Stanford University

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract This is a request for a supplement to our parent grant, “BioPortal: An expansive knowledgebase of biomedical entities and relations.” The supplement would allow us to replace the obsolete server system that provides storage for the more than 1000 publicly accessible biomedical ontologies managed by BioPortal. The BioPortal system provides access to nearly all the world’s publicly available biomedical ontologies and controlled terminologies, and it is used extensively by many secondary resources that are important to the NIH, including the CEDAR metadata management system and the REDCap system for electronic data capture. Our parent grant supports continued maintenance of BioPortal and the development of new features that are important to the biomedical community. Our ongoing work is threatened because the hardware that provides storage of BioPortal ontologies has already reached the end of its anticipated lifespan and is beginning to show signs of failure. We therefore wish to replace the existing server system with modern equipment with an extended period of performance. This singular equipment purchase will enhance access to BioPortal, ensure that we are able to continue to extend the capabilities of the BioPortal resource, and enable us to fulfill the specific aims of our parent grant without fear of server failures. BioPortal is the world’s most widely accessed knowledge base of biomedical concepts and relationships. The availability of this new equipment will support both our enormous user community and the scientific goals of our parent award.

NIH Research Projects · FY 2025 · 2021-09

Abstract Relapse is the most important cause of mortality after allogeneic hematopoietic cell transplant (allo-HCT), but little research progress has been made in several decades. Chimeric antigen receptors targeting CD19 (CD19 CARs) redirect T cell effector functions to eliminate CD19-expressing leukemia and lymphoma cells. However, many patients still relapse. The candidate has preclinical data indicating the feasibility of using genome editing to delete the endogenous T cell receptor (TCR) to reduce the alloreactivity of donor CD19 CAR T cells, but it is unknown how these TCR knockout (KO) cells will function in vivo as anti-tumor agents, to what extent graft- versus-host-disease (GVHD) will result, or how engineering impacts T cell metabolism. This knowledge is essential for progress toward creating readily available “off-the-shelf” CAR T cells for patients with hematologic malignancies. The overall objectives of the proposed research are to determine how donor and third-party TCR KO CD19 CAR T cells impact immune reconstitution, GVHD, and graft rejection in preclinical models, as well as to understand how removing the TCR impacts the immunometabolism of these cells. The central hypothesis is that potent anti-tumor effects as well as negligible GVHD and graft rejection can be demonstrated preclinically by using TCR KO CD19 CAR T cells (either donor or third-party) following allo-HCT, to produce superior outcomes to conventional CD19 CAR T cells following allo-HCT. This hypothesis will be tested in the proposed Specific Aims. This work will provide ideal training for the candidate as she prepares for her long-term career goal to lead an independent laboratory studying cellular therapeutics and allo-HCT. Memorial Sloan Kettering Cancer Center has a renowned immunology program, and Dr. Marcel van den Brink, the candidate’s primary mentor, is a leader in immunotherapy research. Her co-mentor and advisory committee members have diverse and complementary expertise, and all have strong track records of mentoring independent scientists. The candidate and her mentoring team have developed a rigorous training plan designed to increase her knowledge base in: 1) development of next-generation CAR T cells; 2) bioinformatics and programming; 3) cellular metabolism, metabolic flux, mitochondrial function, and metabolic analyses in CAR T cells; and 4) professional development skills. The candidate will undertake training in these areas through coursework, workshops, and mentorship. This research project and training will provide the foundation to establish her future career as an independent physician-scientist. The proposed studies are expected to generate findings that will guide future genome engineering of CAR T cells. The candidate’s aim is to launch an independent research program designing “off- the-shelf” CAR T cells, which are expected to provide much improved therapeutic options for a range of hematologic malignancies.

NIH Research Projects · FY 2025 · 2021-09

Measurements of cortical field potentials are widely used throughout basic and clinical neuroscience, including in electroencephalography (EEG), electrocorticography (ECoG) and local field potential (LFP) recordings. However, the neural origins of field potentials remain poorly understood, due to a lack of techniques for dissecting how different classes of cells contribute to field potential signals. To overcome this longstanding barrier, our project applies fluorescent voltage-indicators and instrumentation for optical voltage-imaging that our team created earlier in the NIH BRAIN Initiative. These new tools will enable us to systematically identify the contributions of 12 different cell-types to neocortical field potential activity. To perform cell-type specific recordings of neural transmembrane voltage dynamics, we will express red and green genetically encoded voltage indicators in a wide set of different transgenic mouse lines, each of which allows selective gene expression in one of the pyramidal neuron or interneuron classes of the neocortex. Concurrent with optical recordings, we will perform traditional electrical recordings of cortical LFPs. These joint optical and electrical measurements will be the first of their kind and will yield important insights into how each neuron-type influences spontaneous and stimulus-evoked cortical field potential activity. Across our collection of mouse lines, we will conduct 3 novel types of recordings, each of which uses cutting-edge instrumentation for optical voltage-imaging in up to 2 cell-types at once in awake behaving mice: a) Fiber-optic voltage-sensing, for tracking the voltage dynamics of genetically defined neural populations; b) Wide-field voltage-imaging of voltage oscillations and waves across the cortex in specific cell-types; c) High-speed (1 kHz) optical voltage imaging of spiking dynamics in up to 2 neuron-types at a time. Further, to test the causal role of each neuron class in shaping cortical field potentials, we will also perform chemogenetic inhibition studies in each of the mouse lines. In these studies, we will silence each of the individual neuron-types and observe how the effective removal of this cell-type from cortical circuitry impacts both LFP activity and the population voltage dynamics of other neuron classes. Together, these groundbreaking studies will propel understanding of cortical field potentials in basic and applied neuroscience by providing fundamental insights into how different cell-types shape field potential dynamics. To help assure that our experiments optimally advance conceptual understanding in the field, our team includes 2 computational neuroscientists whose expertise lies in modeling the biophysics of cortical field potentials. To promote transparency and open-science, we will deposit all of the extensive datasets and analyses from our experiments into public repositories.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract Living cells require a constant supply of nutrients that provide energy and building blocks to support their vital activities and growth. Fluctuations in nutrient availability are inevitable. Thus, to survive, cells need to adapt to these changes by rewiring their metabolism. Studying this metabolic adaptation in lower organisms has revolutionized our understanding of biological systems. For example, the discovery of how prokaryotes respond to changes in the accessibility to lactose and glucose as a carbon source led to the concept of gene regulation after the identification of the lac operon. In eukaryotes the evolution of subcellular organelles provided an optimal environment for biochemical reactions to proceed. Moreover, this system allowed the eukaryotic cell to evolve additional strategies to acquire nutrients besides passive diffusion or transport across the cell membrane. Through the endo- lysosomal compartment, cells can scavenge nutrients from extracellular macromolecules, which provides them with metabolic flexibility to survive various states of nutrient availability by balancing the composition of their microenvironment with their nutrient demands. Nutrient acquisition strategies are fully exploited by malignant cells to survive the harsh tumor microenvironment. Pancreatic cancer, a lethal malignancy, is a paradigm of metabolic adaptation. Hypo- vascularization of pancreatic ductal adenocarcinoma (PDAC) limits the delivery of free nutrients and oxygen to cancer cells. To overcome nutrient scarcity, cancer and stromal cells rely on scavenging nutrients from intra- and extracellular macromolecules via autophagy and macropinocytosis, respectively. Both pathways converge on the lysosome, a cellular organelle that degrades macromolecules to recycle their nutrient content. Despite their essential role in cancer, studying lysosomes in highly heterogenous tumors in vivo is challenging because of the lack of tools that allow the functional profiling of lysosomal content during tumorigenesis at a cell-type-specific resolution. In this proposal, I will describe our novel approach to develop an innovative technology that allows the rapid capturing of lysosomes from specific cell types in the tumor to profile their metabolite, lipid and protein contents to understand how lysosomes in malignant and stromal cells mediate metabolic adaptation. We will also design a modular mouse model system that will allow the selective interrogation of the lysosomal response to major metabolic stressors that exist in the tumor microenvironment. Our innovative approaches combined with functional characterization of the lysosomal components using genetic tools will result in an unprecedent subcellular and cell-type-specific understanding of tumor metabolism. We believe that our work has the potential to revolutionize our understanding of metabolic adaptation in mammalian systems, and to identify vulnerabilities that can be exploited as novel therapeutic targets in pancreatic cancer.

