D. Lansing Taylor - US grants

The proposed research is aimed at bridging the gap of knowledge between the biochemistry, ultrastructure and cell physiology of cell structure and motility especially in amoeboid cells. The investigations center around functional cell extracts from amoeboid cells which can be regulated to exhibit gelation-solation and contraction. Specific proteins will be isolated from the extracts and the interaction of these proteins with actin and myosin will be investigated by standard solution biochemical techniques and fluorescence spectroscopy. Finally, the distribution and interaction of these fluorescently labeled cytoskeletal and contractile proteins will be studied in living cells using our technique of Molecular Cytochemistry. Quantitative spectroscopic techniques will also be tested on living cells using a microspectrofluorometer.

The aim of this program project is to integrate the fields of cell biology, biochemistry, fluorescent probe chemistry, biophysics, and computer science for the study of cell motility and endocytosis. The focus of the approaches is on the application of fluorescence techniques to define the temporal and spatial dynamics of selected cellular physiological events. Fluorescent analogs of specific contractile proteins will be incorporated into living cells and analyzed by digital image analysis, fluorescence recovery after photobleaching, flow cytometry, total internal reflection fluorescence and by standing-wave luminescence microscopy. The processing of ligands through the various stages of endocytosis will be studied by fractionating cells and analyzing the endosomes and various other organelles by flow cytometry. New fluorogenic substrates of specific enzymes, including lysosomal enzymes, will be prepared to study the processing of ligands temporally and spatically. These experiments will be performed on mammalian cells in culture as well as human leukocytes. The projects should help to define the molecular events involved in cell movements and the pathways of processing extracellular ligands starting from the binding of the ligand to the cell surface.

This Biological Facilities Center proposal requests funds to establish a fluorescence imaging facilities center. The fluorescence imaging center will draw on the strengths of the Center for Fluorescence Research in Biomedical Sciences, the Pittsburgh Supercomputer Center, the Center for Art and Technology, and several companies. This facilities center will integrate the technologies of quantitative fluorescence microscopy, imaging technology, computational science, graphics display computer science, and advanced instrumentation developments to construct the next generation quantitative fluorescence microcope/imaging system for the biomedical sciences. One system will be assembled for fluorescence spectroscopy on living cells and tissues and another for immunofluorescence and fluorescence-based in situ hybridization of nucleic acid probes. These systems will have an immediate impact on research in cellular, developmental, and molecular biology. The instrumentation and methodology developed will directly benefit 30 researchers in the Fluorescence Center when fully operational. Researchers outside of the Center will benefit from the development of new sophisticated equipment as it becomes accessible to all biomedical researchers within the next few years.

A video image memory system, acquistion procesor and digitizer, and a cursor generator will be acquired to complete a distributed, yet integrated, imaging facilty for the Center for Fluorescence Research in Biomedical Sciences. Interactive video image acquisition and analysis, and supermini computers for data acquisition and reduction are also part of the imaging facility. Four separate laboratories are networked to allow multiple user applications. A wide variety of projects in cell biology, cell physiology, developmental biology, regulatory biology, and biological chemistry will be supported by in the instrumentation. Among the projects to be supported are analyses of the growth factor stimulation of quiescent cells and macrophage chemotaxis. Ratio imaging will be used as a spectroscopic method for determining the spatial and temporal changes in (cellular) physiological parameters such as pH and pCa. Multiple parameter analysis of two or more separable fluorescent probes will also be used to correlate spatial and temporal dynamics of a variety of cellular funcitons. Three dimensional reconstruction will be carried out on actin networks in living cells. A method of two wavelength total internal reflection image analysis will be used to quantitate the distance from the cell substrate to selected molecules. Methods for mapping the spatial and temporal variations in fluorescence lifetime and fluorescence anisotropy of suitably labeled proteins in living cells will be developed. The first target for mapping with be calmodulin. Overall, the equipment, coupled with other resources in the Center, will permit state-of-the-art measurements greatly extending our understanding of cellular function.

This award provides funds to a group of investigators for the development of a new generation of quantitative fluorescence microscopy/imaging workstations. The techniques required for the project come from fluorescence microscopy, image analysis, computer science, and computer animation. One of the workstations to be developed will be useful in fluorescence spectroscopy of living cells, the other in immunofluorescence studies and in the use of fluorescent probes for in situ nucleic hybridization. In recent years, the application of video recording and computer based image analysis techniques has begun a revolution in the design and use of light microscopes for the examination of both living and fixed cells. Continued progress in these developments is dependent on knowledge in a number of different disciplines. Carnegie Mellon has assembled an outstanding group of investigators and technical staff in its Center for Fluorescence Research in Biomedical Sciences; they will bring the necessary level of skill and diversity to this project.

In the past five years, steady state fluorescence microscopy has revolutionized light microscopy due to the high degree of sensitivity and specificity. The time resolution of steady state fluorescence microscopy when used to measure chemical or molecular parameters has been in the seconds to hundreds of milliseconds range. Fluorescence and phosphorescence lifetimes can attain time resolutions from nanoseconds to seconds depending on the probe and can be used to extend the temporal resolution of measurements. In addition, the chemical and physical environment of a fluorescent or phosphorescent probe influences the lifetime of the excited state of the probe. Thus the decay of the luminescence produced by an excitation pulse is characteristic of the chemical and physical environment so time resolved microscopy can be used to measure chemical and molecular changes in cells. Results with a test microscope system have demonstrated the feasibility of the approach and we are asking for the commercially available components to assemble a complete system. The applications include characterizing the lifetimes of newly developed dyes, measuring changes in ionic environments in cells to measuring the rotational diffusion coefficients of labeled macromolecules. This latter measurement will allow the characterization of assembly and binding processes of macromolecules in living cells. Time resolved microscopy will become a complementary tool to steady state microscopy and will be the method of choice in many applications where the total excitation dose must be minimized. In fact the lower total excitation dose required and the higher sensitivity of rotational diffusion measurements will probably make this the methods of choice over the now popular fluorescence photobleaching redistribution technique in many applications.

