dramatically improve testing throughput and cost-effectiveness (increasing numbers of tested compounds and reduced reagent consumption) and efficiency (reducing hands-on time) to expedite drug discovery. We will accomplish our goals through two specific aims: (i) Generation of cancer cell spheroids with aqueous biphasic systems; (ii) Initial validation of ATPS spheroids for compound testing.
R21 CA177402-01 2013 MALY, DUSTIN J; ONG, SHAO-EN(contact) UNIVERSITY OF WASHINGTON Kinase Profiling with Quantititative Chemoproteomics
The long-term goal of this project is to develop a rapid and quantitative method for globally profiling prognostic signaling cascades in tumor cells. Protein kinases are known to participate in larger macromolecular complexes that transmit extra-cellular signals into phenotypic responses. Because these complexes act as integrators and effectors of cellular stimuli, the abundance and activity of kinases are often used as reporters for the cellular state. By selectively enriching kinase-containing signaling complexes, we will be able to dissect the molecular wiring of cancer cells. The objective of the research proposed in this application is to develop and apply a suite of affinity reagents that can be used to selectively enrich activated signaling complexes. In our first aim, we will generate bivalent affinity reagents targeting signaling complexes containing a kinase and phosphotyrosine residues. Our bivalent affinity reagent will comprise a Grb2-SH2 domain and a kinase inhibitor, which should specifically bind to phosphotyrosine residues and kinase ATP-binding sites, respectively. We will evaluate the abilities of our bivalent inhibitors to selectively enrich signaing complexes over its monovalent components using SILAC-based quantitative proteomics. These studies will guide our understanding of the modularity of our bivalent inhibitor approach and provide design principles for development of future probes. For our second aim, our goal is to design SH2-kinase inhibitors targeting distinct cell signaling pathways and evaluate their ability to enrich pathway-specific protein complexes. Our goal is to develop bivalent affinity reagents that have distinct and targeted affinities to assembled protein complexes instead of single proteins. Our application is to use kinase inhibitor-based affinity reagents to enrich kinases from biological samples overcomes the dynamic range limitations of conventional biochemical analyses. Our inhibitor- resins will be designed to enrich specific kinases in families or signalin pathways and used in combination with quantitative MS to assay both kinase abundance and activity.
R21 CA182335-01 2013 OZERS, MARY SZATKOWSKI; WARREN, CHRISTOPHER L (contact) ILLUMAVISTA BIOSCIENCES, LLC High Density Peptide Arrays for Cancer-Related Post-Translational Modifications
Post-translational modifications of proteins play a pivotal role in cancer etiology and progression by altering protein-protein interactions, enzymatic activity, and protein conformation. Peptide arrays have played a significant role in cancer-related discoveries, such as cancer biomarkers, point-of-care diagnostics, and therapeutics directed at protein-protein interactions. This application will develop an integrated technology, the SNAP-Tide array (Specificity and Affinity for PepTides), to synthesize one million unique peptides on a single glass slide using transcriptional and translational machinery. The integrity of the innovative SNAP-Tide arrays will be validated in a three step process, involving mass spectrometry, in vitro fluorescent labeling of amino acids, and antibody recognition to ensure the peptides are accurately synthesized in this novel process. The peptides on the SNAP-Tide array will then be modified by purified enzymes that confer the common cancer- related post-translational modifications of phosphorylation, sumoylation, and arginine methylation. Lysates prepared from cancer cells will be applied to the SNAP-Tide array to evaluate differences in their ability to confer post-translational modifications on the array peptides. The specific aims o this grant are to: 1. Develop and validate an in vitro method to synthesize high density peptide microarrays that display all peptides in the human proteome. 2. Modify the peptides on the array by conferring phosphorylation, sumoylation, and methylation marks using purified enzymes. 3. Quantitate differences in tyrosine phosphorylation activity between two cancer cell lines. Currently available lithographic or spotted peptide arrays display 100-10,000 peptides, while the SNAP-Tide strategy will synthesize a million unique peptides on a single glass slide offering substantially greater throughput. The SNAP-Tide array will be developed for the cost of a standard DNA microarray (< $1000 per array), which is significantly less than current peptide arrays displaying only a fraction of the human proteome. The SNAP-Tide array is synthesized by a series of carefully timed and precise steps of in vitro DNA replication, RNA transcription, and peptide translation. Once synthesized, the peptides are re-attached in a novel process to specific addresses on the glass slide, and any experiments can occur on the glass slide within the low- volume chamber. This design greatly simplifies peptide array experiments and avoids the need for expensive equipment or complicated procedures, such as mass spectrometry or phage display. Direct applications of the SNAP-Tide array include small molecule screening of pivotal protein drug targets, identification of novel cancer biomarkers, and development of point-of-care diagnostic devices to evaluate cancer patient biospecimens.
R21 CA173243-01A1 2013 PAN, TINGRUI UNIVERSITY OF CALIFORNIA DAVIS Digital one-disc-one-compound array for high-throughput discovery of cancer-targe
The objective of the proposed research is to develop a microfluidics-guided digitally encoded combinatorial method, referred to as digital one-disc-one-compound (ODOC) array, integrating state-of-the-art combinatorial chemistry with emerging microfluidics, microfabrication, encoding and matrix theories, for discovery of cancer-targeting molecules with high throughput, high efficiency and high accuracy at low cost. As an indispensable tool for biology and medicine, combinatorial chemistry has enabled high-efficiency modular synthesis of biomolecules and high-throughput exploration of the lead compounds for specific biological targets. However, existing combinatorial strategies only allow either large-scale synthesis or chemical addressability, but not both. Moreover, high equipment cost and complex chemical processing limit their utility to laboratories only. Under the proposed research, we aim at addressing large- scale combinatorial synthesis, digital molecular identification, synthetic throughput, parallel screening, and quantitative analysis of combinatoria chemistry as a whole, by introducing batch-fabricated microdisc carriers with digital barcodes and matrix-directed combinatorial synthesis in reconfigurable microfluidic networks. Specifically, cancer integrin-targeting peptide libraries will be designed and synthesized on the digital ODOC array, followed by microfluidic quantitative screening of the cell-ligand interactions on the array Comprehensive structure-activity relationship (SAR) data obtained by quantitative cell binding of every single compound-disc facilitates design of focused libraries for rapid optimization of cancer-targeting ligands with higher specificity and affinity than those previously discovered. In brief, the proposed digital ODOC array, once developed, will provide a transformative paradigm for high-throughput high-efficiency screening, optimization, and characterization of biomolecules targeting at various types of cancer cell receptors.