NIH Research Projects · FY 2024 · 2021-09

Cellular Senescence Network: New Imaging Tools for Arthritis Imaging Senescent cells play a key role in the pathogenesis of major musculoskeletal diseases, such as chonic inflammatory joint disorders, rheumatoid arthritis (RA) and osteoarthritis (OA). Cellular senescence in articular joints represents a response of local cells to persistent stress that leads to cell-cycle arrest and enhanced production of inflammatory cytokines, which in turn perpetuates joint damage and leads to significant morbidities of afflicted patients. It has been recently discovered that clearance of senescent cells by novel “senolytic” therapies can attenuate the chronic inflammatory microenvironment of RA and OA, and thereby, prevent further disease progression and support healing processes. In order to identify patients who might benefit from these new senolytic therapies and to monitor therapy response, there is a significant unmet need in identifying and mapping of senescent cells in articular joints and related musculoskeletal tissues. To fill this gap, we propose to develop a new imaging biomarker that will significantly improve our capabilities to identify and characterize senescent cells in human musculoskeletal tissues. We have generated exciting preliminary data that show that 3-D-galacto-2-nitropyridine (PyGal), a known hydrophilic b-gal substrate, can be labeled with 18F-fluorine. Upon intravenous injection, 18F-PyGal enters senescent cells and is selectively cleaved by b- galactosidase, a senescence-specific enzyme in these cells. The trapped radiotracer can be detected with positron emission tomography (PET) and autoradiography, thereby serving as an imaging biomarker for senescent cells. We propose to introduce 18F-PyGal as the first clinically translatable radiotracer which can detect senescent cells in vivo, in bones and joints of animal models and human volunteers. In the initial UG3 phase of our project, we will demonstrate proof-of-principle of this new imaging technology in a mouse model of RA and a large animal model of OA. In the subsequent UH3 phase, we will scale, optimize and validate 18F-PyGal PET for mapping human tissues, first in human joint specimen and second in a first-in- human phase I clinical trial. At the end of the UH3 phase, we will have delivered a novel imaging tool that can visualize and quantify the presence and distribution of senescent cells in multiple musculoskeletal tissues directly, non-invasively and longitudinally in vivo. Results will be catalogized in a planned senescence cell atlas and shared with the cellular senescence network. Our 18F-PyGal-PET imaging tool will significantly improve upon state-of-the-art imaging technologies for the diagnosis of musculoskeletal disorders, can be integrated with other imaging technologies, such as MRI, and is ultimately capable of being scaled to map senescent cells in multiple human tissues in a high-throughput fashion. Since 18F-PyGal targets senescent cells in multiple different tissues and can be easily imaged with widely available medical imaging technologies, our proposed new senescence imaging biomarker can be expected to be used widely by tissue mapping centers and relevant research communities.