9318891 Taylor The vision of the Center for Light Microscope Imaging andBiotechnology (CLMIB) is that understanding the cellular and molecular mechanisms of fundamental biological processes is within reach during the next decade. This will be made possible by integration of three major tools: biochemistry, molecular biology/genetics, and optical techniques which allow exploration of the in vivo dynamics of cell structure and chemistry. The third of these is the focus of the Center. The Center's strengths are in development of, a) novel reagents for detecting, measuring and manipulating the chemical and molecular components of cells, b) new and integrated modalities of light microscopy to detect and measure temporal and spatial changes in cell structure and chemistry, and c) advanced computing methods to record, analyze, display and model complex imaging data. Technique and instrument development is driven by the needs of biological research to answer basic questions in cell and developmental biology, and by emerging applications in biotechnology. The Center's research and training activities are organized into programs in Cell Biology, Developmental Biology, Applied Biotechnology, Fluorescence-Based Reagents, Machine-Vision Microscopy, 3-D Microscopy, Advanced Image Analysis, and Automated Interactive Microscopy. Twenty-five faculty from 6 departments and 26 graduate students from 7 departments are now working on research projects that involve collaborations between biologists, chemists, physicists, engineers and computer scientists. Direct or indirect support from the Center has resulted in 162 publications. A new interdisciplinary course, Applications of Fluorescence Spectroscopy in Biological Research, was initiated, and a biotechnology training laboratory is being constructed for this and related courses. Other knowledge transfer activities include organizing two conferences, supporting a general-use imaging laboratory for outside researchers, and cr eating a scientist exchange program with industrial collaborators. Transfer of technology occurs through interactions with 14 corporations that license the Center's technology and/or collaborate on research projects. These corporations include multi-nationals such as Dupont, Kodak, Carl Zeiss and Procter and Gamble, as well as biotechnology start-ups such as Biological Detection Systems, One-Cell Systems and Cadus Pharmaceuticals. Two new corporations have already been formed in Western Pennsylvania based on technology transfer from the STC. Two patents have issued from Center research, 4 patents have been submitted, and 27 disclosures have been submitted to the University. The STC is involved in two major K-12 outreach projects: a) a group of high school students and teachers worked with the Center for 6 weeks in the summer in a program called "Careers in Applied Science and Technology (CAST), and b) the Center is collaborating with the Carnegie Science Center in Pittsburgh and the Studio of Creative Inquiry at Carnegie Mellon University to produce a planetarium show called "Journey to the Center of the Cell". This project will use advanced visualization technologies, based on work in the Center, to educate the general public, particularly young students, about scientific discovery and the biology of the living cell.***

The Grand Challenge Application Groups competition provides one mechanism for the support of multidiscipinary teams of scientists and engineers to meet the goals of the High Performance Computing and Communications (HPCC) Initiative in Fiscal Year 1992. The ideal proposal provided not only excellence in science: focussed problem with potential for substantial impact in a critical area of science and engineering) but also significant interactions between scientific and computational activities, usually involving mathematical, computer or computational scientists, that would have impact in high-performance computational activity beyond the specific scientific or engineering problem area(s) or discipline being studied. This is a project to research and develop an Automated Interactive Microscope (AIM). The AIM will combine the latest technologies in light microscopy and reagent chemistry with advanced techniques for computerized image processing, image analysis, and display, implemented on high-performance parallel computers. This combination will produce an automated, high-speed, interactive tool that will make possible new kinds of basic biological research on living cells and tissues. While one milestone of the research will be to show the proof-of-concept of AIM, the on-going thrust will be continued development as new technologies arise and the involvement of the biological community.

This award to a group of 13 Faculty from 3 departments (Biological Sciences, Chemistry, and Computer Science) provides 5 slots for an interdisciplinary training program in biophysical approaches to cellular and developmental biology. All faculty are members of the STC for Light Microscope Imaging and Biotechnology. The training program will consist of six required courses, four of them new interdisciplinary courses (fluorescence spectroscopy, biological imaging, quantitative light microscopy and computational biology.) and two of them existing advanced courses in cell biology and developmental biology that are being modified to fit the program's biophysical aims. The training will equip students with the intellectual and experimental skills to pursue both basic and applied research in a multidisciplinary area of great excitement.

The long-term goal of this research is to define the molecular basis of amoeboid movements. The biological system that will be utilized in this grant period will be the polarized migration of cultured Swiss 3T3 cells during wound healing. Two major experimental approaches are used: 1) the quantitation of temporal and spatial chemical and molecular processes in living cells, and 2) the reconstitution of a gel-sol-contract model in vitro. The projects take advantage of advances in molecular cytology, fluorescence spectroscopy, and quantitative light microscopy. The first specific aim is to define the temporal-spatial dynamics of the myosin II-based motor, the gel-sol transformations, and the regulatory chemistry in living Swiss 3T3 cells. The multimode light microscope workstation coupled with new fluorescent probes of calcium, calcium-binding to calmodulin and phosphorylation of the myosin II regulatory light chain will yield the dynamic information. The second specific aim directly tests the role of myosin III in cell locomotion by blocking the function with an injected antibody that blocks myosin II motor activity in vitro. Furthermore, the role of anterior-posterior gradients of gelation, phosphorylation of the myosin II regulatory light chain and calcium in polarized cell movement will be tested with reagents that perturb these conditions. The third specific aim is to reconstitute the gel-sol-contract dynamics in vitro in order to test the solation- contraction coupling hypothesis of amoeboid movement and to understand the molecular interactions that cause gelation, solation and contraction. The fourth specific aim is to define the role of myosin I in polarized cell movement. Results from this study will yield basic information about how amoeboid cells move and should aid our understanding of how wounds are repaired.

