Robert West - US grants

This Organic and Macromolecular Chemistry project is directed towards the development of new carbon-silicon and carbon-boron synthetic methods, which will permit the preparation of unique, new, polymeric materials of theoretical and practical interest. New kinds of multiply bonded molecules of silicon and boron will be synthesized and characterized. The organosilicon and organoboron species to be prepared include (1) Disilynes and their polymerization products; (2) Stable 1,4- and 1,2- disilabenzenes; (3) Stable silanediimines; (4) Silynes; (5) Borenes and Diborenes; and (6) Boranones.

We seek to determine the molecular mechanisms involved in positive and negative control of expression of eukaryotic genes transcribed by RNA polymerase II. We exploit the genetic attributes of GAL (galactose metabolism) gene expression in the yeast Saccharomyces cerevisiae to achieve our goal. We are studying three distinct molecular mechanisms that govern the expression of the adjacent and divergently transcribed GAL1 and GAL10 structural genes. These mechanisms include: repression, antirepression and positive control. Together they comprise a genetic switch that moderates the abundance of the GAL1 and GAL10 gene products over a range of four orders of magnitude. Our first objective is to determine the molecular basis of repression of GAL1 and GAL10 caused by sequences located in the upstream activation site (UASG) region. The function of repression is to reduce the basal level of transcription of these genes by a factor of 1000 fold. We have determined that this particular form of negative control is independent of GAL80 gene product, a previously characterized negative regulator of GAL gene expression, and GAL4 gene product, a positive regulator of the GAL structural genes. Our present efforts are directed at characterizing the putative repressor and its binding sites in UASG, and determining the molecular mechanism of repression. Secondly, our data show that carboxy-terminal truncations of GAL4 product, retaining the DNA-binding domain but lacking the positive control function, can overcome repression. This may suggest that GAL4 product normally competes with a repressor molecule to bind a UASG. We wish to determine the biochemical mechanism that favors activation over repression. Thirdly, we have constructed specific mutations of the GAL4 gene to localize domains rsponsible for its positive control function. Our ultimate goals are to procure missense mutations in such a domain(s) and to obtain trans-acting suppressors of those mutations. By characterizing the latter we may be able to identify the property which causes GAL4 product to induce transcription of the GAL structural genes. Combined, the above projects will reveal several key mechanisms of negative and positive transcriptional control, and how the two may be coordinated to regulate different levels of expression of eukaryotic genes.

The objectives of this proposed research are twofold: 1) to investigate the impact of high spatial density dynamic measurement and rotational degrees-of-freedom measurement on dynamic structural modification, 2) to develop an optimal degrees of freedom in the experimentally derived dynamic model. This work will result in a more informationally complete dynamics model which should be able to predict the response of proposed modifications on the structure. The modifications to the structure will be realized by synthesizing a structural element that generates the desired response based on prescribed design goals and constraints. Results from the field of structural optimization will be applied to the experimental dynamics model to make the design trade-offs.

The Synthetic Organic Program will support the research of Dr. Robert West of the Department of Chemistry at the University of Wisconsin-Madison. Dr. West will continue his innovative studies of compounds compounds containing silicon, with a focus on compounds containing multiply-bonded silicon. The new types of multiply-bonded silicon compounds that will be synthesized and characterized include disilynes, silynes, silanamidides, silandiimines, sila(iso)nitriles and 1,2-disilabenzenes.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of availability data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

This award will provide supplementary support to enable Dr. Robin Tan of the University of Wisconsin, Madison, to conduct collaborative research with Dr. Masato Tanaka for 24 months at the National Chemical Laboratory for Industry in Tsukuba, Japan. Tan and Tanaka will utilize Tan's expertise in silicon chemistry to synthesize new organosilicon compounds. With future applications for lithography, new organosilicon compounds can be used to develop new polymeric materials. Tan and Tanaka propose a new method to synthesize organosilicon species by utilizing low valent metal complexes as catalysts. By varying the starting silicon and unsaturated organic compounds, they will be able to form many novel organosilicon compounds which will serve as important starting materials in the synthesis of silicon-based polymeric materials. Dr. Tanaka's experience in silicon-based polymeric materials and the instrumentation available in his laboratory will complement Dr. Tan's experience in synthesizing organometallic compounds.