NIH Research Projects · FY 2024 · 2021-09

Allergy is a major world health challenge affecting 25% of people with a rising incidence. Peanut allergy alone affects 2.2% of school children in the US and can be life-threatening. Allergy is a complex disease, with both genetic and environmental factors contributing to risk. In order to pinpoint the underlying genetic risk variants, genome-wide association studies (GWAS) have been performed on hundreds of thousands of patients and controls, identifying >100 associated loci. The vast majority of hits are in poorly annotated noncoding regions of the genome and are thought to influence gene regulation. A major challenge for understanding allergy (and all complex diseases) is pinpointing the causal variant(s) and defining molecular mechanisms. The extensive follow up work required is often not undertaken and thus allergy GWAS rarely contribute to our understanding of disease etiology. In order to mine the rich resource of human disease associations, new methods are needed to systematically annotate regulatory effects. Existing catalogs are sparse and biased toward specific cell types (blood), contexts (steady state conditions), molecular mechanisms (perturbation of gene expression), and populations (Caucasians). Given the highly cell type and context specific nature of gene regulation, this limited window is unlikely to be sufficient for identifying most human risk variants. The most comprehensive effort to date to map regulatory effects is the Genotype- Tissue Expression Project (GTEx), which mapped loci that influence gene expression (eQTLs) in 54 tissues using autopsy specimens from hundreds of healthy individuals. Despite its scope, the GTEx catalog thus far explains only 11% of the genetic risk of complex disease, suggesting additional assays and specimens are needed to unearth the majority of regulatory effects contributing to disease. In this application I propose an innovative approach to systematically catalog the gene regulatory effects of genetic variants on a massive scale across diverse allergy-relevant cell types and patient specimens. Crucially, this scalable approach can accommodate cell stimulation conditions (e.g., allergen challenge), inclusion of diverse human ancestry groups, and is deployable on scant human tissue and blood specimens. By leveraging a single cell framework, we are able to probe rare cell populations that play essential roles as mediators of disease. I will apply this method to allergy relevant GTEx tissues as well as a large-scale allergy biobank representing heterogenous cases. This study is expected to provide a greatly expanded window into the biology of genetic loci linked to allergy and elucidate the potential of multi-omic single cell approaches, pathophysiological stimuli, and patient biospecimens to unearth missing complex disease heritability.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Intracortical brain-computer interfaces (iBCIs) can restore lost function for people with severe speech and motor impairment (SSMI) due to neurological injury or disease. Despite tremendous recent progress, iBCI performance remains well below that of able-bodied people. In prior NIH- supported research, our collaborative team developed a high-performance intracortical brain- computer interface (iBCI) that decodes arm movement intentions directly from brain activity. This technology has allowed people with SSMI to control a computer cursor with sufficient speed and accuracy to type at up to 8 words/min and has enabled full control of unmodified consumer devices using only decoded motor cortical activity. In the proposed U01 clinical research, we will take an important next step for the field: investigating neural ensemble encoding during complex tasks that only people are capable of performing (i.e., moving arbitrary combinations of limbs and body parts, and handwriting). This work will build upon decades of experience in studying the motor system in humans and non-human primates, with the end goal of advancing iBCI technology, and will be performed as part of the multi-site BrainGate consortium. We propose to study in detail, at 'coarse' and 'fine' scales, how the Precentral Gyrus (PCG; “motor cortex”) generates complex movements. We will base our investigations on two new key discoveries from our lab: 1) that a small area of the PCG encodes movements of all 4 limbs in a ‘compositional’ way, allowing differentiation of separate limb and movement encoding dimensions, and 2) that complex, dexterous movements such as handwriting can be accurately decoded from the PCG of people with paralysis. The results of these detailed fundamental neuroscience studies will enable us to then design and demonstrate two entirely new iBCIs: a system for helping restore continuous motion of the entire body in virtual reality (‘Whole-Body iBCI') and a system to substantially increase on-screen text generation speed (‘Handwriting iBCI’). Finally, we will continue to evaluate the safety profile of Utah-array based iBCIs through the ongoing BrainGate2 pilot clinical trial, with particular emphasis on critical neuroethics considerations. Upon completion, this project will advance both the capabilities of iBCIs for restoration of lost function and our understanding of the detailed neural mechanisms of complex movements.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT We will use Collaborative Cross (CC) mice to define how genetic traits influence innate and/or IgE-mediated responses of mast cells (MCs) to honeybee venom. In IgE-dependent allergic reactions, crosslinking of MC high affinity IgE receptors (i.e., FceRI) by the binding of bivalent or multivalent allergen to antigen-specific IgE activates MCs to secrete three major classes of products: 1) preformed mediators stored in cytoplasmic granules, 2) newly synthesized lipid-derived mediators, and 3) cytokines, chemokines and growth factors. These MC products are responsible for many of the signs and symptoms of allergic diseases. MC activation, with or without the involvement of IgE, is also thought to contribute to the inflammation, tissue damage, and even fatal shock induced by envenomation. Components of hymenoptera venoms (e.g., honeybee venom), pharmaceutical agents, and foods are the most common triggers for anaphylaxis in humans. Many people have been sensitized to hymenoptera venoms and some unfortunate individuals react after exposure to such insect stings with serious systemic reactions and even fatal anaphylaxis. However, recent experiments in mice and zebra fish demonstrate that MC-derived proteases can degrade animal venoms and diminish their toxicity. Also, IgE/FceRI-mediated MC activation can enhance the survival of mice challenged with honeybee venom or a snake venom, or with S. aureus bacteria. Yet the benefits of “allergic immune responses”, mediated by IgE/MC- dependent mechanisms, have not been widely recognized. While the exact mechanisms determining whether the outcomes of hymenoptera envenomation are detrimental or favorable have been elusive, we know that genetic factors can significantly influence the development, progression, and severity of allergy and anaphylaxis. We hypothesize that genetic traits, by modulating the strength and/or composition of MC responses to honeybee venom and/or the Th2-IgE-MC immune axis, can influence the outcomes of honeybee stings. In this project, we propose to use genetically diverse CC mice to identify genetic modifiers regulating MC functions in insect venom allergy. In Aim 1, we will screen a panel of CC mice for their susceptibility to the toxicity of honeybee venom, development and features of venom-specific type 2 immunity, and induction of MC activation with or without crosslinking of venom-specific IgE/FceRI. We will also perform quantitative trait locus (QTL) mapping of the venom-induced phenotypes in CC mice to identify distinct genetic loci and novel regulators associated with MC-dependent susceptibility vs. resistance to honeybee venom. In Aim 2, we will confirm, in mouse MCs, important regulators of MC functions that are identified by screening CC mice using QTL analysis to assess the involvement of these regulators in innate and/or IgE-mediated MC functions. We think that the identification of genetic modifiers that distinguish beneficial vs. harmful effects of innate and/or allergic immune responses to honeybee venom will improve the treatment of severe reactions to insect venom and probably other disorders in which IgE and MCs have a critical role. 1