9355741 Olejniczak The Carnegie Science Center (of Carnegie Institute) and Carnegie Mellon University (Center for Light Microscope Imaging and Biotechnology, a National Science Foundation Science and Technology Center, and The Studio for Creative Inquiry) have initiated a collaborative project that portends to change in a dramatic fashion the planetarium theater as a tool for informal science education. After several months of preliminary discussions and, now, the beginning of work, the creative team has been assembled that is defining the vision and executing the program of this exciting project. The vision being formulated is the transformation of The Henry Buhl Jr. Planetarium into a new visualization environment to achieve an interdisciplinary and interactive group learning experience. We call this new concept the "Group Immersive Visualization Environment (GIVE). GIVE will accomplish much of the impact of virtual reality by combining "three-dimensional" images generated by Evans & Sutherland's Digistar Projection System with real and animated, high-resolution video computer images and multimedia and by providing direct audience-control of program direction via The Henry Buhl Jr. Planetarium's elaborate 156-seat, electronic response system. While we anticipate the eventual production of a series of programs in a variety of subject fields, the first to utilize GIVE will be "Journey to the Center of the Cell," a 35-minute presentation. The treatment will convey an experience of self-discovery and natural wonder as audiences transport themselves through striking visualizations of the living cell. Production and evaluation of "Journey to the Center of the Cell" and the development of the Group Immersive Visualization Environment will occur under the auspices of staff of The Henry Buhl Jr. Planetarium, key personnel from Carnegie Mellon University, evaluator Harris H. Shettel, and an Advisory Panel consisting of key planetarium and eductional profes sionals. Program production packages, incorporating compatible components of "Journey to the Center of the Cell," will be produced, marketed and distributed to public and school planetariums; and a Teacher Resource Kit containing supplementary educational materials in the form of video tapes, CD-ROMS and computer disks wil extend the program's reach into the classroom. Special relationships and viewing times will be offered at The Henry Buhl Jr. Planetarium targeting Pittsburgh inner city schools and regional districts containing large percentages of underserved and minority students.

This proposal requests funds to support the acquisition of a Hitachi H-7100 transmission electron microscope (TEM) and ancillary equipment. This instrument will be critical for the research activities of the Department and will play an essential role in education and human resources development at all levels. The Hitachi H-7100 instrument has excellent performance characteristics and flexibility in terms of possible future upgrading. We propose to equip this instrument for the collection and storage of digital images. We will be able to take advantage of existing expertise and computational resources in the Center for Light Microscope Imaging and Biotechnology (a N.S.F. Science and Technology Center) and the Pittsburgh Supercomputing Center to assist in the processing, analysis, and 3-D reconstruction of digital images. Both Centers are located in the Mellon Institute, where the Department of Biological Sciences and its Electron Microscope Facility are located. The single existing instrument in the Department of Biological Sciences, a Philips 300 TEM, is more than 25 years old. It is extremely limited in its capabilities because it is difficult or impossible to equip with several kinds of modern peripheral components and accessories, including video/digital imaging devices, goniometer or cooled stages, and STEM-SEM or EDS systems. Moreover, the age of this model raises concerns about the continued availability of parts and even service contracts on the instrument. A new microscope with expanded capabilities is therefore essential for the support of both current and future research activities in the Department of Biological Sciences. Of 18 faculty members in the Department, 7 will be major users of the instrument, and at least an additional 5 are anticipated to be minor users. All 12 of these faculty members, representing two-thirds of the faculty of the Department of Biological Sciences, use electron optical methods as an essential tool in their research programs. The requested instrument will be used in research projects aimed at an understanding of fundamental cell, developmental, neurobiological, and physiological processes. Major-user projects will include: 1) Correlated electron microscopic and light optical studies aimed at understanding cell movements in culture and in living embryos (Ettensohn, Pollock, Taylor); 2) Highresolution immunolocalization studies, focusing on proteins with important functions in embryonic development (Ettensohn, Pollock) and in critical cell biological processes such as protein targeting (Jones), motility (Jarvik, Taylor), and energy metabolism (Koretsky); 3) Ultrastructural analysis of mutant phenotypes; e.g., of cells with altered organelle structure or function (Jarvik, Jones, Koretsky), of normal and mutant visual systems in Drosophila (Pollock), and of tissues in transgenic mice following molecular genetic switching of enzyme isotypes (Koretsky); 4) Determination of the subcellular distribution of mRNAs by electron microscopic in situ hybridization; (Pollock); and 5) Structural studies of the motor protein kinesin, including conformational changes in the protein under varying ionic conditions and kinesin-microtubule interactions (Hackney). The instrument will also have a major impact on education and human resources development at the undergraduate, graduate, and postdoctoral levels. A significant number of the Department's 60 graduate students, 165 undergraduate biology majors, and 20 postdoctoral fellows will be expected to use the requested instrumentation either through an existing, formal course in electron optical methods (Techniques in Electron Microscopy, BSC 03~20) and/or in their independent research projects. 3

