Stephen R Quake, D.Phil. - US grants

This project will develop new applications of DNA in polymer physics, in fields as diverse as adhesion, polymer brushes, reptation, low dimensional systems, confined geometries, and topological statistical mechanics. It is expected that this research will provide significant new progress towards the resolution of open questions in these fields. Specific experiments that will be performed include the following. The kinetics of adhesive fracture will be studied by measuring the dynamic forces as two entangled polymers are separated. The static and dynamic behavior of a DNA brush will be measured, thus testing the predictions of various theories by providing a direct measurement of single molecule configurations and dynamics. The theory of reptation will be critically examined by searching for correlated motion of polymers in a melt. Low dimensional statistical mechanics will be studied by confining DNA in microfabricated structures. Finally, the effects of topological knots on the statistical mechanical behavior of DNA will be investigated by directed knot tying. This research will be integrated with educational activities on many levels. Graduate students will be trained with an interdisciplinary range of skills. An upper level undergraduate/first year graduate course on `The Biophysics of DNA` will also be developed, that will serve to provide an integrated curricula knitting together the various theoretical and experimental applications of DNA to biophysics, and vice versa. This grant is made under the NSF `CAREER` Program. Support is provided jointly by the MPS Division of Physics, the MPS office of Multidisciplinary Activities, and the BIO Division of Molecular and Cellular Biosciences.

DESCRIPTION: Dr. Quake proposes to develop a new method and instrument for sequencing that will increase both the length of the read frame and the throughput for large scale sequencing. The instrument combines standard microfabrication techniques with optoelectronic techniques for single molecule detection. The sequencing scheme is summarized as follows. A microfabricated flow device will be constructed, in which various reagents can be flowed through a chamber of linear dimension 10um-100um. The chamber will have a window to allow optical interrogation. Single stranded DNA molecules with primers will be anchored to the surface of the chamber, with streptavidin-biotin links. Then DNA polymerase and one of the four nucleotide triphosphates (with some fraction fluorescently labeled) will be flowed into the chamber, incubated with the DNA, and flowed out. If the labeled nucleotide is incorporated, a fluorescent signal will be detected. If no signal is detected, the process is repeated with a different nucleotide. Once the signal is detected, the fluorophores will be excited until they photobleach. This prevents previously detected bases from interfering with the current base. This scheme can be iterated ad infinitum, and a read length of at least 3kbp is anticipated. Since the scheme depends crucially on the ability to photobleach the signal before incorporating the next base, it is called fluorescent photobleaching sequencing (FPS). Specifically, Dr. Quake proposes to 1 ) Build a prototype device to implement this sequencing scheme with a microfabricated flow cell and an external electronic valve to control fluid flow. 2) Demonstrate sequencing with the prototype by sequencing all or some fraction of a 3 kbp insert in a single stranded M13 vector. 3) Test the efficiency, throughput and read length of the device. These parameters will be optimized by varying the fraction of labeled nucleotides, the buffer conditions, the number of bound DNA molecules, etc. 4) Build a second generation device with 10um channel depths and integrated microfabricated valves. 5) Show that the second generation device has vastly improved throughput and efficiency for sequencing. 6) Design and fabricate a parallel layout with several devices on a single silicon wafer.

The overall goal of this SGER Proposal is to determine if it is feasible to construct an economical, microfabricated cell sorter for sorting large numbers of bacterial populations generated in "directed evolotion" experiments. An appropriate elastomeric material will be identified that minimizes the adherence of bacterial cells to the sorter surface. A sorting method (identifies and separates tagged cells) will be identified and the appropriate software developed. The viability of the bacterial cells in the apparatus will be determined. This normally major effort is facilitated by the head-start the Quake Laboratory (Applied Physics, Caltech) has with a similar design for sizing and sorting single DNA molecules based on their fluorescent properties.