NIH Research Projects · FY 2024 · 2021-09

Research Summary/Abstract Our goal is to decipher how a molecular-level event or property can create heterogeneous behavior within a population, and how this heterogeneity leads to advantages for the population as a whole that are not available to individual members. We propose to determine how sub-generational gene expression - not only of individual genes, but also of entire operons containing multiple genes with coordinated functions - creates mixed populations that are more fit to respond to various environmental cues. This proposal, which deeply integrates computational modeling and experimental measurement, arose out of our efforts in “whole-cell” modeling of E. coli, which were reported in Science earlier this year. The E. coli model has predicted a number of surprising behaviors; most relevant is the finding that a clear majority of the genes in E. coli are transcribed at a rate of less than once per cell cycle - a phenomenon we call “sub-generational gene expression”. Such expression can have negative consequences for individual bacteria, but benefits the bacterial population as a whole. Because bacteria are unable to reliably anticipate future conditions, the population must always be prepared for any environmental change - but no single bacterium is able to express all of the genes required to respond to any environment at sufficient levels. Instead, our working hypothesis is that the population is heterogeneous, comprised of individual members who are each prepared for a small number of possible environments. Thus, while no single cell is ready for all environments, as a whole the population is prepared for most eventualities. The colony is thus dominated by individuals, emerging stochastically via expression of sub-generationally expressed genes, who are the most fit to survive at any given moment. Our groups combine expertise in both whole-cell and agent-based models, and have been working towards whole-cell population simulations, in which hundreds or thousands of cells each run an instantiation of the E. coli model. Our Aims are to: (1) confirm that model-predicted genes are expressed sub-generationally; (2) computationally predict and experimentally determine the effect of operon structure on sub-generational expression of functionally related gene pairs; and (3) computationally predict and experimentally determine the phenotypic heterogeneity created by operon separation in cell populations. The most impactful and pioneering aspects of our proposal are that we will uncover a fundamental new role for operon structure in prokaryotic gene regulation; that we will produce an expanded whole-cell model of previously unseen complexity, as well as highly innovative new modeling technology; and finally, that this work will be the first to utilize a novel multi-scale simulation platform that combines whole-cell models with agent-based models, including the most exciting experimental demonstration of whole-cell and whole-colony modeling’s major potential: predicting large-scale emergent properties to generate insights into complex cellular behaviors.

NIH Research Projects · FY 2025 · 2021-09

1 ABSTRACT – OVERALL COMPONENT 2 3 The development of brain metastases, experienced by up to 40% of cancer patients, marks a clear inflection 4 point in survival and quality of life. The inaccessibility of brain tumor tissue has stymied progress in our 5 understanding and treatment of brain metastases, and patients are regularly excluded from clinical trials. The 6 Stanford Brain Metastasis Consortium has unified brain and cancer experts in the singular goal of improving our 7 understanding and treatment of brain metastases, a currently increasing yet underserved subset of cancer 8 patients. To accomplish the above goal, we have: (1) designed an organizational structure that supports 9 scientists in our integrated work; (2) developed highly innovative and complementary Projects to understand 10 and disrupt the cancer-neuro-immune axis supporting brain metastases; and (3) created NeuroPathology 11 and ToolKit Cores to make human specimens and cutting-edge technologies readily accessible to participating 12 scientists. We expect to identify and target key mediators of brain metastasis, with therapeutic benefit for 13 patients. 14 Little is known about the distinct mechanisms that drive tumor cells to the brain and allow them to grow in this 15 unique microenvironment, supported in part by normal brain cells. Streamlined access to human brain 16 specimens, combined with innovations in modeling and manipulation of the tumor microenvironment, create this 17 collaborative opportunity for fundamental advancement. Our expert, integrated team of productive collaborators 18 aims to understand how the intrinsic features of tumor cells (Project 1), resident microglia (Project 2), and the 19 systemic immune system (Project 3) contribute to the onset and progression of brain metastases. These projects 20 are facilitated by centralized access to human patient brain metastases samples (NeuroPathology Core), and 21 novel, multiplexed analyses and disease modeling (ToolKit Core). Our multidisciplinary physician Consultant 22 Network provides clinical insight and helps in the rapid translation of our findings into clinical trials for patients 23 with brain metastases. The Administrative and Data Management Core will provide the operational support 24 necessary to successfully achieve the goals of the program. Our Patient Advocates help to integrate and 25 communicate our work to the greater scientific and patient communities. 26 We have formed one of the few groups with the expertise, interest, and capacity to address the underlying 27 mechanisms of and therapeutic opportunities for brain metastases. Only through this combined synergy would 28 this project be possible. These innovative methods will ensure our findings are reflective of and translatable to 29 the human disease, enabling our multidisciplinary team to lay the foundation for diagnostic and therapeutic 30 advancements.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Crohn’s disease and ulcerative colitis are forms of inflammatory bowel disease (IBD) that affect more than 6.8 million patients worldwide. Because no cure is available to date, treatment is limited to reducing IBD symptoms such as severe diarrhea, weight loss, fatigue and pain. Although the exact mechanisms that mediate the pathogenesis of the disease are unclear, excessive proteolysis in the gut combined with dysregulated signaling of protease-activated receptors (PARs) have been identified as important drivers of IBD and additional gastrointestinal (GI) diseases. PARs are a unique class of four eukaryotic G-protein coupled receptors (GPCRs) that are directly regulated by proteolytic cleavage of a peptide sequence in the extracellular N- terminal domain (NTD). Cleavage reveals a tethered activating ligand or alters the receptor conformation to induce activation. PAR-signaling can promote inflammation by disrupting the integrity of the intestinal epithelial barrier, which under physiological conditions allows permeability of nutrients but restricts the entry of bacterial pathogens and toxins. Many GI diseases are accompanied by loss of barrier function and dysbiosis of the gut microbiome. Proteases derived from commensal bacteria are likely to be important regulators of gut homeostasis and pathogenesis, thus making them potential therapeutic targets. However, it is unclear which extracellular proteases are produced by commensal strains in the gut and how these enzymes affect health and disease by proteolysis of co-localized PARs. We hypothesize that beneficial commensal bacteria secrete proteases that keep excessive inflammation in check by basal activation or proteolytic desensitization of PARs. Conversely, pathobiont bacteria species secrete proteases that promote inflammation via increased PAR activation. Therefore, proteases produced by the gut microbiota as well as pathogenic bacteria have the potential to be valuable new therapeutic targets for the treatment of various forms of IBD. To test our hypothesis, we will develop a robust in vitro assay to broadly screen for proteases with PAR-processing activity in both commensals and pathobiont bacterial species. We will then identify specific PAR processing proteases and assess their specific roles in regulating epithelial barrier integrity and inflammation using cell culture systems and gut organoids. Finally, we will establish the therapeutic relevance of the identified proteases by confirming their presence and elevated activity in clinical samples isolated from patients with active IBD. Ultimately, this work will identify specific PAR processing proteases produced by bacterial strains and define the mechanism by which they impact the pathogenesis of IBD.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Lengthening the small intestine is a potentially curative therapy for patients with intestinal failure due to short bowel syndrome. Short bowel syndrome is the end result of devastating diseases that affect the small intestine. Patients with short bowel syndrome have malabsorption and cannot eat normal meals. The current therapy for short bowel syndrome consists of providing parenteral nutrition, optimizing the health of the remnant intestine, and enhancing intestinal adaptation. While parenteral nutrition has dramatically improved their survival, many patients with short bowel syndrome cannot be weaned from parenteral nutrition and develop end stage liver disease. Over the last decade, we developed and refined devices to lengthen the small intestine. By applying a gradual force to a segment of the small intestine separated from continuity, we were able to triple the length of the small intestine in rats and pigs. There was a net growth of the intestinal cell mass and an increase in the absorptive surface area. The lengthened segment was functional when it was restored back into intestinal continuity. These studies demonstrated the potential of mechanical intestinal lengthening as a novel treatment of patients with short bowel syndrome. In this proposed research, we will obtain data on spring devices that will lengthen the intestine that are still in intestinal continuity in different size animals. We will also develop ways to maximize intestinal lengthening with multiple springs applied repeatedly. Lastly, we will employ the spring in a porcine short bowel model. If successful, these studies will lead to a Phase 1 clinical trial of these devices in patients with short bowel syndrome. This approach will revolutionize the treatment strategy of patients with short bowel syndrome.