DESCRIPTION (provided by applicant): A 3D biomimetic liver sinusoid construct for predicting physiology and toxicity Approximately 90% of drug candidates entering Phase 1 clinical trials fail, and one of the main reasons for drug failure is unexpected toxicity. The liver plays a centra role in the human body, contributing to homeostasis and important functions such as biotransformation and metabolism of drugs. The liver is also the most common target for drug-induced toxicity. Existing in vitro models and in vivo animal models have limited predictive power for human liver toxicity. The goal of this project is to construct a microfluidic liver modul which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This human liver model is expected to be the essential elimination organ for modeling human exposure, provide improved predictions of drug induced liver toxicity, and also serve as a disease model for drug discovery. Our approach will be to develop a 3D microfluidic system with human hepatocyte, kupffer, stellate and endothelial cells, to mimic the liver acinus - the smallest functional unit of the liver. A uniue feature of the model will be the oxygenation of the media, and the establishment of an oxygen gradient, which is believed to account for important metabolic, gene expression and functional heterogeneity of the hepatocytes in the sinusoidal space of normal human liver. Hepatocytes in the oxygen rich zone are efficient in oxidative metabolism, fatty acid oxidation, gluconeogenesis, bile acid extraction, ammonia detoxification to urea and glutathione-conjugation while hepatocytes in the oxygen depleted zone are efficient in glycolysis, liponeogenesis and Cytochrome P-450 biotransformation. Another unique feature of the model will be the incorporation of 'sentinel' biosensor cells, a small fraction of cells with engineered biosensors that indicate changes in cellular functions. When combined with other fluorescent probes, standard biochemical and mass spectroscopy readouts, the model will provide a real-time High Content Analysis (HCA) profile to monitor organ function and response. The selection and validation of readouts and performance of the model will be evaluated based on a panel of reference drugs with available clinical data. To facilitate that comparison, a database of drugs with clinical data, and data from other in vitro and in vivo studies will be constructed. The ultimate goal of this project is to develop a microfluidic model of human liver function that will integrate with a series of other human organ modules, to create a microphysiology platform that reproduces human clinical trial results and provides improved predictivity of exposure, safety and efficacy for drug development. The liver plays a central role in human drug interactions, both within the liver and in other organs, as a result of drug metabolism. The performance of the liver module is central to the performance of the microphysiology platform. We believe the design proposed here will optimally recapitulate human liver function on that platform.

DESCRIPTION (provided by applicant): A 3D biomimetic liver sinusoid construct for predicting physiology and toxicity Approximately 90% of drug candidates entering Phase 1 clinical trials fail, and one of the main reasons for drug failure is unexpected toxicity. The liver plays a centra role in the human body, contributing to homeostasis and important functions such as biotransformation and metabolism of drugs. The liver is also the most common target for drug-induced toxicity. Existing in vitro models and in vivo animal models have limited predictive power for human liver toxicity. The goal of this project is to construct a microfluidic liver modul which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This human liver model is expected to be the essential elimination organ for modeling human exposure, provide improved predictions of drug induced liver toxicity, and also serve as a disease model for drug discovery. Our approach will be to develop a 3D microfluidic system with human hepatocyte, kupffer, stellate and endothelial cells, to mimic the liver acinus - the smallest functional unit of the liver. A uniue feature of the model will be the oxygenation of the media, and the establishment of an oxygen gradient, which is believed to account for important metabolic, gene expression and functional heterogeneity of the hepatocytes in the sinusoidal space of normal human liver. Hepatocytes in the oxygen rich zone are efficient in oxidative metabolism, fatty acid oxidation, gluconeogenesis, bile acid extraction, ammonia detoxification to urea and glutathione-conjugation while hepatocytes in the oxygen depleted zone are efficient in glycolysis, liponeogenesis and Cytochrome P-450 biotransformation. Another unique feature of the model will be the incorporation of 'sentinel' biosensor cells, a small fraction of cells with engineered biosensors that indicate changes in cellular functions. When combined with other fluorescent probes, standard biochemical and mass spectroscopy readouts, the model will provide a real-time High Content Analysis (HCA) profile to monitor organ function and response. The selection and validation of readouts and performance of the model will be evaluated based on a panel of reference drugs with available clinical data. To facilitate that comparison, a database of drugs with clinical data, and data from other in vitro and in vivo studies will be constructed. The ultimate goal of this project is to develop a microfluidic model of human liver function that will integrate with a series of other human organ modules, to create a microphysiology platform that reproduces human clinical trial results and provides improved predictivity of exposure, safety and efficacy for drug development. The liver plays a central role in human drug interactions, both within the liver and in other organs, as a result of drug metabolism. The performance of the liver module is central to the performance of the microphysiology platform. We believe the design proposed here will optimally recapitulate human liver function on that platform. PUBLIC HEALTH RELEVANCE: The liver plays a central role in human drug interactions and is also the most common target for drug-induced toxicity, resulting in costly, late stage drug failures. The goal of this project is to construct a microfluidic liver module which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This module will be designed to integrate with other organ models forming a human microphysiology platform to improve drug efficacy and safety testing.

DESCRIPTION: The General Electric Healthcare, InCell 6000 High Content Analysis (HCA) instrument is being requested to allow investigators from the University of Pittsburgh, the University of Pittsburgh Medical Center and Carnegie Mellon University to analyze complex cellular processes in both fixed and living cells, multicellular model systems and research organisms (Zebra Fish and C. elegans). This high instrument combines a high scan-rate confocal imaging device, an environmental chamber, 4-channel fluorescence, transmitted light and on-board fluid addition in a microplate-based system. The InCell 6000 permits automated, high throughput and high temporal and spatial resolution of subcelluar structures and biochemical reactions that are the basis of all cellular and tissue functions. The long-term objective is to extend our present capabilities in acquiring high temporal, spatial and spectral images from a small sample size to the acquisition of large data sets from populations of cells on a cell-by-cell basis, tissue models and experimental animals. This new capability will permit the application of computational and systems biology tools to understand the complexities of life processes based on statistical significance not possible before. The integration of the best biological experimental systems, fluorescence-based reagents and high throughput, automated imaging will enable large, combinatorial treatments of samples to allow the exploration of statistically relevant mechanisms of action in a variety of therapeutic areas. The NIH funded projects that will use this instrument include investigations on human adipocyte differentiation in cancer, the physiology of stem cell derived cardiomyocytes, the heterogeneity of drug responses in head and neck cancer models, the role of the immune response in cancer therapies, live, 3D breast cancer models with the analysis of pathways, cell migration in tumor models, modulation of Huntington's Disease progression in model systems, protein misfolding disease model of alpha 1- antitrypsin deficiency (ATD), and necrotizing enterocolitis (NEC) models in C. elgans. In addition, a kidney regeneration model in Zebra fish to identify small molecules that stimulate stem cell proliferation, as well as the application of a new generation of genetically encoded biosensors, standards for imaging and flow cytometry and the further application of active machine learning optimization of experimental strategies. The application of this platform to fundamental biomedical research and translational research programs will ultimately lead to better success in drug discovery and development, while helping to define the mechanisms of normal and disease processes.