Tan This award will provide supplementary support to enable Dr. Robin Tan of the University of Wisconsin, Madison, to conduct collaborative research with Dr. Masato Tanaka for 24 months at the National Chemical Laboratory for Industry in Tsukuba, Japan. Tan and Tanaka will utilize Tan's expertise in silicon chemistry to synthesize new organosilicon compounds. With future applications for lithography, new organosilicon compounds can be used to develop new polymeric materials. Tan and Tanaka propose a new method to synthesize organosilicon species by utilizing low valent metal complexes as catalysts. By varying the starting silicon and unsaturated organic compounds, they will be able to form many novel organosilicon compounds which will serve as important starting materials in the synthesis of silicon-based polymeric materials. Dr. Tanaka's experience in silicon-based polymeric materials and the instrumentation available in his laboratory will complement Dr. Tan's experience in synthesizing organometallic compounds. Dr. Tanaka's laboratory is participating in a national Japanese government project on silicon chemicals and silicon-based materials.

The focus of this research is the preparation of silaallenes, silynes, borasilenes and diborenes. Attempts will be made to prepare fullerenes containing one or more silicon atoms in the cage and to construct polymers containing fullerene clusters linked by siloxane or polysilane moieties. %%% With this renewal award the Synthetic Organic Program is supporting the research of Dr. Robert West of the Department of Chemistry at the University of Wisconsin, Madison. Professor West will focus his work on the synthesis of multiply-bonded silicon and boron compounds, the preparation of silicon derivatives of the fullerenes and polymers containing fullerene clusters.

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West
This project will support a program of research on organosilicon polymers. Materials to be synthesized and studied will include (a) new types of polysilanes with properties which may make them useful as fluorescent materials for radiation detection, as electroluminescent materials for display devices, as photoconductors, and as photorefractive materials for holographic data storage; (b) polysiloxanes which will complex alkali metal ions and may be useful as electrolytes in high energy-density batteries; and (c), polymers containing fullerene (C60) molecules which may be superior photoconductors and organic semiconductors.
In addition, the electronic properties of polysilanes will be investigated. UV, Raman, NMR and fluorescence spectroscopy, combined with thermal measurements and X-ray crystallography, will be employed to elucidate the effects of conformational change on the properties of polysilanes.
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This research is in the field of novel, silicon-based materials, which can have important potential applications in the areas of electronic and photonic devices.
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We have characterized the process of spindle pole body (SPB) duplication in wild-type Schizosaccharomyces pombe. (Ding et al., 1997, Mol Biol. Cell). We have also studied various strains of fission yeast that are mutant in genes required for normal mitosis, in order to use 3-D fine structural analysis to characterize mutant phenotype and to understand the functions of the corresponding wild type gene products in the normal cell division process. Cells carrying the mutation, cut11-2 have been reconstructed in 3-D, showing that while the SPB duplicates, one SPB is non-functional. A monopolar spindle is usually formed, but it often fails to attach properly to the nuclear envelope. The cut11+ gene has been cloned, sequenced, and deleted, showing that it is a novel, essential gene encoding a predicted protein with 7 membrane spanning domains. Its product, Cut11p, has been localized to the nuclear envelope in the light microscope, using a fusion protein with the green fluorescent protein (GFP). We have now localized this chimera by immunoEM, using antibodies to GFP and secondary antibodies labeled with 10 nm gold; it is concentrated at the margins of the nuclear pores and of the SPBs during mitosis. We conclude that this protein is an essential part of the mechanism by which objects bind to openings in the nuclear envelope. (In press in Molecular Biology of the Cell) Note that this was the first successful labeling with antibodies to the GFP, yielding a signal-to-noise ratio of about 200. It has served as the model for a series of localization collaborations that are described below. C1