NIH Research Projects · FY 2025 · 2021-09

CLCs (the “Chloride Channel” family) are anion-selective transporters and channels ubiquitous in all organisms. Among them, CLC-Ka and CLC-Kb are essential for Cl– and water handling in the kidney. CLC-Ka is localized to the thin ascending limb, where it helps to establish the steep solute gradient in the inner medullary interstitium that drives renal water reabsorption. As such, CLC-Ka is a potential drug target for treating pathologic water retention (hyponatremia) that frequently complicates the management of patients with hypertension, heart failure, or cirrhosis. A specific CLC-Ka inhibitor would be invaluable for validating CLC-Ka as a drug target for manipulating renal water excretion. In this project, we leverage recent breakthroughs to develop selective CLC-Ka inhibitors. The first breakthrough is our discovery of BIM1, a substituted benzimidazole that displays >20-fold selectivity for CLC-Ka over its closest homolog CLC-Kb. The synthetic accessibility of BIM derivatives makes them well suited for further development. The second breakthrough is the revolution in cryo-electron microscopy, which enables high-resolution structure determination of challenging targets, including ion channels. A molecular structure of the BIM/CLC-K complex will identify which regions of the BIM molecule must be retained for potency/selectivity and which may be modified to improve pharmacokinetic properties. Guided by this information, we will use a medicinal chemistry approach to develop BIM derivatives with optimized potency, selectivity, and pharmacokinetic properties. Optimized BIM derivatives will be tested for in vivo efficacy.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY. Peripheral metabolic tissues engage in pervasive inter-organ crosstalk to maintain systemic glucose and lipid homeostasis. This long-range intercellular communication is mediated by blood borne, secreted polypeptides. Over the last decade, there has been renewed interest in identifying additional proteins secreted from metabolic tissues. This is because the collection of secreted proteins (e.g., secretome) from metabolic cell types is large and also poorly characterized, and therefore many additional polypeptides that mediate peripheral tissue crosstalk likely remain to be discovered. It is not unreasonable to imagine that many of these orphan factors represent new signaling pathways and consequently potentially new therapeutic targets for obesity, diabetes, and related metabolic disorders. Typically, approaches to this problem have relied on surrogate methods that attempt to predict, rather than directly measure, in vivo polypeptide secretion events. In recent work, we have introduced an in vivo chemical methodology that enables a radically different strategy: to measure metabolic tissue secretomes directly in living animals (Wei et al., Nat. Chem. Biol. 2020). Importantly, this chemical strategy provides unique insights into the composition and dynamics of secretomes in mice that could not have been predicted by existing in vitro or computational approaches. This proposal seeks to further develop these chemical methodologies with the goal of generating a complete endocrine map of the secreted polypeptides that mediate peripheral metabolic tissue crosstalk. To achieve this goal, we will (1) produce a 6 organ, 15-cell type atlas of peripheral metabolic tissue polypeptide secretomes and determine how these secretomes are dynamically altered by metabolic perturbations such as obesity, diet, environmental temperature, and physical activity; (2) develop new in vivo chemistries that enable high-resolution mapping of secreted polypeptide fragments produced via proteolytic cleavage events; and (3) integrate metabolic tissue secretomes into endocrine circuits through in vivo chemical pulse-chase approaches. Successful completion of this high-risk, high-reward project will provide a chemical toolbox for dissecting cellular secretomes, open potentially important new areas in tissue crosstalk, and ultimately enable the long-term vision of “capturing” the pathways of tissue crosstalk to combat obesity, type 2 diabetes, and related metabolic disorders.