Principal Investigator: Stephen R. Quake, CalTech

Proposal Number: 0088649

Abstract:

In this research, chips will be fabricated by monolithic integration of replication molded fluidics with optics and active detectors. These devices will consist of flow channels, including valves and pumps, made from silicone elastomer. In the simplest design, these will be aligned onto micro-optic detector chips with filtered p-n diode detectors and diffractive optic lenses for coupling light into and out of the chip in order to perform fluorescence measurements and manipulation of sub-nanoliters volumes of fluid. The integration of optics with replication molded microfluidics is expected to lead to the construction of very compact but versatile biological testing systems.

The main application for these chips will be rapid analysis of cellular information, either of gene expression pattern by RNA analysis or of protein levels through antibody assays. It is also anticipated that this effort will enable the construction of inexpensive and disposable multifunctional bio-sensor chips compact enough to ultimately be implanted into a host. Since valves and pumps are already integrated into the microfluidic system, the resulting chips can concentrate and measure pathogens or toxins as well as deliver drugs in-situ to the host, or perform complex chemical and biological analyses.

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Scherer
The objective of the proposed research is to develop and build compact absorption spectrometers and fluorescence measurement systems with the capability to test and identify the composition of picoliters of analyte. The approach is to integrate microfabrication techniques for both microfluidic and photonic devices in order to create monolithic analysis "chips" for bio-analysis applications. Specifically, three optical technologies will be developed and integrated into microfluidic systems: (1) Fabry-Perot optical resonators with high-finesse for optical spectroscopy, (2) photonic crystal nanocavities for interferometric sensing with high sensitivity, and (3) development of manufacturable solid immersion lenses for high numerical aperture examination of microfluidic samples.

DESCRIPTION (provided by applicant): This partnership will bridge the technology development efforts of Caltech faculty in Applied Physics and Bioengineering with clinical needs of faculty at the USC Keck School of Medicine. We will apply technology for nanofluidic chips that has been developed at Caltech to problems of biological and medical interest. Using the combined resources of the Applied Physics and the Bioengineering programs, we will develop new nanotechnology to solve a number of bioengineering obstacles that presently exist for single cell genomic analysis on a chip. We will develop microfabricated chips with the ability to manipulate nanoliters of fluid; these chips will be used to perform highly parallel biochemical manipulations and genetic analyses of rare populations of cells. The chips will be able to create unique reagents that can be analyzed using conventional functional genomics techniques. A chip foundry will be created in order to produce research quantities of these chips that can be shared among collaborators. The chip technology will be used to investigate the following two problems of particular medical importance: factor and marker discovery in haematopoietic stem cells, and the discovery and characterization of unculturable pathogens in the human gut.

All the cells of a developing organism carry the same instructions for making proteins, yet the execution of the genetic program is systematically varied to produce the specialized tissues that comprise the fully-formed adult. While the character of each cell reflects the expression of many thousands of genes, this expression pattern is itself determined by a smaller network of regulatory genes, or 'transcription factors.' The causal basis for cellular differentiation can be traced back to the activity of these regulatory networks. For example, in response to diffusive signals called cytokines, self-renewing hematopoietic stem cells give rise to daughter cells that commit to progressively more specialized fates, ultimately becoming terminally differentiated cells, such as erythrocytes, macrophages, and the B and T cells of immunity. This project will develop and apply a new technology - a digital RT-PCR assay using silicone elastomer microfluidic chips - to take a focused look at the behavior of genetic regulatory networks in individual developing blood cells. This research will profile the activity of networks of genes implicated as hematopoietic regulators with single-cell resolution. A database of such profiles will reveal how network activity varies within ostensibly uniform populations of progenitor cells and support comparisons between distinct, developmentally-staged populations. RT-PCR readings will be collated with flow cytometry data to facilitate population analysis that encompasses both surface marker phenotype and internal regulatory network state. Sibling assay studies will be conducted to explore the possibility that metastable regulatory network states bias lineage choice responses to instructive signaling from cytokines.

Broader Impact: This project will further creation of new, interdisciplinary approaches to biological investigations by integrating mainstream developmental genetics, advanced 'lab-on-a-chip' technology, and a quantitative, engineering-oriented approach to the analysis of complex systems. Software developed for the project, including a powerful new program for designing multiplexed PCR primers, will be made available to the research community on an open source basis. In addition, this project will provide opportunity for cross-disciplinary education and training of graduate students; undergraduate students will be involved through a CalTech- sponsored summer research fellowship program (SURF). Valuable research experience will also be available to underrepresented groups through investigator involvement with the Pasadena City College Biotech Program that sponsors research science internships by providing opportunity for interns to work on this cross-disciplinary project.