The green fluorescent protein (GFP) from jelly fish of the genus Aequoreus has been isolated and characterized by several workers, and the gene that encodes this protein has now been cloned and expressed in numerous organisms to serve as a marker for aspects of cell physiology. We are interested in converting the fluorescence of GFP to an electron scattering stain so that fine structural studies can be used to complement and extend the work in labs that use GFP as a reporter for protein position. Previous work in our lab has led to only limited success in photoconverting GFP by fixing cells that express it, bleaching their fluorescence with either an argon ion laser or a mercury vapor lamp in the presence of diaminobenzadine (DAB), followed by further fixation with OsO4, then embedding, sectioning and electron microscopy. We are now pursuing two alternative approaches: screening for mutant alleles of the GFP that photoconvert better than the wild type; and finding a small molecule, like eosin, that can serve as an acceptor for fluorescence resonance energy transfer (FRET) of the energy from excited GFP. Acceptors that are in the immediate vicinity of GFP will then be excited and can oxidize DAB. This approach circumvents the difficulty of getting free radicals from GFP photobleaching out of its ~-barrel without their reacting with the protein's many functional groups.

With the support of the Organic Synthesis Program, Professor Robert West, of the Department of Chemistry at the University of Wisconsin, studies the synthesis, structure, and bonding of multiply-bonded and low-coordinate silicon and germanium compounds. Specific classes of compounds under investigation include silynes, disilynes, metallole dianions, and metallacalicenes, as well as other compounds containing new types of double bonds between silicon or germanium and other atoms. Professor West is also exploring the chemistry of stable dicoordinate silicon compounds (silylenes) and their complexes with transition metals and the vaporization of carbon in the presence of silicon tetrachloride, producing silicon-containing fullerene derivatives.

Many questions remain unanswered about the nature of chemical bonding, particularly with regards to the bonding between elements other than carbon. Professor Robert West, of the Department of Chemistry at the University of Wisconsin, is supported by the Organic Synthesis Program for his studies of the synthesis and structure of compounds containing silicon and germanium atoms displaying unusual chemical bonding motifs. Of particular interest to Professor West are those compounds which display multiple bonds to these elements (e.g., silicon-carbon and silicon-silicon triple bonds) and those in which these elements bear fewer bonds than usual (e.g., dicoordinate silicon compounds). Finally, by inclusion of chemical sources of silicon atoms in the carbon vaporization synthesis of fullerenes ("Bucky balls"), Professor West is preparing silicon-containing fullerenes. Observations from each of these studies promise to afford fundamental advances in our understanding of the nature of chemical bonding.

With the support of the Organic and Macromolecular Chemistry Program, Professor Robert West, of the Department of Chemistry at the University of Wisconsin, Madison, is exploring mulitply-bonded and low-coordinate compounds of silicon and germanium. Professor West synthesizes new types of organosilicon and organogermanium compounds containing multiple bonds, including silynes (RSiCR'), germynes (RGeCR'), 1,3-disilaallenes (R2Si=C=SiR2), 1,3-digermaallenes (R2Ge=C=GeR2), polysilyne polymers [(RSi=CR')n], and silafullerenes such as C58Si2. He then studies the chemical reactions of these unusual molecules, including their activity as polymerization catalysts and as precursors to stable silylenium ions, and their halophilic reactions.

Although our understanding of the chemistry of compounds containing carbon has become quite sophisticated, the chemistry of carbon's closely related relatives, silicon and germanium, is comparatively undeveloped. Professor Robert West, of the Department of Chemistry at the University of Wisconsin, Madison, is studying the synthesis and reaction chemistry of a series of compounds in which one or more carbon atoms are replaced by silicon or germanium. These studies are of fundamental importance in developing our understanding of the chemistry of the other elements in the periodic table. In addition, the unusual structures of Professor West's target compounds promise potential advances in practical reaction chemistry, including polymerization reactions.

This project addresses the fundamental chemistry of unusual heavy-metal congeners of carbon compounds. Metallole dianions - five-membered ring compounds containing silicon, germanium or tin atoms - bear two negative charges which are delocalized over the rings. Reactions of these unique dianions are expected to give rise to a wide range of novel compounds containing metal-to-carbon double bonds, as well as to a remarkable series of stable diradicals, containing two unpaired electrons. The synthesis and chemistry of stable silylenes - compounds of silicon in the unusual oxidation state of 2 rather than the customary 4 - will also be explored. The process by which some stable silylenes serve as catalysts for the polymerization of unsaturated organic compounds will be investigated.