NIH Research Projects · FY 2025 · 2021-09

Overall - Interaction of external inputs with internal dynamics: influence of brain states on neural computation and behavior Project Summary A central challenge in neuroscience involves understanding how assemblies of cortical neurons, comprised of different cell types and inhabiting different layers, work together to generate coherent dynamical internal states, that then interact with external sensory inputs to generate state-dependent behaviors on a moment-by-moment basis. Key impediments to meeting this foundational challenge include lack of adequate technological and computational tools to monitor, control, identify and model neural state dynamics emerging from cortical cell assemblies spanning multiple cortical cell-types and layers. We propose to develop an unprecedented confluence of technology and computation to achieve such capabilities by building on our team’s significant prior work. In particular, our combined technology and computation platform will enable us to: (1) perform volumetric imaging of thousands of cortical cells during behavior to collect both relevant spatiotemporal activity patterns and 3D positioning; (2) simultaneously write arbitrary spatiotemporal patterns into tens to hundreds of individually identified cells at millisecond temporal resolution using 2-photon multiSLM methods; and (3) using hydrogel tissue-chemistry and single-cell sequencing methods, obtain deep molecular cell-type information in the same neurons that were both measured and controlled during behavior. This unprecedented simultaneous read/write/cell-typing technology will be tightly integrated with computational methods that can: (1) employ state of the art systems identification methods to identify and extract neural states and the dynamical laws governing their interactions with external inputs; and (2) amongst the astronomical number of possible spatiotemporal stimulation patterns, predict interesting ones that can best refine models, yield conceptual insights, and yield the capacity for optimal control of cortical circuit dynamics, with potential clinical relevance. This combined technology and computation will empower next-generation experiments that allow us to learn the dynamical language (in terms of state space dynamics) of cortical circuits, play back modified versions of this language for both insight and control, and understand how this language emerges from the concerted activity of multiple cell-types across layers. Our technology/computation platform will be validated in multiple experiments across species and brain regions, guided by deep and long-standing theories of internal state dynamics in computational neuroscience. Throughout, new methods will be collaboratively validated in the diverse preparations of our experimental labs (such cross-cutting interactions are shown in blue text). In particular we will focus on testing theories underlying several foundational classes of neural computation: (1) ability of sensory networks to generate accurate percepts by detecting and amplifying weak sensory inputs amidst spontaneous background activity; (2) Bayesian integration of multisensory inputs to convert sensorimotor experiences into internal estimates of external state variables and their uncertainty; and (3) triggering and maintenance of discrete internal attractor states capable of controlling stable behavior.

NIH Research Projects · FY 2025 · 2021-09

The US is in the midst of a maternal health crisis. The US has worse maternal mortality (MM) than any other high-income country and is the only one for which the rate is increasing. Severe maternal morbidity (SMM), which encompasses conditions that put pregnant women most at risk of dying, doubled in the last two decades. This proposal focuses on SMM, as a sentinel outcome leading to MM, yet 100 times more common. Most prior research on SMM has focused on proximal clinical factors (primarily related to co-morbidities and obstetric management); these factors alone are insufficient for understanding SMM. The objectives of this proposal are: 1) develop a causal pathway framework to understand how social determinants and more proximal health-related factors together contribute to SMM risk, and 2) use this framework to identify actionable strategies to reverse current trends. We will create a unique dataset that harmonizes 4 years of data from 6 states on 4.4 million births and 66,000 women with SMM. These states collectively include 1 in 4 US births and sufficiently varied social environments to disentangle complex multi-level drivers of maternal health. Our focus is on social determinants from two specific domains: community resources and health care access, characterized at the county, neighborhood (census tract), and individual level. Health-related intermediaries include birth hospital quality of care, mode of birth, and maternal morbidities (eg, hypertension, anemia). These domains and intermediaries were selected for their known relevance to maternal health. In addition, we will evaluate impacts of 3 Quality Improvement (QI) collaboratives on SMM, which were designed to improve specific aspects of hospital quality of care and implemented by state-wide perinatal quality collaboratives (PQCs). Our Specific Aims are: 1) Assess the relative and joint contributions of multi-level social determinants to SMM; 2) Identify potential health-related mechanisms by which multi-level social determinants affect SMM risk by conducting causal pathway analyses; and 3) Evaluate the impact of 3 QI collaboratives designed to reduce SMM (3a), and apply what we learn from the causal framework developed in Aims 1 and 2 to these state-specific contexts, to identify state-specific strategies for preventing SMM (3b). We currently have very limited understanding of how social determinants contribute to SMM. The proposed research will fill this important knowledge gap, which is an essential part of realizing sustainable improvement of maternal health.