Abstract: Chemical Biology Facility (ChBF) The Chemical Biology Facility (ChBF) is a major, cancer-focused component of the University of Pittsburgh Drug Discovery Institute (UPDDI) with the following Specific Aims: 1) provide assistance to designing, developing, and implementing cost effective screening assays as well as access to large compound libraries for probe and lead identification. This includes assisting with early toxicity assays for profiling compounds; 2) guide collaborators in designing effective drug discovery projects/programs for the discovery of novel cancer therapeutics; and 3) provide access to the state-of-the-art use of QSP in implementing novel drug discovery campaigns. The ChBF provides guidance on designing and executing drug discovery and development projects from target and lead discovery through preclinical development. The ChBF provides support services to UPCI researchers for: 1) high throughput screening and high content screening assay design, development, validation, and implementation; 2) lead characterization and optimization including early safety assessment; 3) data analysis; 4) accessing companion diagonstic opportunities; 5) assisting investigators in establishing connections with pharmaceutical companies; and 6) designing comprehensive drug discovery/development programs. The ChBF has added several early safety assessment assays to its armamentarium to aid researchers in the early triage of compounds during drug development including a zebrafish cardiotox assay, liver toxicity assay, and a microsomal stability assay. We have also implemented zebrafish imaging assays to enable higher throughput vertebrate models for drug discovery and development, as well as tissue engineered, human 3-D, biomimetic models (e.g. liver). A key addition made to the ChBF services is the implmentation of Quantitative Systems Pharmacology (QSP) as a new approach to drug discovery. QSP is an approach to drug analysis and development that combines computational and experimental methods to elucidate, validate, and apply new pharmacological concepts to the development and use of therapeutics. Additionally, the ChBF has developed and implemented methods to assess heterogeneity in biological systems to help researchers better understand the activity of compounds in their models. During the current project period investigators in 8 of the UPCI Research Programs used the ChBF.

DESCRIPTION (provided by applicant): A 3D biomimetic liver sinusoid construct for predicting physiology and toxicity Approximately 90% of drug candidates entering Phase 1 clinical trials fail, and one of the main reasons for drug failure is unexpected toxicity. The liver plays a centra role in the human body, contributing to homeostasis and important functions such as biotransformation and metabolism of drugs. The liver is also the most common target for drug-induced toxicity. Existing in vitro models and in vivo animal models have limited predictive power for human liver toxicity. The goal of this project is to construct a microfluidic liver modul which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This human liver model is expected to be the essential elimination organ for modeling human exposure, provide improved predictions of drug induced liver toxicity, and also serve as a disease model for drug discovery. Our approach will be to develop a 3D microfluidic system with human hepatocyte, kupffer, stellate and endothelial cells, to mimic the liver acinus - the smallest functional unit of the liver. A uniue feature of the model will be the oxygenation of the media, and the establishment of an oxygen gradient, which is believed to account for important metabolic, gene expression and functional heterogeneity of the hepatocytes in the sinusoidal space of normal human liver. Hepatocytes in the oxygen rich zone are efficient in oxidative metabolism, fatty acid oxidation, gluconeogenesis, bile acid extraction, ammonia detoxification to urea and glutathione-conjugation while hepatocytes in the oxygen depleted zone are efficient in glycolysis, liponeogenesis and Cytochrome P-450 biotransformation. Another unique feature of the model will be the incorporation of 'sentinel' biosensor cells, a small fraction of cells with engineered biosensors that indicate changes in cellular functions. When combined with other fluorescent probes, standard biochemical and mass spectroscopy readouts, the model will provide a real-time High Content Analysis (HCA) profile to monitor organ function and response. The selection and validation of readouts and performance of the model will be evaluated based on a panel of reference drugs with available clinical data. To facilitate that comparison, a database of drugs with clinical data, and data from other in vitro and in vivo studies will be constructed. The ultimate goal of this project is to develop a microfluidic model of human liver function that will integrate with a series of other human organ modules, to create a microphysiology platform that reproduces human clinical trial results and provides improved predictivity of exposure, safety and efficacy for drug development. The liver plays a central role in human drug interactions, both within the liver and in other organs, as a result of drug metabolism. The performance of the liver module is central to the performance of the microphysiology platform. We believe the design proposed here will optimally recapitulate human liver function on that platform.