With this award, the Organic and Macromolecular Chemistry Program is supporting the research of Professor Robert West, of the Department of Chemistry at the University of Wisconsin - Madison. Although our understanding of the chemistry of compounds containing carbon has become quite sophisticated, the chemistry of carbon's closely related relatives, silicon and germanium, is comparatively undeveloped. Professor West is studying the synthesis and reaction chemistry of a series of compounds in which one or more carbon atoms are replaced by silicon, germanium or tin. These studies are of fundamental importance in developing our understanding of the chemistry of the other elements in the periodic table. In addition, the unusual structures of Professor West's target compounds promise potential advances in practical reaction chemistry, including polymerization reactions.

The Small Business Innovation Research (SBIR) Phase II project proposes the development of ultracapacitor devices that combine the use of nanostructured carbon electrodes with organosilicon electrolytes. These innovative ultracapacitor devices are expected to provide higher working voltages than existing devices, yielding significantly increased energy and power density. This Phase II project will use laboratory results to develop prototype devices and address issues associated with scale up and development of procedures for creating prototype devices. These ultracapacitor devices will be characterized for long-term use by evaluating their physical properties and stability.

The size of the ultracapacitor market, already surpassing $200M, continues to grow at a compound annual growth rate of more than 15%. The development of improved ultracapacitor energy storage devices should accelerate this growth by facilitating the commercial development of low-emission vehicles, which should reduce the overall demand for energy. Organosilicon-based electrolytes should improve the overall safety profile of ultracapacitor devices due to their low flammability and low vapor pressures. The improved safety and improved physical characteristics will expand opportunities for the use of ultracapacitors as robust energy storage devices in consumer electronics and industrial applications. This work will also assist in the development of a trained workforce by involving graduate students and postdocs in the research and development effort.

This REU Site program for Tire Engineering is a multidisciplinary research program for undergraduate students. This site will train 10 undergraduate students each year by providing them with research as well as hands-on training that will help them with their future education as well as career opportunities. The students are subjected to all aspects of tire engineering and are provided with individual challenging research projects that include conceptual, theoretical and numerical design and analysis, and hands-on components in state-of-the-art research laboratories. The individual research projects are integrated into an interdisciplinary, team-based environment where research results will be used to prove a hypothesis. Talented community college and university undergraduate students, particularly underrepresented and women, are recruited to conduct research in this field. Through this REU Site, a diverse program is created and students are motivated to continue their studies towards a bachelor degree (for the community college students) and a graduate degree (for the university students). This project is fully supported by the National Science Foundation Division of Engineering Education and Centers.

PROJECT SUMMARY/ ABSTRACT (RESEARCH PROJECT) Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms (LUTS) in men. Given the clinical and pathological heterogeneity in disease presentation, it is likely that BPH represents a molecularly heterogeneous disease. Furthermore, distinct molecular subtypes of BPH are likely driven by different pathways, encompassing epigenetic regulation, cell signaling, stromal-epithelial interactions and other causes. Identification of these BPH subtypes and drivers will lead to novel targets for treatment. To address this clinical challenge in LUTS through the Center's vision of precision medicine, the Scientific Research Project has two broad aims. The first aim is to apply sequence-based transcriptional profiling to define a new molecular classification of BPH, identify BPH biomarkers and associated microbes, and discover disease drivers and therapeutic targets. The second aim is to develop novel BPH cell culture models using conditionally reprogrammed cell (CRC) technology, and to investigate putative known and novel BPH drivers. Findings will support follow-on translational studies of BPH and LUTS, and provide key resources in minable data and BPH cell models. These studies bring together leaders from fields outside of Urology to apply innovative methods and approaches to problems in LUTS.