[unreadable] DESCRIPTION (provided by applicant): The Human Genome Project took several years to complete, yet it is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required for medical diagnostics, forensics, and developmental biology. Conventional sequencing technology has limitations in cost, speed, and sensitivity and the demand for sequence information far outstrips the current ability to obtain it. We recently demonstrated the first proof of principle experiments for a new technology that will provide a fast, low cost, and highly parallel technique for DNA and RNA sequencing. This technology uses single molecule detection of fluorescently labelled nucleotides after DNA polymerase incorporates labeled dNTPs into immobilized individual DNA molecules. A major advantage of this technique over current sequencing methods lies in its ability to obtain sequence information from millions of independent molecules in parallel. Here we propose to develop reagents and methods for single molecule sequencing runs with longer read lengths and higher accuracy, ultimately reaching the NIH gold standard of 99.99%, while reducing the cost of sequencing a mammalian genome to below $100,000. [unreadable] [unreadable]

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Scherer
This proposed instrumentation acquisition is that of a dual-beam focused ion beam scanning electron microscope (FIB/SEM) system, with which the involved research team intends to investigate electromagnetic phenomena at the nanometer scale. The involved research 'team' includes three collaborative elements. CalTech will take the lead, JPL will provide supplemental input, and the FIB/SEM vendor (i.e., FEI Inc.) will not only furnish the basic hardware but will interactively assist CalTech students and faculty in their efforts with software optimization.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A computational model was introduced to predict optimal growth rates of E. coli on several carbon substrates including glucose, glycerol, malate, and acetate (1). Experimental testing of the model showed that E. coli grew optimally on all carbon substrates tested except glycerol. E. coli was subject to growth on glycerol over several generations and it evolved to the optimal predicted growth rate. The metabolic genes were sequenced to identify mutations responsible for the optimized growth rates. In total, eleven different point mutations were identified, and eight of the eleven mutations were in the glycerol kinase gene. The mutations were mapped to highly regulated domains of glycerol kinase. The glycerol kinase mutants have been expressed and structural analysis is underway to determine how these mutations caused an increased growth rate of E. coli on glycerol.

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DESCRIPTION (provided by applicant): DNA sequencing is currently in the midst of disruptive technological shifts, with 454, Illumina, and Solid providing us with enormous throughput increases and large reductions in cost per base. Massively parallel technologies deliver a few Gbp of sequence per week as short fragments, or reads. New applications of sequencing only recently considered impractical are enabled: personal genome sequencing, "metagenomics" analysis of 'soups'containing several, to hundreds of unique organisms, and finally, de novo sequencing of novel genomes of complex organisms. No matter how the sequencing is done, reads must be assembled computationally, if they are to be useful. Given the read length and read quality limitations of new instruments and the massive volume of data generated, the computational assembly problem is becoming critical, with the cost of computational infrastructure and personnel exceeding reagent and instrument-related costs. Moreover, the results of assembly are currently far from ideal;for example, much of the human genome remains invisible due to high percentage of repeats. We propose to develop a new "front end" to next-gen sequencers for DNA preparation, the "Read-Cloud Method", which can reduce computational cost of genome assembly by 2-3 orders of magnitude, produce more complete and accurate genomes, and make metagenomics tractable. We propose a hierarchical sequencing approach, without any need for bacterial cloning. We will achieve this by handling single DNA molecules, tiled across the genome with high redundancy, on microfluidic devices. We will design, prototype, and thoroughly test technology to (i) shear genomic DNA into 200- kbp fragments with narrow size distributions;(ii) randomly amplify each individual, 200-kbp DNA in isolation, within a porous gel microcontainer that will be formed around the dsDNA molecule within a microdevice;(iii) digest micro-encapsulated DNA into small fragments, of tunable size;(iv) bar-code the progeny of each 200-kbp DNA with a 12mer oligonucleotide, to identify each read as associated with a particular 200-kbp DNA. A planar microfluidic device will be fabricated to allow one unique bar- code sequence to be blunt-end-ligated to both DNA termini. Bar-coded DNA is pooled, and next-gen sequencing is done. The results are a highly reducible data set. The method and algorithm are applicable universally, to next-generation platforms. The PIs (Batzoglou, Barron, Shaqfeh, Quake) will collaborate to make an efficient approach to hierarchical sequencing in microfluidic devices. PUBLIC HEALTH RELEVANCE: Project Narrative Gene sequencing is important to medicine. Our DNA sequencing method has the potential for reducing computational cost by orders of magnitude while making the assembled genomes significantly more complete and accurate. The key to this step is using microfluidic handling technologies to subdivide genomic DNA into 200kbp fragments, which are then amplified in isolation from each other and uniquely-labeled to form a highly reducible dataset for genomic assembly.