R21 CA177526-01 2013 QUINN, THOMAS P (contact); ROBILLARD, MARC UNIVERSITY OF MISSOURI-COLUMBIA In vivo metal-free cycloaddition chemistry driven pretargeted cancer radiotherapy
The goal of the proposed research is to develop novel metal-free ""click chemistry"" pretargeted approaches for cancer radioimmunotherapy (RIT). It is hypothesized that novel in vivo cycloaddition chemistry will greatly facilitate the use of pretargeted antibodies as cancer treatment agents, overcoming their relatively slow in vivo pharmacokinetic properties and yielding high tumor to normal tissue localization. The monoclonal antibody (mAb) CC49 will be used as the targeting agent. CC49 binds the-TAG72 mucin antigen that is over expressed on a number of cancers including colon carcinomas. The CC49 mAb will be derivatized with trans-cyclooctene (TCO) and used to target the tumor prior to injection of the therapeutic probe in a 2-step pretargeting approach. A bis(pyridinyl)tetrazine (tetrazine) conjugated metal will be radiolabeled with the ?-emitter 212Bi or its parent 212Pb or the ?-emitter 177Lu and injected hours to a few days post injection of the tumor-avid antibody. In vivo, a rapid and highly specific cycloaddition reaction will occur between the TCO on the tumor- targeted antibody and the tetrazine moiety of the radiolabeled chelator-tetrazine yielding a covalent conjugate. Unreacted radiolabeled chelator-tetrazine will be rapidly cleared from the body resulting in a high tumor to normal tissue localization of radioactivity. The aims of the proposed research are as follows. Aim1, pretargeting chemistry development, which encompassed second generation cyclooctene, chelator-tetrazine conjugate and clearing agent synthesis and characterization. Aim 2, radiochemistry and conjugation studies, will focus on the radiochemical labeling efficiencies and radiochemical stabilities of the chelator-tetrazine conjugates that will be radiolabled with 212Bi, 212Pb and 177Lu. The bioacitivties of TCO-CC49 and the labeled tetrazine-chelators will be evaluated with TAG72 expressing cell lines. Aim 3, evaluation of new pretargeting components, will optimize the dose to the tumor while reducing the dose to normal tissues, including blood kinetics and biodistribution of newly developed tetrazine probes. Aim4, therapy studies, will take the best combination of ? and ? emitter pretargeting molecules and examine their therapeutic efficacy in a colon cancer xenograft mouse model. Milestones for the project include, improving in vivo cycloaddition reaction by 10 fold, targeting a greater that five-fold improvement in dose to tumor versus normal tissue using pretargeted ? RIT and a five-fold improvement in targeted ? RIT. The final milestone will be to demonstrate that pretargeted therapy studies will yield statistically significantly extension in survival in a mouse colon cancer model. This application of novel in vivo cycloaddition chemistry in a pretargeted approach for cancer radiotherapy is highly innovative. The cycloaddition reaction of olefins with tetrazines in vivo is the first of its kind to be used effectively at clincally relevant conditions in vivo due to its unprecedented reactivity, and for the first time has the potential to extend this type of organic chemistry in to man. This project would be the first of it kind in vivo application of TCO-tetrazine cycloaddition chemistry with pretargeted ?-particle radiotherapy.
R21 CA173279-01A1 2013 SOPER, STEVEN ALLAN UNIVERSITY OF NORTH CAROLINA CHAPEL HILL Acute Myeloid Leukemia: MRD Analysis Using Modular uFluidics and uFlow Cytometry
Acute myeloid leukemia (AML) can be cured through allogeneic stem cell transplantation (SCT). Unfortunately, 25% of patients will experience relapse after SCT that is usually diagnosed by histologic evaluation of peripheral blood or bone marrow. This method is insensitive and leads to diagnosis of relapse with a high disease burden, which is more difficult to successfully treat. Multi-parameter flow cytometry (MFC) can detect lower burden of disease (0.1-0.01% AML blasts from a mixed population); however, it is expensive and impractical for use in diseases that require frequent monitoring due in part to the need for analyzing bone marrow. In this R21 project, a novel processing strategy will be carried out by an inexpensive, easily manufactured, and highly automated fluidic bio-processor used to select and identify rare AML blasts directly from whole blood to allow more frequent testing to detect MRD at an earlier stage compared to MFC. The bio- processor will consist of modules poised on a fluidic motherboard. The modules and motherboard are made from thermoplastics with the prerequisite microstructures generated via replication. Three modules will be used to affinity-select AML blasts from whole blood using a capture bed comprised of surface immobilized antibodies tethered to the selection channel walls via single-stranded DNA bifunctional linkers. The antibodies will target CD33, CD34 and CD117 expressing blasts. The selection modules will consist of an array of 50-250 microchannels that can process large input volumes (2-10 mL) in < 20 min. The AML blasts will be released from the capture bed by engineering a cleavable unit into an oligonucleotide bifunctional linker. Following blast release, they will be detected using an impedance sensor to direct them into a containment reservoir possessing a fabricated filter to permit immuno-staining of the blasts. The final module will consist of a micro- flow cytometer cell fabricated from an amorphous fluoropolymer, CYTOP, which has excellent optical properties and a refractive index (~1.3402 @ 546 nm) close to that of water (1.3331 @ 546 nm). This module will allow for sheath-less operation by matching the flow cell channel dimensions to near the diameter of the AML blasts and overfilling the flow cell channel with the laser excitation beams to produce a uniform intensity profile. Using a 3-color laser-induced fluorescence system, further immuno-phenotyping of the selected AML blasts will be secured. The fluidic bio-processor will be used to test the hypothesis: Detection of MRD following SCT will assist clinicians in administering proper therapies at an earlier stage of AML relapse to achieve higher cure rates. A pilot study will be performed to measure MRD status in AML patients and associate that with the onset of hematologic relapse using the designed fluidic bio-processor with results compared to MFC.