DESCRIPTION (provided by applicant): Every cell present in our bodies is related to every other cell by mitotic division, and the history of each of our somas is a bifurcating cell lineage tree whose root is the zygote. We will use this concept to establish lineage relationships among neoplastic lesions and carcinomas from each of 100 HER2-positive breast cancer cases. We will accomplish this by sequencing the genomes of several distinct tissue samples (normal, neoplastic, carcinoma) from each case and by performing expression profiling. The somatic variation we will identify (single nucleotide variants, structural variants, and aneuploidies) willbe used to build lineage trees that serve as roadmaps to determine when during evolution genomic driver events (HER2 overexpression and/or amplification, aneuploidies, and mutations in key cancer genes) and gene expression changes occurred. Several additional neoplastic samples that are too small for whole-genome sequencing will be identified and typed by targeted PCR and sequencing, for 192 of the identified somatic mutations from each case. These additional samples will substantially broaden the phylogenetic tree and facilitate finer resolution as to which types of mutations and other genomic changes happen first during neoplastic evolution. They will also allow us to determine if there are mutations that recur within the same case. Remarkably, in our previous work we have shown, on the basis of such tree analyses, that H1047R in PIK3CA has arisen multiple times within several patients. We will identify additional such mutations, if they exist. Our proposed work is distinct from other studies of tumor evolution, which have so far focused exclusively on within-tumor subclone evolution or metastatic changes, and which cannot order the earliest driver changes. Our study will distinguish drivers of the initial proliferative phenotype from those that cause a full-blown carcinoma. This can only be done by comparative analyses of early neoplasias with normal tissue and with carcinomas. We note that this concept is well-established in species phylogenetics and evolution, where past events are routinely inferred by comparison among extant species, and which have broadly facilitated insight into both gene function and evolutionary mechanisms. Just like evolving species, cells in our somas are governed by inheritance, change, and divergence, and our understanding of the origins and evolution of neoplasias towards tumors will benefit from a phylogenetic and evolutionary perspective. Our proposed study is one of the first that is dedicated to examining cancer in the light of evolution. We believe that a fuller understanding of mutational mechanisms, the order of driver changes during progression, and the role of hypermutable sites, will be essential for improving diagnostics, prediction, and drug development of this fundamentally evolutionary disease.

? DESCRIPTION (provided by applicant): Recent large scale genomic studies have identified and confirmed numerous recurrent single nucleotide variations and aneuploidies in invasive breast cancer (IBC). In contrast to IBC, little is understood about the genomic changes associated with progression to breast cancer, from normal tissue to early neoplasias to ductal carcinoma in situ (DCIS) to IBC. The clinical evaluation of DCIS found in screening breast biopsies relies solely on morphologic criteria and the light microscope that were developed decades ago. These morphologic criteria do not perform well in identifying DCIS lesions that are associated with or are destined to progress to IBC. Among newly diagnosed breast cancer cases in the United States, 20% will be DCIS. With the advent of screening mammography, there has been a remarkable rise in the diagnosis of DCIS among asymptomatic women without the expected reduction in breast cancer mortality, leading to concerns about both over- diagnosis and over-treatment. Clinical trials are emerging to analyze active surveillance as a clinical strategy for patients screen detected with low grade DCIS. Many patients with screen detected DCIS are now left in a quandary, wondering what is their risk of progression to IBC if their lesion were left untreated at initial detection. Because IBC represents the accumulation of recurrent, common genetic changes, we hypothesize that the identification of these mutations, the degree of their accumulation in DCIS, and associated nuclear changes will help us identify cases that have a high likelihood of progressing to IBC. This would allow us to stratify these lesions for risk of developing IBC. We have demonstrated the feasibility of this approach with preliminary data from three independent studies: one involving whole genome sequencing of neoplasms in the progression to IBC, a second involving targeted DNA copy number measurements in a cohort of DCIS, and a third examining the nuclear morphometric differences between DCIS and hyperplasias. We propose a case-control study design using three distinct longitudinal cohorts. We will perform targeted analysis of genomic and nuclear phenotypic features with a case-control study design on two large independent cohorts, including cohorts from the Nurses' Health Study (NHS), Washington University (WashU), and a smaller cohort from Stanford University (SU) to create Discovery, Training and Cross-validation, and Test sets. In our discovery cohort, we will use whole exome sequencing, FISH, and nuclear morphometric analysis to identify genomic and phenotypic changes in DCIS that develop IBC versus cases of DCIS that do not develop IBC. Biomarkers identified from these studies will be used to construct a genomic predictor of breast cancer risk in DCIS. The predictive model will be built using DCIS samples from cases and controls in the Nurses' Health Study. The genomic predictor of future IBC risk generated in the NHS will then be validated on an independent large cohort of DCIS samples with long-term clinical follow-up from Washington University. Improved knowledge of risk stratification for DCIS will help reduce IBC incidence and mortality by improving our ability o stratify patients with DCIS into molecularly defined subgroups of high- and low-risk patients. The high-risk patients may benefit from more aggressive clinical treatment, like complete surgical excision, chemoprophylaxis, and/or intensive surveillance with techniques such as breast MRI, while the low-risk patients may not require or benefit from these measures.