Cancer stem cells in solid tumors have been recently isolated and appear to be primarily responsible for the growth and spread of the disease. The presence of a stem cell population in a tumor has implications for the diagnosis and treatment of cancer, as it is these cancer stem cells that must be targeted to achieve a cure. Preliminary evidence demonstrates that there is a host of genes differentially expressed by the cancer stem cells and their non-tumorigenic progeny. Many of these genes are thought to play a role in essential cancer functions including proliferation, survival, self renewal and resistance to standard therapeutics. Uncovering the true functional stratification of the superficially uniform population of stem cells in leukemia, breast cancer and other solid cancers is a challenge which requires new kinds of measurements at the single-cell level. Based on recent discoveries by the Clarke and Weissman groups, we believe that gene expression in normal stem cells, as well as cancer stem cells is significantly regulated at the epigenetic level. Epigenetic regulation of gene expression is in part controlled by the posttranslational modification of histone proteins, as well as methylation of DMA, both of which result in the alteration of chromatin structure. These modifications are examined using Chromatin immunoprecipitation (ChIP) assays. Epigenetic analysis is a necessary complement to gene expression analysis in order to understand the control of normal and cancer stem cell self-renewal and discover new therapeutic targets. The microfluidic tools developed by the Quake lab for single cell gene expression (aim1), chip (aim 2) and high throughput in vitro cell culture (aim 3) will allow the Clarke and Weissman laboratories to perform gene expression and epigenetic analyses on rare, purified cells from model mice and primary human cancer or cancer xenografts. These assays will all use microfluidic platforms which have already been developed and validated in the Quake laboratory or are commercially available. New systems will also be designed as needed in the course of this project. Microfluidic epigenetic and genetic assays will allow the study of highly purified, homogeneous, rare cell populations that were previously inaccessible with the standard techniques. Microfluidic cell culture platforms will allow finding appropriate conditions to culture cancer stem cells in vitro and test new therapeutic targets

Technical Development Project 1: Measuring the Immunome: Genomic Approaches to B Cell Repertoire. In this project, we propose to develop new technological applications of next generation DNA sequencing, high throughput PCR chips, and microfluidic single cell processors to perform systemic analyses of the human B cell repertoire. Using B cells purified from human peripheral blood, we will apply these technologies to try to understand the effects of three phenomena on immune repertoire: aging, genetic background and vaccination history. Four groups of subjects will be studied: 1. age groups - children, young adults and elderly;2. genetic background - identical twins vs fraternal twins vs not-related subjects;3. vaccination history - pre, post and multiple immunizations;4. vaccination type - the trivalent inactivated influenza vaccine (TIV) and the live attenuated influenza vaccine (LAIV). This study will provide the first comprehensive and systematic measurements of B cell repertoire at three different anatomic levels, V/D/J/C exon usage, immunoglobulin (Ig) gene deep sequencing, and single cell gene expression analysis. Our specific aims are: Aim 1. Develop a quick and quantitative assay to measure the V, D J, and C exon usage landscape. Aim 2. Develop a high throughput sequencing protocol to perform deep sequencing of Ig heavy chain gene diversity. Aim 3. Develop a microfluidic parallel processor for single cell analysis of B cell repertoire. Aim 4. Dissect the influences of aging, genetic background, and vaccination history on the human B cell repertoire.