R21 CA174611-01A1 2013 VAN DAM, ROBERT MICHAEL UNIVERSITY OF CALIFORNIA LOS ANGELES Compact microfluidic PET probe concentrator for preclinical and in vitro imaging
Positron emission tomography (PET) is a molecular imaging modality that utilizes radiolabeled probe molecules to target, image and quantify biological processes in vivo. PET probes can be used to study disease mechanisms, to develop novel diagnostics and therapeutics, detect early stage disease, and monitor response to therapy. Due to the high cost of equipment, infrastructure, and personnel currently required to produce PET probes, the availability and diversity of probes is severely limited (especially for research purposes), hindering both research that depends on this imaging tool and the translation of novel PET probes into medical practice. This challenge is being addressed by efforts to develop miniaturized PET probe production technology based on microfluidics with the eventual goal of an affordable, automated, user-friendly system with built-in radiation shielding that operates on a bench top instead of in a ""hot cell"". Such a system would enable on-demand production of diverse probes at affordable cost. Current miniaturization efforts have focused primarily on the synthesis itself, and not on downstream processes such as purification and formulation. Most PET tracers require a concentration process during formulation to reduce the volume after HPLC purification so that a sufficient amount of probe is contained in the limited volume that can be injected into small animal models such as mice without adversely affecting their physiology. Concentration is currently achieved by rotary evaporation, using bulky equipment occupying valuable real estate inside the hot cell. To prevent the concentrator from becoming the size-limiting factor in miniaturized radio synthesis, there is a need for development of miniature concentration technologies. In preliminary studies, a compact proof-of-concept microfluidic device to evaporatively concentrate aqueous solutions was developed, and successful concentration of the PET probe 1-(2'-deoxy-2'-[18F]fluoro- arabinofuranosyl) cytosine ([18F]FAC) dissolved in 1:99 EtOH : 10mM NH4H2PO4 (HPLC mobile phase) was demonstrated. This proof-of-concept chip will be further developed in this application into a robust, automated, compact system for routinely concentrating diverse probes. Aim 1 focuses on the development of a microfluidic chip with performance increased to at least match that typically achieved by rotary evaporation. In Aim 2, the chip parameters and operating conditions will be characterized to enable further performance optimization. The concentrated sample collection process will be optimized in Aim 3. In Aim 4, an upstream module will be developed to enable concentration of non-aqueous solutions, thereby extending this technology to all PET probes. A fully automated system (sample loading, concentration, and recovery) will be developed in Aim 5. This application will result in the development of a prototype microfluidic concentrator that will be a critical part of emerging benchtop production platforms for diverse PET probes that will accelerate preclinical research and translation of diagnostics and therapies to the clinic by increasing access to molecular imaging with PET.
R21 CA182322-01 2013 WANG, ANDREW ZHUANG UNIVERSITY OF NORTH CAROLINA CHAPEL HILL Development of 3D organ-specific models of colorectal cancer metastasis
Understanding the biology of cancer metastases is critical to improving the treatment of cancer. A key challenge in these efforts has been the lack of easy-to-use tumor models that can recapitulate the metastatic disease condition or process. Current models are either too difficult to study or unable to replicate the complex microenvironment of tumor metastasis. Our application aims to address the need for models of cancer metastasis by applying recent advances in tissue engineering. A recent breakthrough in tissue engineering has been the development of decellularized tissue. One novel technique for generating decellularized tissue, developed by Dr. Reid, preserves growth factors and cytokines that are matrix-bound in addition to the extracellular matrix. Decellularized tissue generated using this technique has been termed biomatrix scaffolds. The Reid group has shown that biomatrix scaffolds are tissue-specific but not species-specific both chemically and functionally. Using biomatrix scaffolds, we have obtained exciting preliminary data. We have found that colorectal cancer cells, HT29, SW480 and CaCO2, can spontaneously form 3D colonies on tissue culture dishes coated with liver and lung biomatrix scaffolds. More importantly, we have demonstrated that treatment responses to chemotherapy and radiotherapy are different between cells grown on liver and lung biomatrix scaffolds. Such organ-specific responses have not been observed with other 3D culture systems. Lastly, we have shown that human primary tumor cells from hepatic metastases of colorectal cancer form significantly more colonies when grown on liver biomatrix in vitro compared to that on lung biomatrix, collagen or plastic. Based on our preliminary data, we hypothesize that we can use biomatrix scaffolds to generate 3D in vitro and ex vivo models of cancer metastasis. In this application, we plan to use colorectal cancer as a model disease and develop models of colorectal cancer with liver and lung metastases. We theorize that our proposed models can recapitulate the biology of colorectal cancer metastasis to liver and lung as well as predict treatment responses of metastases. Our application has two specific aims. The first aim will focus on the development of in vitro organ-specific 3D models of colorectal cancer metastasis using tissue-specific biomatrix scaffolds only. Our second aim will focus on the development of 3D ex vivo models of colorectal cancer liver metastases using liver organoids prepared by recellularization of liver biomatrix scaffolds. Success with our research can lead to the development of novel in vitro/ex vivo models of cancer metastasis that can better mimic the disease process. These can become powerful tools for studying the biology of metastasis including: mechanisms of metastasis; roles of physical forces on metastasis; and identification of matrix components controlling metastatic potential. Furthermore, models can be useful for in vitro therapeutic screening assays targeted towards cancer metastasis to a specific organ. Our strategy can also be applied to other types of cancers and metastasis to other organs.
R21 CA181859-01 2013 WANG, ZHENGHE CASE WESTERN RESERVE UNIVERSITY Next-generation mouse gene-targeting technology to model tumorigenesis
The goal of this application is to develop a very fast and cost-effective method to generate gene-targeted mice to model tumorigenesis. Gene-targeted mice are invaluable tools to determine the roles of oncogenic mutations in cancer development. However, conventional gene targeting is slow, expensive and prone to failure. While nuclease-mediated targeting may speed the production of mutants, there remain significant concerns about off-target mutations, relative ease of use and access to the entire genome. In preliminary studies, we have successfully developed an innovative method to directly and efficiently target mouse fertilized eggs using recombinant adeno-associated virus (rAAV)-mediated homologous recombination. Using this approach, we were able to generated germ-line-transmitting mice with at least 10% targeting frequency in a month. We believe that our technology is superior to nuclease-mediated gene targeting approaches (e.g. ZFN, TALEN and CRISPR/Cas). In contrast to nuclease-mediated approaches, off-target mutations are infrequent, embryos can be processed en masse without individual microinjection, and all regions of the genome are accessible to manipulation. Here we propose to further develop this technology to generate gene-targeted mice to model tumorigenesis by determining: (a) if gene-targeted mice generated by our method are suitable for modeling tumorigenesis, (b) if our approach is generally applicable to create gene-targeted mice of various tumor suppressors and oncogenes, and (c) if our approach can be used to generate conditional knock-out and knock-in mice. Successful development of these technologies will revolutionize generation of genetically engineered mice to model tumorigenesis. It will have huge impacts on basic cancer biology as well as cancer drug development.