DESCRIPTION (provided by applicant): A 3D biomimetic liver sinusoid construct for predicting physiology and toxicity Approximately 90% of drug candidates entering Phase 1 clinical trials fail, and one of the main reasons for drug failure is unexpected toxicity. The liver plays a centra role in the human body, contributing to homeostasis and important functions such as biotransformation and metabolism of drugs. The liver is also the most common target for drug-induced toxicity. Existing in vitro models and in vivo animal models have limited predictive power for human liver toxicity. The goal of this project is to construct a microfluidic liver modul which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This human liver model is expected to be the essential elimination organ for modeling human exposure, provide improved predictions of drug induced liver toxicity, and also serve as a disease model for drug discovery. Our approach will be to develop a 3D microfluidic system with human hepatocyte, kupffer, stellate and endothelial cells, to mimic the liver acinus - the smallest functional unit of the liver. A uniue feature of the model will be the oxygenation of the media, and the establishment of an oxygen gradient, which is believed to account for important metabolic, gene expression and functional heterogeneity of the hepatocytes in the sinusoidal space of normal human liver. Hepatocytes in the oxygen rich zone are efficient in oxidative metabolism, fatty acid oxidation, gluconeogenesis, bile acid extraction, ammonia detoxification to urea and glutathione-conjugation while hepatocytes in the oxygen depleted zone are efficient in glycolysis, liponeogenesis and Cytochrome P-450 biotransformation. Another unique feature of the model will be the incorporation of 'sentinel' biosensor cells, a small fraction of cells with engineered biosensors that indicate changes in cellular functions. When combined with other fluorescent probes, standard biochemical and mass spectroscopy readouts, the model will provide a real-time High Content Analysis (HCA) profile to monitor organ function and response. The selection and validation of readouts and performance of the model will be evaluated based on a panel of reference drugs with available clinical data. To facilitate that comparison, a database of drugs with clinical data, and data from other in vitro and in vivo studies will be constructed. The ultimate goal of this project is to develop a microfluidic model of human liver function that will integrate with a series of other human organ modules, to create a microphysiology platform that reproduces human clinical trial results and provides improved predictivity of exposure, safety and efficacy for drug development. The liver plays a central role in human drug interactions, both within the liver and in other organs, as a result of drug metabolism. The performance of the liver module is central to the performance of the microphysiology platform. We believe the design proposed here will optimally recapitulate human liver function on that platform.

? DESCRIPTION (provided by applicant): Informatics Tools for Tumor Heterogeneity in Multiplexed Fluorescence Images Comprehensive genetic profiling has revealed intrinsic molecular variability, or intra-tumor heterogeneity (ITH), in multiple cancers. Heterogeneity is rooted in both genetic and non-genetic factors and evolves within the context of a tumor microenvironment (TME). Not surprisingly, genetic, phenotypic, and TME heterogeneity present major obstacles to optimal cancer diagnosis and treatment; however, the importance of spatial patterning in ITH has been largely overlooked. The spatial distribution of heterogeneity can be critically analyzed with imaging of tissue sections or tumor microarrays (TMAs) using methods such as immunofluorescence (IF) for proteins and fluorescence in situ hybridization (FISH) for DNA and RNA. These fluorescence imaging techniques probe the tumor and surrounding tissue for the expression of proteins, DNA, and RNA in the context of individual cells, sub-cellular domains and clusters of cells within tissue sections. Typically IF/FISH has been restricted to no more than 4-7 proteins/nucleic acids labeled per slide (multiplexed), but new technological advances now allow up to 60 proteins and a few RNA or DNA probes to be labeled on the same multicellular tissue section of up to ca.10 mm (hyperplexed). Larger tumor domains can be analyzed by stitching together images from tissue sections taken from adjacent regions of the tumor. However, the ability to analyze spatial relationships between proteins and nucleic acids at this scale raises several new informatics challenges, such as how to quantitate and characterize spatial ITH and how to interpret ITH data. To address these challenges, a collaborative team of computational biologists, cancer biologists and pathologists at the University of Pittsburgh and engineers and computer scientists at the General Electric Global Research Center (GRC) will develop software for use by cancer biologists and clinicians to quantitate, interpret and visualize spatial ITH in the context of their particular application and s a first step toward constructing diagnostics based on both cancer biomarker expression levels and spatial relationships between cancer and stromal cells.

We propose to apply four complementary technologies in a Quantitative Systems Pharmacology approach to create a human experimental model of non-alcoholic fatty liver disease (NAFLD), the most rapidly growing disease, and to use the model to test novel therapeutic strategies:1) Implement a vascularized, liver acinus microphysiological system (vLAMPS) constructed with human patient-derived, liver cells, as an experimental model to recapitulate early NAFLD phenotypes and as a platform to experimentally test novel therapeutics; 2) Building on our experience in computational and systems biology, we will use RNAseq data from normal and NAFLD patients to infer pathways of disease progression, to identify the potential molecular protein targets that are in the inferred pathways, and to use our latent factor modeling approach and 3D similarity models to identify drugs that statistically interact with the targets in these pathways; 3) We will employ our highly efficient processes for generating mature iPSC-derived hepatocytes combined with gene editing to incorporate disease engineered iPSC hepatocytes (conditional gain/loss of function) into the vLAMPS to begin testing patient specific therapies; and 4) Apply phenotypic drug screening technologies. NAFLD encompasses a spectrum of liver damage ranging from simple steatosis (NAFL) to more serious non- alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Cirrhosis and HCC resulting from progressive damage to the liver have become the third most common causes of liver transplants. The disease pathogenesis of NAFLD is complex and confounded by the considerable inter-individual differences in disease susceptibility, progression and complications, suggesting the need for a patient specific approach. Studies have identified NAFLD associated gene signatures and single nucleotide polymorphisms (SNPs). In particular, the SIRT1 gene that is downregulated in NAFLD, has been identified as a key regulator of lipogenesis, gluconeogenesis, ER stress, fatty acid oxidation, urea cycle and the antioxidant response in hepatocytes. A SNP in the patatin-like phospho-lipase domain-containing 3 (PNPLA3) gene is strongly associated with hepatic steatosis, fibrosis, cirrhosis, and HCC. However, there continues to be major gaps in our understanding of the pathogenesis of NAFLD. For example, despite its strong association with NAFLD, the functional significance of the PNPLA3 variant is unknown. A major limitation in the elucidation of a mechanistic role of PNPLA3 in NAFLD has been the interspecies differences in its expression and tissue-specific distribution, suggesting the need for human cell models. This combination of the technologies and approaches is expected to lead to new strategies for development of repurposed and new therapeutics with the potential to slow or halt the progression of early NAFLD to the more advanced, life threatening stages.