Project Overview: Cancer therapeutics targeted against the Epidermal Growth Factor Receptor (EGFR) have demonstrated great potential in lung cancer;however, these agents are effective in only a subset of patients. Furthermore, tumors tliat are initially responsive frequently acquire resistance over time. Though it is straightforward to measure molecular (DNA, RNA, protein) and biophysical (mass, density, charge) characteristics of tumors in bulk, recent studies have shown wide cell-tocell variability and the importance of characterizing that variability in estimating patient outcome^^^. We hypothesize that molecular and biophysical characterizations of circulating cells can discriminate cells that are responsive to therapy from those that are resistant. When analyzing cells collected from the circulation, or from other bodily fluids (e.g., pleural effusions, ascites), typically only a small number of cells are available. To asses the cell-to-cell heterogeneity of this limited number of cells, ive propose to develop and to apply quantitative, comprehensive single-cell analysis devices for assessing the DNA genome (e.g., single nucleotide polymorphisms, fusions, deletions), RNA expression, protein abundance (cell surface, intracellular, and secretome abundance), and biophysical properties of single cells for the dual purposes of predicting a patient's likely response to EGFR-targeted therapies and for monitoring a patient's acquisition of resistance to EGFR-targeted therapies (Fig. N3.3.1). We propose two specific aims for the development, testing, and application of our comprehensive analysis platform (Table N3.3.1).

DESCRIPTION (provided by applicant): Our goal is to develop and test a novel donor-specific, genomic approach to the non-invasive, early diagnosis of rejection and graft dysfunction after solid organ transplantation, and addresses two targeted thematic areas: 1) Applying Genomics and Other High Throughput Technologies;2) Translating Basic Science Discoveries into New and Better Treatments. Organ transplantation saves the lives of patients with end-stage organ failure, yet in many cases, the transplanted organ is rejected by the recipient, causing a life-threatening situation. Previous attempts to develop a non-invasive marker of graft rejection have focused on recipient-specific immune responses, and thus have inherent limitations in sensitivity and specificity, especially for distinguishing rejection from infection. Our novel approach is the first to focus on a donor-specific marker of acute rejection. We will use high throughput next generation sequencing to monitor the proportion of cell-free donor DNA to recipient DNA, in the recipient's blood stream as a marker of rejection. This approach is enabled by the fact that an organ transplant is also effectively a genome transplant, and by monitoring single nucleotide polymorphisms that are specific to the donor's genome one can measure the relative health of the transplanted organ. RATIONALE/HYPOTHESIS: during rejection, apoptosis of donor cells releases donor DNA into the recipient circulation;this DNA can be distinguished from recipient DNA and quantified using high-throughput sequencing techniques;and dynamic changes in donor DNA levels will predict (a) acute rejection;(b) graft dysfunction;and (c) chronic rejection. PRELIMINARY STUDIES: using banked blood samples from heart transplant recipients, we showed that donor DNA levels rise during episodes of acute rejection but remain at stable, low levels in the absence of rejection. AIM 1: Develop a donor DNA monitoring approach for the non-invasive detection of allograft rejection. We will develop and test: (1) next generation DNA sequencing methods to perform low cost, noninvasive analysis of donor DNA load;(2) bioinformatic algorithms that maximize the sensitivity to discriminate donor and recipient DNA;(3) sensitivity of a defined single nucleotide polymorphism (SNP) panel that differentiates donor from recipient. AIM 2: Evaluate the utility of donor DNA monitoring for detection of clinical events (acute rejection, chronic rejection, and graft dysfunction) after heart transplantation. We will conduct a prospective cohort study of 110 consecutive heart transplant recipients, collecting blood samples during and between endomyocardial biopsy (EMB) procedures (yielding 1,516-paired EMB/blood) to determine whether donor:recipient DNA ratio can detect rejection in its early stages and before the development of graft dysfunction. AIM 3: Test this approach in lung transplantation and determine whether donor:recipient DNA ratio can differentiate between graft dysfunction due to rejection versus pulmonary infection. If achieved, these specific aims will provide the foundation for validation studies of a donor-specific, genome-based approach to non-invasive, early detection of rejection after solid organ transplantation. PUBLIC HEALTH RELEVANCE: Organ transplantation saves the lives of patients with end-stage organ failure, yet in many cases, the transplanted organ is rejected by the recipient, causing a life-threatening situation. We propose to develop and test a new and innovative approach to the non-invasive, early diagnosis of rejection and graft dysfunction after heart and lung transplantation. By simply monitoring the blood of a transplant recipient for level of the donor genome appearing during follow-up after receiving a transplant, we hope to establish a diagnostic tool that has potential to save hundreds of millions of dollars in health care costs annually, and reduce patient morbidity and mortality.