R21 CA174608-01 2013 WILLIAMS, JOHN CHARLES CITY OF HOPE/BECKMAN RESEARCH INSTITUTE Optimization of multivalent ligands by super-resolution microscopy to treat cancer
Monoclonal antibodies (mAbs) represent an important and rapidly growing class of therapeutics to treat cancer and other diseases, and their success has lead to extensive re-engineering efforts to improve and extend their functionality. Recently, we have uncovered a completely novel and highly specific interaction between a therapeutic antibody and a small peptide (a meditope). We hypothesize that this interaction can be exploited to more effectively target diseased tissue, potentially reduce adverse side effects, and lower the cost compared to current treatments involving combination of monoclonal antibodies. Towards these goals, we have demonstrated that we can couple this meditope to an antigen binding scaffold and target cells overexpressing the tumor antigen EGFR that have been pre-treated with the therapeutic monoclonal antibody against EGFR (cetuximab). This application leverages a new super-resolution protocol we recently developed which allows for quantitative investigation of single-molecule distribution on the plasma membrane. We will use super-resolution microscopy to systematically optimize multivalent meditopes as leads for cancer therapy and imaging. This combination of unique reagents and single molecule detection is highly innovative, and its successful demonstration will set the stage to develop and optimize new multivalent ligands to treat multiple cancer types.
R21 CA182341-01 2013 WILSON, JAMES NORBERT UNIVERSITY OF MIAMI CORAL GABLES Kinase Binding Fluorescent Probes for Assaying Cellular Receptor Populations
Receptor tyrosine kinases are primary targets for small molecule and monoclonal antibody chemotherapies given their key role in intracellular signaling pathways, regulation of cell growth and cell survival. Immunohistochemical (IHC) analysis of receptor levels is an essential component for cancer classification and attempts to predict the treatment regimens, however, IHC has significant limitations in its predictive value due to the fact that the basic science of receptor signaling has moved well beyond using simple protein levels as an indicator. We now appreciate the importance of kinase activation states that are controlled by multiple factors that have been individually confirmed to be highly relevant for their oncogenic potential. Yet our ability to translate this knowledge into analytical readouts tha are robust and clinically useful is lagging far behind our understanding of signaling events. Our goal is to generate protein kinase specific molecular probes, which will enable detection, quantification and detailed analyses of cancer-relevant signaling pathways alongside dynamic, real time analysis of systemic cellular perturbations. To achieve this goal, we will utilize chemical synthesis to generate candidate probes, characterize their performance as reporters by optical spectroscopy, evaluate their pharmacological properties against relevant cancer signaling pathways and validate their performance against current ""gold standard"" analytical methods. Successful execution of this research plan will provide a set of probes that provide an optical ""fingerprint"" with far more informational content and predictive power while being robust in its application for clinical staging, individualized evaluation of drug response, drug development and cancer pathway dissection.
R33 CA177462-01 2013 BAILEY, RYAN C (contact); JOHNSON, MARK D UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN Meso-plex miRNA and protein profiling for cancer diagnostics using chip-integrate
Armed with an increasingly clear picture of the complex biomolecular mechanisms of disease onset and progression, clinical oncology is poised to realize the promise of personalized medicine by applying multiplexed analytical tools for improved diagnostic, prognostic, and theranostic capabilities. However, there are, in general, a lack of suitable technologies to support multiplexed analyses in the clinic-particularly with respect to the analysis of disease-relevant miRNA and protein panels, despite their clearly established utility. Complex and aberrant mechanisms underlying cancer onset and progression can only be unraveled through the measurement of multiple biomarker signatures and at both the miRNA and protein level, a wealth of putative biomarkers have been identified that show enhanced predictive value when considered together with panels of other markers. However, many discovery technologies are not amenable to the clinic. For miRNAs, qRT-PCR assays are incredibly sensitive, relatively rapid, and cost effective, yet are only able to quantitte expression of a single target per assay. Conversely, microarrays are readily multiplexable, yet quite slow and expensive. Thus, there exists a pressing need for meso-plex diagnostic capabilities whereby focused panels of 10s of miRNAs can be simultaneously interrogated using rapid, cost effective, and highly scalable technologies. Again, there is a striking gap in analyticl capabilities that limit the translation of multiplexed proteomics into the clinic. The gold standar enzyme-linked immunosorbent assay (ELISA), is typically very sensitive, selective, and cost effective, though most often single-plex. Protein microarrays are highly multiplexable, but generally far less sensitive, less selective, and not amenable to the clinical setting. Emerging multiplexed analysis methodologies offer some improvements but have yet to find widespread clinical utility. Chip-integrated silicon photonic sensor arrays have recently emerged as an inherently scalable and multiplexable biomolecular analysis technology, and this application aims to robustly validate this powerful technology for meso-plex cancer diagnostics. Silicon photonic microring resonator arrays, having up to 128 uniquely address-able sensor elements, have been previously utilized to quantitatively detect nucleic acid and protein signatures in multiplexed assay formats and from within complex, clinically-relevant sample matrices. Importantly, the technology has its origin in well-established methods of semiconductor processing and sensor array chips can be scalably fabricated to allow low cost assays (< $1/measurement). Furthermore, the molecular generality of this methodology will be utilized to simultaneous profile micro-RNA (miRNA) and protein expression from the same clinical sample-a transformative capability. Although applicable to any cancer, the lethal brain cancer glioblastoma multiforme will be the laboratory and clinical model. Well-established miRNA and protein biomarkers exist for glioblastoma, thus keeping the proposed efforts entirely on technology validation.
R33 CA182377-01 2013 BROWN, BRIAN DAVID (contact); SACHIDANANDAM, RAVI MOUNT SINAI SCHOOL OF MEDICINE Sensor-seq: A genome-wide biological measure of microRNA activity.
microRNA (miRNA) are a recently uncovered class of regulatory RNAs which help to regulate gene expression, and control cell function. Over 2,000 human miRNAs have been discovered. Many miRNAs have tumor suppressor or oncogenic functions, and miRNA dysregulation has been shown to play a key role oncogenesis, metastasis, and even chemoresistance. Although much has been learned about miRNA biology, as the number of new miRNA genes have been discovered it has become increasingly challenging to annotate relevant miRNA regulatory networks. A critical limitation has been the lack of high-throughput, biological approaches for genome-wide analysis of miRNA behavior. In this R33 proposal, we will develop and validate a new technology and methodology that can be used to: (1) Measure the activity of each and every miRNA within a cell, at single cell resolution, and (2) Identify optimal synthetic miRNA binding sites that can be used to improve the targeting of suicide vectors and oncolytic viruses being developed for cancer therapies. Our technology will have major utility for expanding our understanding of cancer biology, for generating tools to study and even track specific cancer cell subsets, such as cancer stem cells, for screening for miRNA modulating drugs, and for developing novel therapies that better target tumor cells for destruction.