Project Summary The standard for assessing the effectiveness of drugs to treat metastatic melanoma is the patient's response, but there is a pressing clinical need for a human surrogate model that could support prediction of drug efficacy, thereby saving the patient from trial and error treatments, and that would ultimately serve as a guide for the selection of patient-targeted drug therapies. Today, there is significant interest in the use of patient-derived xenografts (PDXs), in which a patient's tumor is implanted into an immune-deficient mouse, to create in the mouse a model of the patient's tumor. Unfortunately, this process is slow and expensive and is based upon an animal microenvironment rather than a human one. Microphysiological systems (MPS), which encompass organs-on-chips, tissue chips, and engineered organoids, can be constructed using human cells to create an in vitro microenvironment. The proposed research would build upon a strong collaboration at Vanderbilt University, the University of Pittsburgh, and the University of Wisconsin to develop powerful MPS to address the need for models of a patient's response to cancer therapy. This project will study how the tissue microenvironment affects the growth of metastatic melanoma cells and their response to drugs by using the Vanderbilt neurovascular unit tissue chip, the Pittsburgh liver-on-chip, and the Wisconsin engineered organoids for brain and liver, each of which includes multiple cell types. The research will focus on the final stage in the metastatic cascade ? the growth of tumor cells at sites distant from the primary tumor. This growth is governed by ?seed and soil? interaction between the tumor ?seed? and the tissue microenvironment ?soil.? Instead of using a mouse as the soil, patients' cancer cells will be planted into the soil provided by brain and liver MPS constructs derived from human induced pluripotent stem cells. The aims are 1) Implement a common set of human organ constructs (liver-on-chip, neurovascular unit, and engineered organoid from a single human stem cell source), 2) Demonstrate successful seeding of these human organ constructs with metastatic cutaneous melanoma or uveal melanoma cells derived from Vanderbilt and Pittsburgh patients, and 3) Compare the response to drugs by patients' cancer cells that have been seeded into the organs-on-chips and engineered organoids with the response to the same drugs by existing PDX lines. This project will provide guidance as to which in vitro human model might be more predictive of patient outcome when translated to the clinic, based in part upon the type of tumor, the nature of the patient sample, and the patient genotype. It will also test the hypothesis that the human MPS devices and models developed at Vanderbilt, Pittsburgh, and Wisconsin will provide a more realistic, in vitro, three-dimensional human microenvironment to study tumor metastasis than mouse PDXs. The final phase will be a proof-of-concept demonstration of precision medicine in which the microenvironment of the brain and liver could be from the patient's induced pluripotent stem cells.

Human Microphysiology Systems Disease Model of Type 2 Diabetes Starting with Liver and Pancreatic Islets Over 30 million Americans have diabetes, constituting about 9.4% of the adult population. An additional 84 million adult Americans have pre-diabetes, both amounting to an economic cost of $322 billion annually. The underlying cause of all forms of diabetes is an inadequate insulin secretion relative to the metabolic needs. While there is an absolute loss of beta cells in type 1 diabetes (T1D) due to an autoimmune destruction, the pathogenesis of type 2 diabetes (T2D) is much more heterogeneous with preceding insulin resistance being present in many tissues, principally the liver, ?-cells in pancreatic islets, white adipose tissue and skeletal muscle. The insulin resistance and the metabolic consequences vary between tissues and more importantly, vary enormously in the population. Furthermore, evidence from human and model organism studies has demonstrated the importance of organ crosstalk including the role of myokines, adipokines, hepatokines and cytokines from inflammatory cells, as well as the exosomal transfer of miRNA in the pathophysiology of diabetes. Interspecies differences between human and model organism physiology limits the translatability of many findings (e.g. from transgenic mouse studies), such as those from beta cells. All of these make it necessary to devise in vitro systems to study human physiology that allow organ crosstalk interrogation. Understanding the pathophysiology of T2D in a human microphysiology system (MPS) will help understand the progression of the disease, identify biomarkers and develop therapeutic strategies that can prevent, mitigate or reverse the morbidity associated with diabetes and improve patient outcomes. Our proposal focuses on two of the critical organs: liver and pancreatic islets. We will first demonstrate the relevant physiology and pathophysiology in the vascularized liver acinus MPS (vLAMPS) and the vascularized pancreatic islets MPS (vPANIS) using primary human cells/tissue (Aim 1). The full power of MPS disease models will utilize patient- derived, adult iPSCs of all of the key cells in the organs and include real-time fluorescent biosensors of key physiological parameters and conditional knock-downs of selected genes. Our proposal has a strategic plan to optimize the migration from primary human cells in the UG3 phase to iPSC-derived cells in the later stages of the UH3 phase, including collaborative integration of relevant progress in the iPSC field (Aim 2 and 4). The initial use of human primary, cell-based MPS?s will define the optimal normal and disease metrics in each MPS model to begin the investigation of the disease and to serve as a functional reference to test the physiological relevance of the iPSC-derived models. We will functionally and then physically couple the vLAMPS to the vPANIS to test the hypothesis that factors from the insulin resistant liver can potentiate beta cell dysfunction in the context of hyperglycemia and hyperinsulemia (Aims 3 and 4). We will use our microphysiology database as a platform for sharing data, protocols, reagents, the vLAMPS and vPANIS models and results (Aim 5).