DESCRIPTION (provided by applicant): This project covers 3 thematic areas: Applying Genomics and Other High Throughput Technologies, Translating Basic Science Discoveries into New and Better Treatments and Reinvigorating the Biomedical Research Community. Somatic cells are highly stable in adult animals due to robust gene expression patterns, which are stabilized by epigenetic mechanisms. The seminal invention of induced pluripotent stem (iPS) cells, however, provided the surprising conclusion that the differentiated state can be reversed by simple expression of four transcription factors (TFs). This finding proved that even supposedly stable epigenetic modifications of genes are essentially controlled by TFs. We asked whether this concept can be extended to trans-differentiation of one cell type into another, and recently succeeded in converting mouse fibroblasts directly into functional neurons, referred to as induced neuronal (iN) cells, by overexpression of only three lineage-specific TFs. Our findings indicate that TFs suffice to not only reverse a particular pathway of differentiation, but also to redirect the transcriptional regulatory network in a cell into a completely different pathway. This fundamental result answered one of the key open questions in the field, and is the basis of the current proposal. Apart from documenting the dominance of TFs over epigenetic modifications, iN cells could represent an attractive way to derive patient- specific neurons from skin fibroblasts. This may be used to model various neurological diseases or for cell transplantation therapy. This proposal aims to characterize the process of iN cell generation on the molecular level, with the expectation to gain fundamental insights into the biology of the underlying trans-differentiation process. In addition to identifying the molecular events underlying the fibroblast-to-neuron conversion, this study will in particular assess the epigenetic stability of the iN cell state as well as their safety with respect to their potential tumorigenicity, key prerequisites for clinical application of iN cell technologies. Our multidisciplinary approach entails state-of-the art high-throughput sequencing technologies for genome-level interrogation of epigenetic states and transcription, newly developed microfluidic devices enabling genome- wide analyses of small cell populations as well as multiplex gene expression on the single cell level allowing the determination of cellular heterogeneity, electrophysiology, and neurodevelopmental techniques. PUBLIC HEALTH RELEVANCE: This application will develop methods to generate neurons directly from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. Patient-derived neurons could be used for modeling neurological diseases or as cell grafts to treat neurodegenerative diseases like Parkinson's disease.

The overall objective of the proposed center CCHI is to investigate the adaptive immune response in influenza vaccination and immunity. The genomics core will serve two research projects (Projects 2 and 3), and the purpose of the core is to provide leading edge genomic techniques, tools, and equipment to aid in the completion of the research objectives. We will 1) provide research projects with convenient and rapid access to highly multiplexed qRT-PCR as well as next-generation sequencing (NGS) including immune repertoire sequencing; 2) provide a novel platform and methodology to enable studies of immune cell responses to infection and vaccination at single-cell resolution; 3) optimize the single-cell approaches for each research project; 4) bridge the gap between research projects and the bioinformatics core by turning raw samples into pre-processed data ready for in-depth statistical and analytical analysis; and 5) serve as a source of information to research projects with respect to genomic technology and tools. The specific aims of the genomics core are: Specific Aim 1 - Provide access to genomics technologies. The Genomics Core will carry out NGS sequencing on the samples generated by the research projects, as well as provide access to the single-cell analysis platform. The core also has a variety of peripheral equipment for sequencing library preparation and quality control, which will also be available to the projects. The core can also process raw sequence data ready to be interfaced with the bioinformatics core's analytical tools, if needed. Specific Aim 2 - Optimize single-cell approaches for whole transcriptome analysis of immune cells. The methodology and pipeline for single-cell whole transcriptome profiling allows elucidation of the transcriptional profile for all genes from each single cell in an automated fashion without complex hands-on workflows. This platform can be coupled with either NGS for whole transcriptome analysis, or with highly multiplexed qRT-PCR for targeted gene expression analysis. The core will optimize this methodology to work with immune cells, which are smaller in size and lower in RNA content than the epithelial cells that were used to initially validate the technology.