PROJECT SUMMARY/ABSTRACT Macrophages constitute a major subset of cells in the cancer tumor microenvironment (TME), but despite decades of research in murine models and in vitro studies on human blood monocyte-derived macrophages there are no reliable markers to detect the various macrophage functions in the human TME and no systematic study on human macrophage in vivo functional diversity has been performed. Current understanding of macrophage biology is based on findings from murine studies and ex vivo experiments on human blood monocyte-derived macrophages but these have not translated well into human tumor physiology. A convergence of three novel technologies (Smart-3SEQ, CIBERSORTx and MIBI) now offers an opportunity to discover novel markers by studying macrophages in vivo at the microscopic level in the actual TME rather than in a model thereof. We hypothesize that those studies do not represent the true physiological state in vivo because they lack the complex context of the human TME. New technologies will enable us to discover subtypes/phenotypes associated with different functions by directly studying human tumor samples where macrophages are known to have different activities. Our proposal represents an entirely novel approach to study TAM by comparing them in the setting of two distinct carcinomas, breast cancer and colon cancer; two tumor types in which TAM fulfill opposing functions. We will also analyze TAM at the microscopic level within distinct regions of the TME, thus identifying genes that distinguish distinct macrophage subsets based on their location within tissue. Finally, we will study changes in their expression profiles during different stages of tumor progression. This project will provide fundamental new biological insights into the diversity of macrophages in the context of human cancer. It will provide new biomarkers that can be used to provide a rational decision for the choice of novel macrophage- targeting therapy.

PROJECT SUMMARY Benign Prostatic Hyperplasia (BPH) is the benign enlargement of the prostate gland that occurs in older men, obstructing bladder outflow. The resultant lower urinary tract symptoms, such as urgency, frequency and incomplete emptying, have considerable morbidity, and carry annual healthcare costs in the Billions. Current BPH treatments are not very effective because the drugs target normal prostate physiology but not BPH pathophysiology, which is still poorly understood. Next generation, disease-targeted therapies will require a more detailed knowledge of BPH pathogenesis. In genomic studies of BPH clinical samples, we discovered a singular, massive overexpression of Bone Morphogenetic Protein 5 (BMP5) in BPH. BMPs, members of the TGF-beta superfamily, have important roles in tissue morphogenesis, though BMP5 itself has not been extensively studied. In pilot single-cell analyses, we identified a poorly characterized fibroblast cell type enriched in BPH stroma as the principle cell expressing BMP5. Remarkably, addition of recombinant BMP5 to prostatic cells in culture alters their gene-expression profiles to more closely resemble the profiles that we observe in the BPH clinical specimens. From these data, we hypothesize that BMP5 orchestrates key molecular and cellular changes underlying the BPH disease process. As such, therapies that abrogate BMP5 signaling may provide a new precision approach to prevent or treat BPH. Towards that goal, the proposed studies aim to Define the prostatic cell type and triggers of BMP5 production in BPH; Define the cell types and receptors for BMP5 signal transduction in BPH; and Determine whether blockade of BMP5 signaling reverses BPH disease phenotypes. Study findings will provide important new insight into the prostatic cells and signaling orchestrated by BMP5, and key validation data towards new disease-targeted therapies for BPH.