PROJECT SUMMARY The University of Pittsburgh Drug Discovery Institute (UPDDI) is requesting funds to purchase the Perkin Elmer OPERA PHENIX high speed, high resolution spinning disk confocal High-Content Screening (HCS) device. The Opera Phenix will replace two Molecular Devices ImageXpress Ultra high content readers purchased in 2008, which are critical to multiple NIH-, DoD-, and Foundation-funded projects at the University of Pittsburgh, but are no longer supported by the manufacturer and have been decommissioned. We have determined that one Opera Phenix instrument can replace the two IXUs. The Phenix is a third generation HCS instrument that will be essential to satisfy the diverse needs of users that the UPDDI serves. No comparable instruments exist at the University of Pittsburgh, the University of Pittsburgh Medical Center, and Carnegie Mellon University. Over the last decade, HCS has become a standard in the pharmaceutical industry for target identification, phenotypic screening, as well as toxicology, and in academia for large-scale biological studies, where cell-by- cell quantitation is critical. The UPDDI has been an academic pioneer in the application of HCS and serves an extensive number of collaborators across campus that require and rely on HCS, ranging from neurodegeneration, organ regeneration, cancer, liver diseases, organotypic model development, and traumatic brain injury. Our diverse user groups? needs emphasize discovery models of physiological relevance and high complexity, and therefore require fast, high resolution 2D, 3D, and kinetic imaging and maximum flexibility in image analysis. The large number of HCS users working in the UPDDI further demands a fast system to permit effective sharing of instrument time, and an integrated database with off-site user access to perform off- line analysis. Key requirements for an HCS imager therefore are superior speed in acquiring z-series of images at high resolution of thick specimens in aqueous matrices, mature yet flexible image algorithms, and seamless integration of instrument software with system, public,and custom-developed UPDDI databases. The only instrument that meets all of these criteria is the Opera Phenix because it has 1) fast laser-based illumination and the ability to acquire multiple channels simultaneously 2) water immersion objectives that eliminate non-matching refractive indices, which limit spherical aberrations of air and oil objectives at longer working distances and require adjustment of correction collars depending on imaging depth; 3) a powerful suite of user-friendly yet flexible image analysis routines including a 3D module, advanced texture and morphology analysis, and intuitive and user-friendly machine learning; and 4) the ability to perform seamless ?adaptive high-resolution imaging?, i.e., pre-scanning a large area at low magnification, followed by automated ?on-the- fly? switching to higher magnification to acquire high resolution images of user-defined regions of interest. The Opera Phenix is the only instrument on the market that is capable of fulfilling the demands of the University of Pittsburgh?s diverse drug discovery community.

A vascularized patient-derived iPSC liver acinus microphysiology system (vLAMPS) is an innovative precision medicine platform for optimizing clinical trial design for nonalcoholic fatty liver disease (NAFLD). Non-alcoholic fatty liver disease (NAFLD) is a major health crisis with no approved therapeutics and many failures in the clinic. The prevalence of NAFLD is estimated to increase from 25% of the US population in 2015 (~83 million) to over 100 million by 2030, accompanied by an increase in nonalcoholic steatohepatitis (NASH), the progressive form of the disease, that can lead to cirrhosis with liver failure and hepatocellular carcinoma (HCC). Despite its public health importance, there is currently no FDA-approved therapy for any stage of NAFLD. NAFLD/NASH is a complex heterogeneous disorder involving multiple molecular pathways. Development of efficacious pharmacotherapy has been hampered by the limited utility of preclinical drug testing models. Simple cell culture and animal models do not recapitulate the spectrum of NASH phenotypes in humans. Highlighting these species differences, knock-in murine models with the high-risk NASH-associated genetic polymorphism, PNPLA3 I148M, develop hepatic steatosis but do not recapitulate the progressive disease seen in humans. Additionally, heterogeneity in risk of progression of NASH, individual genetic variations modulating risk of fibrosis progression, and presence of NAFLD-associated metabolic comorbidities such as Type 2 diabetes mellitus (T2DM), adds additional complexity. We will implement the vLAMPS to initially characterize both a ?normal? and a NAFLD/NASH vLAMPS generated from primary human liver cells (hepatocytes, liver sinusoidal endothelial cells, stellate and Kupffer cells) and then reproduce the results with induced pluripotent stem cells (iPSCs). We will ultimately generate patient-specific iPSCs of the four cell types from patients in our NAFLD clinic to create patient-specific vLAMPS. We will test two cohorts: 1) patients with the PNPLA3 I 148M variant and 2) patients with the wild-type PNPLA3 to identify the patients who respond to two NAFLD drugs that have or are now going through clinical trials and two control drugs. Importantly, this paradigm circumvents the conundrum of high-risk patients being enrolled in large prolonged studies with a high likelihood of failure being simultaneously disqualified from other potentially beneficial studies/treatments. This approach will prove transformational for clinical trial design by enriching for subjects most likely to benefit from a therapy, and in the future, after more than one currently investigational drugs are approved, for precision medicine to identify the most efficacious therapy for high-risk subgroups.

Abstract The Micorphysiology Systems Database Center (MPS-DbC) developed and implemented the Microphysiology database (MPS-Db) enabling the management, analysis, sharing, integration of diverse information and computational modeling of data in one platform, improving the systematic way MPS users work. Aggregation of metadata, experimental data, and references provides for robust and relevant interpretation of the results, and having the data centrally located facilitates data sharing among user-defined collaborators and groups. Ready access to experimental data, metadata, and reference data in a mineable format provides a convenient platform for statistical analysis of MPS performance, and building computational modeling tools to predict PK, identify compound mechanisms of actions, and infer pathways of disease progression. We have been assisting users in capturing and managing MPS data, and the MPS-Db is the central repository for the Tissue Chip Testing Center program data. We propose to build the research and commercial value of the MPS-Db by: 1) continuing to support MPS users to build database content; 2) enabling on-line preclinical/clinical concordance analysis capabilities; 3) enhancing the suite of data mining and computational modeling tools; and 4) augmenting methods for ensuring data quality and the secure, controlled release of data to user-defined groups. We will implement a commercial version of the MPS-Db that can reside on a controlled network behind corporate firewalls for the management of proprietary data while still providing access to public MPS resources. Finally, we propose to establish a self- sustaining MPS-Db and experimental analytics company that will generate revenue from commercial users for the maintenance and further development of the MPS-Db for all users.