? DESCRIPTION (provided by applicant): We are developing an innovative, minimally invasive method to study the genetic spectrum of tumors metastatic and primary to the central nervous system (CNS). Cell free DNA (cfDNA) acts as a tumor fingerprint, allowing detection of genetic alterations that mark the variation and progression of disease. As a clinical tool, it may supplant the currently available and relatively insensitive methods of brain tumor diagnosis. As a scientific tool, novel tumor cfDNA mutations identified in CSF will allow for great insight into tumor biology, and more faithful animal modeling of these diseases. Specific Aim 1: Develop novel microfluidic chips to isolate and characterize cfDNA within the CSF of patients with primary and metastatic brain tumors. Specific Aim 2: Refine new sequencing techniques to characterize tumor cfDNA in CSF from patients with CNS tumors. Determine the feasibility of supplanting cytology with tumor cfDNA sequencing in the diagnosis of leptomeningeal metastasis. Specific Aim 3: Subtype primary brain tumors through the development of single-gene copy number variation sequencing of cfDNA in CSF. Summary: cfDNA in CSF from brain tumors shows great promise to track cancer's mutation profile, diagnose disease, and inform treatment. The techniques accelerated here may revolutionize the recognition of leptomeningeal metastases, and deepen our scientific understanding of brain tumor progression.

Abstract In type 1 diabetes (T1D) insulin producing ?-cells of the pancreatic islets of Langerhans are lost and secretion of the glucose-raising hormone glucagon from ?-cells is dysregulated, contributing to hyperglycemia and impaired counter-regulation. Recent studies demonstrate appreciable heterogeneity within the ?-cell and ?-cell populations both in vitro and in situ. Emerging single-cell approaches have established ?-cell sub-groups that differ in their Ca2+ signaling and transcriptomic profiles and may represent ?pacemaker? cells or replication niches. Evidence is also accumulating, including preliminary data in the present application, to suggest that the pancreatic ?-cells are both heterogeneous and malleable ? the altered function of human ?-cells in type 1 diabetes (T1D) is consistent with a shift towards a ?-cell phenotype. This could contribute to the dysregulation of glucagon secretion. Others have shown the persistence of ?resistant? or surviving ?-cells in T1D, both within islets and throughout the pancreas, although the nature and function of these remain unclear. Understanding the variability and malleability of human islet cell function, and the relationship of this to components of the islet microenvironment such as vasculature or nerves, is important since this may provide avenues for correction of glucagon secretory dysfunction, protection of ?-cells, or the regeneration of ?-cell mass. The present proposal will combine in-depth transcriptomic, proteomic, functional phenotyping on a cell-by-cell basis to understand the underlying regulation of islet cell functional heterogeneity and will map these in situ in relation to other islet cells types and components of the local environment. The Aims are to (1) examine human islet cell functional phenotypes, and the linkage of phenotypic variability to single-cell gene expression; (2) map the markers that define islet cell heterogeneity and sub-populations within the 3D islet microenvironment in health and T1D using approaches that span a range of resolutions and scales; and (3) link islet cell function, single-cell gene expression, single-cell metabolism, and single-cell proteomics in situ to understand islet cell pathophysiology. Integration of an in-house human islet isolation program, multi-dimensional cell imaging expertise, and single-cell dual functional and transcriptomic profiling using electrophysiology (Patch-Seq) on isolated cells and in situ using live human pancreas slices will help accomplish the goal of obtaining a high resolution understanding of islet cells within the local tissue architecture in health and diabetes.