R33 CA177446-01 2013 CUNNINGHAM, BRIAN T. (contact); ZANGAR, RICHARD C UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN Photonic Crystal Enhanced Fluorescence: Development of Sensors Structures and Det
There is a clinical need for rapid, multiplexed, and cost-effective detection of soluble protein-based cancer biomarkers. Photonic crystal (PC) surfaces have been recently demonstrated as an effective approach for increasing the output and collection efficiency of fluorescent dyes that are used for protein microarray biomarker detection. Using a quantitative sandwich fluorescent enzyme-linked immunosorbent assay (ELISA) format, the combined effects of PC- enhanced fluorophore excitation and PC-enhanced extraction have been used to reduce the limits of detection of breast cancer biomarkers in plasma, compared to performing the same assay on an ordinary glass surface, resulting in the ability to detect biomarkers in the 0.1 - 10 pg/ml concentration range, as is required for low abundance proteins. In the proposed project, we will develop several innovative approaches for the instrument design that will further reduce the limits of detection for PC-based fluorescent ELISA assays, while integrating the PC into a microfluidic format that will minimize assay volume and allow the assay process to be performed on a droplet of plasma. The PC will be fabricated from low autofluorescence silicon materials, with a design that enables a high quality-factor resonant optical mode to simultaneously provide enhanced excitation and enhanced extraction of fluorescence. The detection instrument will utilize a novel laser scanning approach that optimally couples laser illumination into the PC and matches the resonant coupling condition. The proposed effort seeks to translate photonic crystal enhanced fluorescence (PCEF) technology developed to the proof-of-principle stage under previous NIH funding towards clinical applications. In our previous work, PC device structures, fabrication methods, and detection instruments were developed and first demonstrated for DNA microarrays and protein microarrays. While detection of a panel of breast cancer biomarkers will be evaluated in the proposed project, the detection platform can be applied to multiplexed arrays of biomarker assays for other cancers and diseases. To validate the new sensor and detection instrument, we will detect biomarkers spiked into plasma to establish calibration standards, directly compare against conventional ELISAs, and subsequently quantify biomarker concentrations in blood from breast cancer patients with known Her2 and estrogen receptor statuses. A suite of bioinformatics tools will be modified for automated data analysis and interpretation. The overall goal is to develop a highly sensitive, multiplexed, rapid, and automated assay platform for fluorescent microarray ELISA that can perform biomarker analysis on a droplet of plasma.
R33 CA177456-01 2013 DI CARLO, DINO UNIVERSITY OF CALIFORNIA LOS ANGELES Isolating circulating tumor cells using a centrifuge on a chip
The coming generation of personalized anti-cancer treatments will increasingly require companion diagnostic tests to ascertain the presence of specific genetic mutations for which drug targets are available. Cancer tissue or disseminated cancer cells in the circulation or other bodily fluids will be required to carry out these assays. Tissue directly from the tumor is ideal because it is highly pure and can yield sensitive mutational analyses, but this is not available in certain cases as the primary tumor may have already been resected, micrometastases are too small to biopsy, or tumor is present near sensitive anatomical sites thus preventing safe surgical access. In such cases, disseminated tumor cells present in the blood can be highly useful as this is an easily accessible source. A major problem with using cells from blood in companion diagnostics is the presence of a large background of white blood cells within unprocessed samples; these cells prevent the counting and overpower the molecular signal from rare cancer cells and must therefore be removed for accurate diagnosis. We are developing the ""Centrifuge on a Chip"" technology to address this challenge and enrich rare circulating cancer cells from blood samples to high purity. This device innovatively utilizes inertial fluid physics at high fluid flow rates to generate microscale vortics which size-selectively and passively isolate larger cells and releases them into a small volume of fluid off chip. We have conducted a pilot study with this device in which we have isolated putative tumor cells from patients with advanced malignancies of the prostate and bladder and these cells can be processed for downstream molecular assays. This technology is easy to use and an order of magnitude faster than current microfluidic cell separation technologies. We aim to validate the Centrifuge Chip technology to enumerate circulating tumor cells as a function of cancer stage and to evaluate whether cells purified with this approach yield higher quality genetic and mutational analyses. Ultimately, such a device may provide a cost-effective and rapid 'liquid biopsy' sample which can be utilized for downstream genetic analyses in order to more effectively personalize anti-cancer therapy.
R33 CA174616-01A1 2013 FRANCIS, MATTHEW B; HSIAO, SHIH-CHIA(contact) ADHEREN, INC. The development of a microscopy-based cell-array toxicity assay for quantifying C
Live cell screening represents a major paradigm shift for the development of new pharmaceuticals and biologics. Despite the many automation and data analysis advances for this purpose, however, many challenges still hinder the implementation of this approach. As one set of examples, the most common cell cytotoxicity assays used to evaluate the efficacy of an antibody drug, the complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays, only focus on end-point results. As a result, they yield little information about which cells are killed or resitant in a heterogeneous tumor population. In addition, none of the assay products offered today are compatible with the use of high content imaging to study the onset and rate of cytotoxicity, despite the rich quantity of data that this approach can provide. Adheren's new cell-array toxicity (CAT) assay platforms promise to advance this field by minimizing false positives, providing earlier cytotoxicity data, and allowing the visualization of cell death in real time. These producs are based on a core DNA-based cell attachment platform, which allows strong and programmable cell binding for a very wide range of cell types. The adhesion method allows both live and killed cells to be retained on the microarray, providing improved cytotoxicity statistics. This technology also allows the cell microarray to be washed without cell loss, and it enables the direct visualization of cell cytotoxicity over time using fluorescence imaging. This can providing unprecedented levels of real-time cell-specific data for the elucidation of complement- and effector cell-based cytotoxicity mechanisms. We propose to scale up and further develop these cell array-based ADCC and CDC microscopy assays by validating their effectiveness for a wide range of cell types and biological assays. In addition, the assay platform will be extended from currently the used glass bottom 96 well plates to include low-cost industry standard polystyrene 96 well plates.