NIH Research Projects · FY 2025 · 2021-09

Co-Clinical Research Resource for Imaging Tumor Associated Macrophages ABSTRACT The development of quantitative imaging (QI) methods for monitoring cancer therapy response has been transformative for the development of novel cancer therapeutics. QI efforts have positioned imaging as a key element in the design of clinical trials for cancer therapy response assessment. Hence, there is increased interest by the academic and industry sectors to use web accessible research resources and develop consensus approaches to validate QI methods for the next generation of clinical trials. This is particularly relevant for assessment of cancer immunotherapy, since immunotherapy does not lead to a decrease in tumor size, at least not in the immediate post-treatment phase. Therefore, we urgently need new QI tools that can monitor tumor response to novel immunotherapies. The overall goal of our project is to optimize and validate preclinical and clinical imaging techniques for in vivo quantification of tumor associated macrophages (TAM) in osteosarcomas. Recent evidence has shown that the abundant TAM response in the microenvironment of bone sarcomas can be employed to directly attack cancer cells. Blockade of the cell surface molecule CD47 expressed on sarcoma cells resulted in activation of phagocytic anti-cancer activity from TAM and efficiently eradicated tumor cells in mouse models of osteosarcoma. 26 Preclinical studies have been finalized and a multi-center phase I clinical trial is currently being planned, with expected start date in 2021. To solve the unmet clinical need for a QI tool to monitor response to new TAM- modulating therapies, our team developed a quantitative TAM imaging test, which relies on intravenous injection of the iron supplement ferumoxytol. Ferumoxytol is composed of iron oxide nanoparticles, which are phagocytosed by TAM and can be quantified with T2*-weighted MRI 45. Since ferumoxytol is FDA-approved and can be used “off label” as a TAM biomarker, it is immediately clinically available. We showed that ferumoxytol-MRI can detect TAM in osteosarcomas in mouse models 21 and patients 25. Through this project, we will (a) optimize and validate pre-clinical quantitative imaging methods for TAM imaging in an established mouse model of osteosarcoma, (b) implement the optimized methods in a co-clinical trial in patients with osteosarcoma who are undergoing immunotherapy with CD47 mAb, and finally (c) populate a web-accessible research resource with all the data, methods, and results collected from the co-clinical investigations. Developing the proposed imaging test could represent a significant breakthrough for clinicians as a new means for treatment stratification and new gold-standard imaging test for predicting treatment response of novel immunotherapies. Our QI imaging test could be utilized to compare the efficacy of different immune-modulating therapies in preclinical settings and translate the most primising candidates to the clinic. Since the development of new therapeutic drugs is expensive and take years to complete, the immediate value and heath care impact of our QI tool could be immense.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT – OVERALL COMPONENT Benign prostatic hyperplasia (BPH) is the most common cause of urinary symptoms in older men, yet we understand little about its origins, drivers of growth, and how it causes lower urinary tract symptoms. Since BPH-caused Lower Urinary Tracts Symptoms (LUTS) appears unique to man, we propose a highly integrated project to create an atlas encompassing the molecular, cellular, microenvironmental, histological and macroscopic dimensions of human BPH. Definition of the features responsible for growth and progression of BPH could ultimately lead to new therapeutic approaches to treat or prevent BPH. Our overall goal is to expand research in benign urology to improve our understanding and treatment of urological diseases. The components of the Stanford O’Brien Urology Research Center include: The Administrative Core is based in the Department of Urology and directed by Dr. James Brooks, an experienced clinician and translational scientist in prostate disease who will administer the Center to ensure the scientific and training goals are realized and interface with the NIDDK and Urology Research Consortia. He will be advised by an Internal and External Advisory Board, to ensure progress is made and to provide scientific advice to ensure success. He will meet with the Investigator Committee to formulate plans, integrate findings between projects and allocate Project and Core resources to ensure projects succeed. The Biospecimen/Bioimaging Core, directed by Dr. Robert West provides critical support to projects of the Center by providing human BPH tissues with deidentified data, generates histological images and manages these and the MRI images and provides Multiplexed Ion Beam Imaging (MIBI) and data analysis for Projects 1, 2 & 3. The Core also provides this service to the O’Brien Urology Centers and Urology Disease Centers. Project 1 seeks to define the role of fibroblast subtypes in the development and progression of BPH. Project 2 characterizes the immune microenvironment and investigates how it is shaped by the stromal cells and how it influences the stromal and epithelial compartments of BPH. Project 3 uses MR Images with associated International Prostate Symptom Scores (IPSS) and Bothersome Indices (BI) to construct 3D models of BPH overlayed with histology. These models serve as an atlas for integrating stromal and immune microenvironment data and gene expression subtypes and will provide a means to test how molecular, cellular, microenvironment, histological and radiologic features and their heterogeneity relate BPH to LUTS. These projects serve as the nucleus for training of undergraduate, graduate, and post graduate students to become the next generation of leaders in urological science.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Dilated cardiomyopathy (DCM) is a leading cause of heart failure and the leading reason for heart transplantation. Major gaps exist in our understanding of the pathophysiology of DCM and mutations in the gene that encodes the nuclear envelope proteins lamin A and C (LMNA) are considered to be the most common cause of DCM. However, the molecular mechanisms that underlie “cardiolaminopathy” remain elusive, and it is unknown why mutations in this ubiquitously expressed gene have such a disproportionate effect on the heart. Using induced pluripotent stem cell (iPSCs)-derived endothelial cells (iPSC-ECs), we recently studied a family affected by DCM due to a frameshift variant in LMNA, which showed endothelial dysfunction (Sayed et al. Science Translational Medicine, 2020). This EC dysfunction could be reversed by upregulating Krüppel-like Factor 2 (KLF2) by treatment of iPSC-ECs with a subset of statins, including lovastatin. Importantly, this improvement in EC dysfunction had a positive effect on co-cultured iPSC- cardiomyocytes (iPSC-CMs) from cardiolaminopathy patients, indicating an intricate crosstalk between the ECs and CMs in LMNA cardiomyopathy. Despite impressive progress, little attention has been given to the potential importance of cell-to-cell signaling between ECs and CMs, despite the fact that ECs serve a paracrine function to enhance signaling in CMs, especially in context to pharmacological stimulation. This knowledge gap impedes our comprehensive understanding of organ dysfunction at a multi-cellular level. The overarching goal of our proposal is to use a multidisciplinary approach that integrates human iPSCs, bioengineering tools, genome editing, and NGS to gain novel insights into the pathogenesis of DCM. Using human iPSCs, we propose to decipher the impaired cross-talk between ECs and CMs in LMNA cardiomyopathy and elucidate the beneficial class effects of statins in improving the EC-CM signaling as a key factor in regulating cardiac function. We will pursue three specific aims. In Aim 1: we will establish an experimental platform to study the genotype-phenotype association of LMNA mutations on ECs and CMs. For this, we will recapitulate the EC-CM crosstalk in LMNA iPSC-derived cells with 3D engineered heart tissues (EHTs). In Aim 2: we will decipher the mechanism of EC-CM crosstalk in LMNA iPSC-derived EHTs using single-cell approaches (scRNA-seq and scATAC-seq). In Aim 3: we will validate the key regulatory players of EC-CM crosstalk in LMNA cardiomyopathy by using CRISPR technology and zebrafish animal model. We have provided compelling preliminary data to support the soundness of our hypothesis-driven research proposal, and we are well positioned to achieve the project goals within five years. If successful, our studies will provide a new paradigm for understanding the pathogenesis and treatment of familial DCM.