ABSTRACT ? PROJECT 2 Benign prostatic hyperplasia affects the majority of men in later life and leads to considerable dysfunction. Current treatments do not address the pathophysiology of the disease but rather target the overall prostate physiology. The goal of our proposal is to identify BPH (Benign Prostatic Hyperplasia) specific treatments by uncovering its pathophysiology. BPH is marked by proliferation of both epithelial and stromal cells in the transition zone of the prostate. This expansion of tissue surrounding the prostatic urethra presumably gives rise to many of the lower urinary tract symptoms (LUTS) associated with BPH. A number of biologic pathways have been proposed to drive BPH including epithelial or stromal senescence, inflammation possibly related to infection, and aberrant activation of developmental pathways. We have recently performed gene expression profiling of BPH and identified at least two subtypes of BPH with possible therapeutic implications. One of the two most significantly differentially expressed genes in our study is CXCL13 which modulates the immune response. The CXCL13 finding represents an important lead in understanding how the immune response is established in BPH. We hypothesize that immune-related pathways play a significant role in BPH pathophysiology and that pathways associated with CXCL13 are drivers. The Aims of this proposal undertake to identify differences in immune response between BPH and normal prostate and to uncover important pathophysiology relationships that arise from these differences. In Aim 1, we will profile immune cell types in BPH and normal prostate using MIBI-TOF (Multiplexed Ion Beam Imaging by Time-Of-Flight) to measure 14 different immune cells and understand their spatial relationships with the epithelium and stroma. In Aim 2, we will identify spatial relationships and interactions between immune and other cell types. We will do this by combining RNA profiling of the stromal and epithelial compartments with multiplex IHC and computational modeling. In Aim 3, we will determine whether CXCL13 expression and accompanying immune cells is part of an immune response or a senescence response. We will examine whether senescent cell accumulation correlates with CXCL13 expression and BPH subtypes. We will also look for the presence of B and/or T cell clonality.

For many cancer types, the wide-spread adoption of cancer screening has increased the detection of pre-malignant lesions (PML). Despite such efforts, screening has had limited impact on overall survival. Clinical guidelines vary widely from watchful waiting (e.g., prostate) to radical surgery and adjuvant treatment (e.g., breast). In absence of reliable progression risk biomarkers and models, these interventions may have deleterious consequences at the two clinical extremes: delay in life-saving treatment or overtreatment. The study of pre-malignant lesions (PML) at molecular level present significant challenges: PML are small, generally archived in formalin. Moreover, the clinical significance of any identified marker can only be assessed after long follow- up, limiting the translational studies to retrospective collections. These hurdles have prevented the development and application of precision medicine approaches and unbiased biomarker to develop models of progression. The current proposal will extend the MCL Pre-Cancer Atlas Pilot Project (PCAPP initiated in September 2017) with the goal to build multi-modal profiles of highly characterized pre-malignant lesions from the 4 target organs (Lung, Breast, Prostate and Pancreas). The four organs included represent a diverse spectrum of histology - pure histology or mixed with invasive lesions - and clinical settings - treatment or active surveillance. Similarly, the selected profiling methods are as comprehensiveas for invasive tumors atlas (whole transcriptome gene expression or DNA mutations) but also innovative, focusing on micro- environment and exploring spatial heterogeneity (multiplex IHC) and, for a few cases, cellular heterogeneity (single-nuclei sequencing). The propose extension will support the completion of the PCAPP and enable a uniform data analysis and sharing with the community.

ABSTRACT ? BIOSPECIMEN / BIOIMAGING CORE The Biospecimen/Bioimaging Core leverages infrastructure of the Stanford Urology and Pathology departments, but augments services to benefit O?Brien Center investigators. Led by a qualified team, the centralized resource provides qualitative advantages and efficiencies of scale. The Core procures and provides Center investigators with needed biospecimens, including fresh, freshly-frozen, and formalin-fixed paraffin- embedded (FFPE) prostate issues. The Core also performs less visible but equally important activities, including patient consent, quality control/assurance, database management, clinicopathologic annotation, and regulatory compliance. In addition, the Core provides tissue characterization services, including histology, tissue microarrays, laser microdissection, and multiplex immunohistochemistry (IHC) using MIBI-TOF (Multiplexed Ion Beam Imaging by Time of Flight). MIBI-TOF is a powerful and robust technology that harnesses metal-tagged antibodies for the simultaneous quantification of 40 or more antibody targets, with superior spatial resolution and dynamic range. The Core also stores and provides portal access to images including prostate MRIs, whole-mount histology and IHC, and MIBI-TOF data, as well as an integrated multi- scale prostate Atlas spanning MRI to histology, cells and molecules. The Biospecimen/Bioimaging Core provides essential tissue procurement, characterization and imaging services to support the three Center Projects, the Education Enrichment and Opportunity Pool programs, as well as outside O?Brien Centers, P20 grant programs, and the broader benign urology community, .