R33 CA177461-01 2013 FURDUI, CRISTINA(contact); POOLE, LESLIE B WAKE FOREST UNIVERSITY HEALTH SCIENCES New Reagents for Tracking Protein Oxidation in Cells by MS and Imaging Methods
The association of reactive oxygen species (ROS) with the initiation and progression of cancer, including stimulation of tumor growth and metastasis, is well established; paradoxically, ROS are also important players in many anti-cancer treatments involving ionizing radiation and chemotherapies. Yet we have only a limited appreciation for the molecular mechanisms involved in the many normal and disease-associated functional roles played by ROS, largely due to the limited tools available for studying the molecular targets of ROS. Our research team at Wake Forest University has pioneered the development of highly specific chemical probes, with previous support from the IMAT program, which enable detection and identification of oxidized proteins, targeting the initial sulfenic acid(-SOH) product of cysteine thiols undergoing oxidation. While these probes have been used successfully to identify targets of oxidation within specific proteins such as Akt2 (in the context of PDGF signaling) and specific lipid raft-associated protein tyrosine phosphatases involved in angiogenesis, they have not yet proven amenable to wide-scale identification of such sites using high- throughput mass spectrometry (MS) analysis. As demonstrated in our preliminary data, factors which interfere with MS have been identified and circumvented with new probe designs; for example, acid-base properties of these 1,3-dicarbonyl probes which interfere with the charge states needed for MS detection can be blocked by post-labeling cyclization of the products, and new linear probes exhibiting much higher reactivity with the low abundance sulfenic acids have been generated. This application describes additional new strategies to overcome the remaining issues that limit detection and analysis of the oxidized proteome. The first aim describes new chemical probes for more efficient trapping of electrophilic and nucleophilic sulfenic acids. With the second aim we will investigate new imaging and MS technologies to visualize selective protein -SOH modification in situ and identify sulfenic acid sites in endogenously expressed proteins. Successful completion of this project will have high impact, enabling a much deeper understanding of redox-controlled intracellular processes involved in normal and cancer signaling, angiogenesis and metastasis, as well as chemotherapeutic and radiation-based treatments. In the long term, it may enable the design of selective agonists or antagonists to modulate the activity of target proteins in tumors.
R33 CA174575-01 2013 JI, HANLEE STANFORD UNIVERSITY Oligonucleotide-Selective Sequencing for integrated and rapid cancer genome analysis
Personalized cancer medicine involves identifying clinically actionable cancer mutations and genomic aberrations in any given tumor. As we recently published in the November 2011 issue of Nature Biotechnology, Oligonucleotide-Selective Sequencing (OS-Seq) is a novel targeted resequencing approach that fundamentally improves the detection of cancer mutations from clinical samples. This technology has the potential for enabling the rapid, accurate detection of cancer mutations for both translational research studies and potentially, ""personalized"" cancer diagnostics. OS-Seq provides a number of advantages to make personalized cancer analysis accessible, rapid, robust and accurate. The overall workflow is simplified such that the majority of preparative steps take place on a standard fluidics device and the actual experimental manipulation is limited. The performance is improved compared to the current commercially available methods for targeted, gene-specific cancer sequencing analysis. Based on our empirical analysis and subsequent refinements in designing capture probes, we demonstrate very specific capture of genomic targets with less variance than other methods. We achieve a high level of sequencing coverage on our targets that permit sensitive and specific detection of cancer mutations. With recent improvements in sequencing technology speed, OS-Seq can potentially be adapted to analyze large number of cancer genes in a matter of days which includes the time that genomic DNA is extracted from a biopsy to the completion of the targeted sequencing run. This holds the possibility of rapidly identifying cancer mutations from clinical samples. Our proposal is focused on development of the OS-Seq technology for identifying cancer mutations, rearrangements, copy number alterations and potential cancer-related infectious agents from clinical tumor samples. To achieve this goal, we will develop key aspects of OS-Seq technology for integrated detection of cancer mutations and genomic aberrations with simple protocols that are reliable, rapid and with high accuracy.
R33 CA174550-01 2013 KAMM, ROGER D. MASSACHUSETTS INSTITUTE OF TECHNOLOGY Microfluidic 3D Assays for Metastatic Cancer
Migration from the primary tumor and through extra-cellular matrix (ECM), intravasation across a cellular barrier, and extravasation and recolonization in a remote site comprise the critical steps of cancer metastasis. Physiologically relevant and well-controlled models that mimic the in vivo tumor microenvironment will enable better understanding of the steps of metastasis and evaluation of potential therapy efficacy. In vivo models have physiological relevancy, yet inherently lack a high level of control. In vitro cancer models provide control, yet lack critical components of the tumor microenvironment. We propose a new technology, a microfluidic metastasis assay (uMA) that replicates essential components of the in vivo tumor microenvironment, including a 3D ECM and vasculature, while providing tight control of biochemical and biophysical parameters. The objective of the proposed work is to extend our previous work under the R21 IMAT program to further develop and evaluate our uMA. Several extensions are proposed including: (i) creating a controlled hypoxic environment, (ii) introducing realistic levels of shear stress in the vascular compartment, (iii) use of tumor spheroids to simulate EMT, and (iv) expanding the range of ECM materials currently being used (Aim 1). Another novel direction is to develop a similar assay to investigate extravasation and recolonization (Aim 2). Finally, to promote use of the uMA by other researchers and for high throughput studies, the platform is multiplexed and methods are developed for manufacturing in plastic (Aim 3). As developed, the uMA has separately addressable communicating regions for cancer cells and other tumor-associated cells seeded in a 3D collagen gel, and for endothelial cells (EC) that line a second channel to simulate the vasculature. The configuration permits migration of cancer cells from tumor spheroids within the gel toward the EC-lined channel. The EC layer mimics the in vivo vascular barrier allowing observation of cancer cell intravasation. A similar device allows cancer cells seeded in the channel to extravasate across an EC layer into ECM. Excellent optical access will permit real time observation of cancer cell migration, intravasation and extravasation. The optical access combined with image processing techniques will quantify cancer cell morphological and migratory parameters, leading to identification of novel invasion metrics that will quantify the metastatic potential of cancer cells. Finally, we will leverage the capability of the uMA for use a a functional screen for anti-metastatic drugs. These aims will establish the uMA as a useful model for quantitative research of biological mechanisms governing cancer cell metastasis. Therapies that address multiple steps of the metastatic process would clearly benefit from using the uMA as a development platform, as the system provides a well-characterized EC layer under tightly controlled microenvironmental conditions. Future development will enable the uMA to serve as a cancer cell diagnostic device and a high throughput drug development tool.
R33 CA173373-01A1 2013 KUHN, PETER SCRIPPS RESEARCH INSTITUTE Clinical validation of the HD-CTC fluid biopsy in early detection of lung cancer.
Currently, the clinical diagnosis of non-small cell lung cancer (NSCLC) relies on a number of imaging modalities followed by a tissue confirmation with an invasive procedure. This process is somewhat imprecise because a patient's pathologic diagnosis after surgery may be discordant from pre-operative assessment and may contribute to poor patient prognosis since accurately staging early cancers is essential to good outcome. We hypothesize that rigorously validating the recently developed High Definition Circulating Tumor Cell (HD- CTC) Fluid Biopsy in a controlled clinical cohort of patients undergoing evaluation for lung cancer will improve the accuracy of detection and prognosis in early-stage, malignant nodules of the lung with non-small cell lung cancer histology. We will capitalize on NIH-supported proof of concept data, which has demonstrated that the HD-CTC Fluid Biopsy is capable of detecting disease derived cells in the majority of patients at the time of diagnosis. These data now motivate us to investigate the performance of the assay ""the day before diagnosis"" with the expectation of achieving similar results in a more realistic, prospectively designed, clinical study with matched controls consisting of patients who have non-malignant lung nodules. Our aim is to accurately establish the test characteristics of the HD-CTC fluid biopsy in a group of patients undergoing evaluation for lung cancer and to identify those patients with malignant nodules that have detectable HD-CTCs to determine whether the fluid biopsy can augment 1) clinical staging prior to definitive treatment and 2) prognosis after definitive diagnosis and treatment. We will significantly enhance the test by developing single cell molecular analysis to provide definitive characterization. Integrating non-invasive, reliable and accurate circulating biomarkers with the current standard of care will not only allow for the development of more accurate clinical prediction models that are cost-effective, but may also augment our understanding of tumor biology and metastasis using a clinical, in vivo model of early non-small cell lung cancer.
R33 CA174554-01A1 2013 LIU, YU-TSUENG ; SIMBERG, DIMITRI(contact) UNIVERSITY OF CALIFORNIA High quality CTC isolation using microbubbles for downstream molecular analysis
Early cancer detection and intervention are crucial for long-term survival of either primary or recurrent tumors. Circulating tumor cells (CTCs) could be isolated for the diagnosis, prognosis, and treatment planning of cancer. Genetic profiling and expression analysis of CTC could provide additional invaluable information for accurate diagnosis and prognosis. Moreover, in vitro culture of CTC can be applied for functional analysis of cancer cells, such as drug sensitivity test. To achieve this goal, a transformative technology must be able to unmistakably isolate a few cancer cells using cost-effective and non-invasive procedures. Under the previous R21 IMAT award, we developed a novel technology for rare tumor cell isolation. Lipid shell-perfluorocarbon buoyant microbubbles (MBs) coated with anti-EpCAM antibody immediately attached to tumor cells in unfractionated blood, and isolated the cells after a quick centrifugation step (Shi et al., PLoS One, 2013). A short video clip to demonstrate this technology is included in this application. In conclusion, MB technique offers unique advantages over the existing immuno-enrichment technologies: (a) Short processing time can avoid RNA and protein degradation; (b) Scalability (isolation of cells from large volume sample) can help obtain large numbers of CTCs; (c) Specificity (minimal carryover of leukocytes) can improve sensitivity and specificity of molecular analysis; (d) Flexibility (cells are collected in a very concentrated volume of 5-10 ¿l and could be used for cell growth, immunostaining and/or molecular analysis. The R33 phase of this proposal is focused to further develop MB isolation method to achieve high purity isolation of CTCs from metastatic brain cancers for enumeration and molecular and functional analyses. We set the following specific aims of the study: (1) Optimize MB method to achieve high speed, high purity harvesting of tumor cells spiked in 7-20ml blood sample; (2) Applications of MB isolated CTCs for PCR analysis and culturing; (3) Validate the MB method for sensitivity and specificity of CTC enumeration from blood of metastatic brain tumor patients using CellSearch system as a benchmark. Simple but robust CTC isolation technologies that result in high quality CTC sample for downstream analysis will have transformative impact on clinical management of malignancies and early diagnostics of asymptomatic cancers.
R33 CA173300-01A1 2013 PAULOVICH, AMANDA G FRED HUTCHINSON CANCER RESEARCH CENTER Advanced development of immuno-MRM technology to analyze archived cancer tissues
Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new tissue biomarkers have been translated into clinical use. Clinical validation studies must be performed on large numbers of candidates for a single novel biomarker of clinical utility to be identified. The handful of biomarkers that have successfully reached the clinic were identified mostly through retrospective analysis of archival formalin-fixed paraffin embedded (FFPE) biospecimens. The current gold standard for detecting proteins in FFPE tissues is immunohistochemistry (IHC), but this technology is wholly inadequate to support large-scale testing of hundreds of candidate biomarkers in retrospective validation studies, due to the high costs and long lead time for the development and analytical validation of new IHC assays. Furthermore, even with multi-parameter fluorescence detection, the multiplex capabilities of IHC remain limited and would only allow testing of small numbers of candidate biomarkers in each assay. Additionally, multiple sources of variation in IHC-based clinical assays have resulted in poor inter-laboratory concordance. Furthermore, as currently deployed, IHC assay results are semi-quantitative at best, leading to difficulties interpreting intermediate results, and hampering the ability to assemble multivariate panels as diagnostics. An emerging technology that has the potential to overcome this barrier is a targeted form of mass spectrometry called multiple reaction monitoring mass spectrometry (MRM-MS). While MRM enables specific, precise quantification of polypeptides at high multiplex levels, sensitivity is limiting for many analytes. To address thi limitation, we have developed a novel platform that couples peptide immuno-affinity enrichment to MRM, resulting in highly sensitive immuno-MRM assays. We recently established the feasibility of using this emerging immuno-MRM technology for large-scale testing of cancer biomarker candidates in plasma, and in this application we will perform advanced development of the immuno-MRM technology platform for application to small numbers of human cells derived from FFPE tissues. In Aim 1, a standard operating procedure will be developed that supports analytically robust multiplex MRM and immuno-MRM quantification in FFPE cancer tissues. In Aim 2, analytical validation of the immuno-MRM technology will be performed in an emulated retrospective biomarker validation study using archived human breast cancer tissues.
R33 CA177466-01 2013 SCHLEGEL, RICHARD(contact); WELLSTEIN, ANTON GEORGETOWN UNIVERSITY Conditionally reprogrammed cells as a novel tool for biobanking
Our recent discovery of conditional reprogramming (CR) to generate cell cultures from human tissues offers new and exciting opportunities for biospecimen repositories. This cell technology makes it possible to rapidly generate cell cultures from surgical specimens and small biopsies, thereby providing an unlimited amount of patient material for genetic and proteomic analysis. However, this technology goes further: it will allow the functional analysis of tumor cells and comparison with the patients' normal cells from the same tissue. In this proposal we will extend and validate aspects of the CR technology and optimize its usage for biobanking. It is important to verify that the genotype and functional responses of CR cells mimics that of the primary tumor and experiments will address these issues using exome sequencing and TruSeq analysis. We will also examine whether the CR cells can predict patient responses to therapies, as we have recently described for a single case in the New England Journal of Medicine. Indeed, we believe that this technology will alter how Pathology departments and tissue repositories freeze patient specimens. Rather than simply quick freezing samples for future molecular analysis, tumor samples will be frozen in cryopreservative, which will additionally permit the generation of cell cultures for diagnostic and therapeutic evaluation.
R33 CA173264-01A1 2013 TACKETT, ALAN JACKSON UNIVERSITY OF ARKANSAS FOR MED SCIS Development of MassSQUIRM to Quantitatively Measure Lysine Methylation
The methylation of histone lysine residues has been correlated to numerous phenotypes of cancer. Histone lysine residues can have up to three methyls added and each state can have clearly different cellular roles. For years, researchers in epigenetics (including those focused on epigenetic processes in cancer) have used techniques that rely on antibodies or radioactivity to measure the methylation state of a given lysine; however, these are neither comprehensive nor quantitative. What has become an alternative approach to get around these limitations is the direct use of mass spectrometry, but the problem with this approach is that differentially methylated peptides do not ionize the same and CANNOT be directly compared as a measurement of activity. Therefore, the field of cancer epigenetic research and epigenetics as a whole needed a comprehensive method to simultaneously monitor demethylase/methyltransferase reaction intermediates (i.e., different methyl states on a lysine) in a quantitative manner. In 2011, we provided a novel approach for the comprehensive and quantitative measurement of lysine methylation states, which is called MassSQUIRM (Mass Spectrometric Quantitation Using Isotopic Reductive Methylation). MassSQUIRM utilizes the chemical incorporation of isotopically heavy methyl groups on lysines to convert all reaction intermediates (un- and monomethyl) to fully dimethyl lysines (differing only by hydrogen and deuterium - which does not affect ionization properties in mass spectrometry). A comparison of peptide intensities of the mixture of heavy and light species allows for comprehensive (un-, mono- and dimethyl states) quantitation of lysine methylation. We recently published the MassSQUIRM technique, and in this application we outline how we will evaluate it in a cancer relevant context in order to ultimately develop a kit for cancer research. Our overall goal is to provide a MassSQUIRM kit to cancer researchers to assay demethylation and methylation (un-, mono- and dimethylation specifically) of lysine residues in proteins correlated to particular cancer phenotypes. To validate the MassSQUIRM approach for its use in cancer research, we will pursue the following Aims: Aim 1. Determine the general applicability of MassSQUIRM by assaying a panel of histone lysine demethylases and methyltransferases. Aim 2. Evaluate the effectiveness of using MassSQUIRM to assay LSD1 activity from cell lysates. Aim 3. Optimize MassSQUIRM for lysine demethylation screening with a panel of LSD1 inhibitors.
R33 CA174562-01 2013 TSENG, HSIAN-RONG UNIVERSITY OF CALIFORNIA LOS ANGELES Molecular and Functional Analysis of Single Circulating Melanoma Cells
The long-term objective of this research proposal is to i) develop a single-cell isolation technology by coupling a NanoVelcro Chip with Laser MicroDissection (LMD) techniques to enable highly efficient enumeration and specific isolation of viable/preservative-free circulating melanoma cells (CMCs) from blood, and ii) to demonstrate the feasibility of performing molecular and functional analyses of the isolated single CMCs. In collaboration with the UCLA melanoma team, we will validate the clinical utility of the proposed single-CMC molecular assays for dynamically monitoring disease progression, treatment outcomes and drug resistance in melanoma patients treated with BRAF inhibiters (BRAFi). Our team at UCLA has demonstrated a highly efficient, inexpensive circulating tumor cell (CTC) assay capable of enriching, identifying and isolating CTCs in whole-blood samples collected from patients with different solid tumors. First, we pioneered a unique concept of ""NanoVelcro"" cell-affinity substrates, by which capture agent (antibodies or aptamers) -coated nanostructured surfaces were utilized to immobilize CTCs in a stationary device setting. Second, by integrating the NanoVelcro substrate with an overlaid microfluidic component that can generate vertical flows, further improved CTC capture efficiency (>85%) has been achieved as a result of the enhanced collisions between CTCs and the substrate. Side-by-side analytical validation studies using both artificial and patient CTC samples suggested that the sensitivity of NanoVelcro CTC Assay outperformed that of CellSearchTM. CTCs and CMCs are cancer cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. Enumeration of CTCs/CMCs has established clinical utility in patients with metastatic solids tumors, in whom the CTC/CMCs number becomes an independent and accurate predictor for a patient's response to chemotherapy, disease free/overall survival. It is conceivable that a minimally invasive blood-based diagnostic technique could allow repeated characterization of CTCs/CMCs, providing insight into tumor biology during the critical window where intervention could actually make the difference. Currently, FDA- cleared CellSearchTM Assay is costly and inefficient in capturing CTCs/CMCs without contamination of surrounding white blood cells, thus the diagnostic values of CTCs/CMCs are not fully utilized. Herein, we will explore the combined use of new NanoVelcro Assay and LMD technique for isolating viable/preservative-free single CMCs from blood samples collected from melanoma patients over the course of BRAFi treatment. We will then subject the isolated CMCs for molecular and functional analysis. We envision the variation of CMC number and resulting CMC-based molecular signatures can be used to better investigate and monitor evolution of resistance mechanisms during BRAFi treatment, and to guide development of next- generation kinase inhibitor-based melanoma treatments.
R33 CA174560-01A1 2013 WEISSLEDER, RALPH ; XIE, XIAOLIANG SUNNEY(contact) HARVARD UNIVERSITY Whole Genome Amplification and Sequencing of Single Cancer Cells
This proposal brings together two research teams with expertise in single cell manipulation, amplification strategies, sequencing, and translational cancer research to advance technologies to tackle important questions in cancer biology. It responds to RFA-CA-12-003, and proposes to create and validate a platform for robust single cell sequencing of human samples based on our most recent advance in single cell whole genome amplification. The immediate goal is to combine different technical approaches and then use them to determine how individual cancer cells differ in populations. Our focus will be on sequencing single cancer cells in peripheral blood, as well as from primary and metastatic cancer tissues by fine needle aspiration. Aim 1 will develop and validate integrated approaches so that immunocytochemical analyses can inform decisions on which cells to sequence. Aim 2 will focus on applying the single cell amplification and sequencing technology to primary human samples with phenotypic identification achieved in Aim 1. Overall, our goal is to adapt and further develop a suite of recently developed and highly promising single cell analytical technologies to gain an understanding of genomic variations in primary and shed cancer cells. Success of the project will result in an integrated platform that will serve to overcome prevailing impediments in cancer research. The research is expected to have a broad impact on basic research, clinical practice, and the development of emerging anti- cancer drugs.