NIH Research Projects · FY 2025 · 2021-09

Abstract Speed of processing training (SOPT), practicing to enhance the information processing efficiency while performing various perceptual and cognitive tasks, is the widest examined type computerized cognitive training among aging populations, including those with mild cognitive impairment (MCI). However, the efficacy of existing SOPTs in maintaining or improving older adults' cognitive health greatly varies across individuals. Our preliminary studies identified that the flexibility of autonomic nervous system (ANS) is associated with learning and cognitive and neural gains in existing SOPT in older adults with MCI. Our premise is that adaptation capacity, which is primarily reflected by ANS flexibility, is a key contributor to the neuroplasticity underlying broad and sustained effects of cognitive interventions. In this proposed study, we will combine this ANS response profile with the traditional learning index to develop a “personalization engine”, called pSOPT, for better reflecting individual adaptation capacity, and to test pSOPT's feasibility (R21 phase) and preliminary effect (R33 phase) in MCI. In R21 phase (intervention refinement, Stage Ia), we will establish a “personalization engine” for the SOPT by taking advantage of unique information derived from ANS assessment that links to learning and test the feasibility in MCI. Advanced time-series data analysis methods (e.g., shapelet analysis) will be used to develop a prototype of pSOPT based on the previously identified ANS shapelet. Compliance and usability of the pSOPT will be examined using interviews, questionnaires, and recorded performance data using a single group design in older adults with MCI (n = 10). Specific aims include (1) Use the identified ANS shapelet to develop a “personalization engine” that can modulate SOPT according to real-time measures of ANS; (2) Examine the feasibility of administering the pSOPT. In R33 phase (pilot test, Stage Ib), we will test the preliminary effects of the pSOPT in MCI. A pilot double-blinded randomized controlled trial (RCT). An MCI group (N = 50) will be randomized into a 6-week pSOPT (n = 25), or attention control (n = 25). Cardiac monitor-based ANS signals will be recorded throughout training sessions across groups. Learning is indexed by performing accurately across a consecutive set of trials in training tasks. Cognitive battery (measuring cognitive gains) and BOLD fMRI-based brain function (measuring neural gains) will be assessed at baseline, post training (7-week), and short-term follow-up (3-month); neurodegeneration (T1MRI and blood- based Alzheimer's pathology) assessed at baseline. Specific aims include (1) Compare changes of cognitive and neural gains between groups; (2) Explore whether pSOPT will enhance ANS flexibility in supporting cognitive gains against baseline neurodegeneration. This study is a prerequisite to efficiently launch an efficacy trial of pSOPT in slowing dementia progress.

NIH Research Projects · FY 2025 · 2021-09

Project Summary The overall goal and singular focus of our proposed Center Without Walls is to unravel the mechanisms of FTLD-TDP. We have formed a diverse interdisciplinary team to tackle this challenge. Our team brings together experts in genetics, genomics, neuroscience, neurology, and pathology. We have FTLD experts as well as outsiders who bring new perspectives and key resources and approaches to the field. Our team has also recently made an unexpected discovery of a new splicing target of TDP-43, which provides a direct and surprising connection to FTD human genetics and will be a launching pad for defining the mechanisms of FTLD-TDP. We posit that mis-splicing events caused by TDP-43 dysfunction may well be the earliest events in the process. Our vision is to create a Center dedicated to providing unprecedented access to TDP- 43 function, even before it is depleted from the nucleus. Rather than have human genetics as an afterthought or addendum, we endeavor to have the genetics deeply integrated in our program from Day 1. Our Center will make all of the data and code we generate freely available via a web portal that contains high resolution images of human brains across different subtypes of FTLD- TDP showing, at cellular resolution, TDP-43 localization along with a panel of cryptic splicing readouts as sensitive beacons of TDP-43 activity in different brain regions. This will empower the broad FTLD research community to generate (and test) new hypotheses about disease mechanisms and to have at their disposal sensitive biomarkers. Our Center will launch multimodal efforts to 1) comprehensively discover the TDP-43 splicing targets relevant to human FTLD-TDP; 2) define the mechanisms by which TDP-43-dependent cryptic exon splicing events contribute to neurodegeneration, using model systems and human tissues; 3) harness these novel cryptic exons to generate highly sensitive and specific biomarkers for the FTD field; 4) innovate genomics analysis methods to integrate human genetics data and RNA sequencing data and make these resources available to the community to discover how genetic risk factors for FTD contribute to cryptic exon splicing and vice versa. We strongly suspect that we will discover the cryptic exon splicing code that serves as the Achilles’ heel to drive neurodegeneration in FTLD-TDP.

NIH Research Projects · FY 2025 · 2021-09

The broad objective of this project is to develop imaging instrumentation and algorithmic technology to perform non-invasive, real-time, in-vivo, 3D, virtual H&E biopsies. One in four people worldwide will ultimately be affected by cancer. Surgical removal is the main treatment for most solid cancers. The surgeon is tasked with the delicate balancing act of excising enough tissue to avoid leaving behind residual cancer cells while not removing too much tissue, which can harm organ function. This is particularly important for brain tumors, the most common type of solid tumor in children and the leading cause of pediatric cancer mortality. The gold standard for detecting most solid cancers and confirming tumor margins is hematoxylin and eosin (H&E) stained tissue sections, which require an invasive biopsy procedure. Unfortunately, current non-invasive in-vivo imaging modalities do not produce images of comparable usefulness. We propose a novel imaging modality called a "virtual H&E biopsy'' that would generate H&E-like images of living tissue in real time. non-invasively up to 1 mm into the tissue. This imaging modality would be able to provide real-time diagnosis of tumor margins and invasiveness by scanning a large tissue area for residual cancer cells. Such information would guide treatment decisions for diseases such as brain and skin cancer. Beyond its clinical benefits, this technology can also be used for research into tumor development and tumor responses to treatment by providing in-vivo H&E-like images of healthy and tumorous tissue microstructures changing over time. To generate virtual H&E images, we will optimize a new imaging instrument we have developed based on optical coherence tomography (OCT) and image translation by a generative adversarial neural network (GAN). The key breakthrough enabling us to train a GAN to generate virtual H&E images is a technique called optical barcoding, which we used to obtain a dataset of OCT images and corresponding real H&E images aligned to single-cell precision. We have demonstrated this virtual H&E system with ex-vivo human skin tissue samples. For the proposed project, we will first train a GAN to generate virtual H&E images of healthy mouse brain tissue and glioblastoma mouse brain tissue ex-vivo (Aim 1 ). Second, we will use transfer learning to retrain the GAN to generate virtual H&E images of mouse brain tissue of in-vivo OCT scan (Aim 2a), and track for the first time how H&E images change as a mouse glioblastoma tumor develops (Aim 2b}. Finally, we will assess whether the GAN can be retrained across species by applying transfer learning on the mouse-brain trained GAN and use it to generate a virtual H&E biopsy of ex-vivo low-grade human glioma (Aim 3). To the best of our knowledge, this will be the first time transfer learning has been applied across species for biomedical images. Such transfer learning can accelerate virtual biopsy research since mouse samples are significantly easier to obtain and handle, thereby opening up applications in locations where acquiring a human dataset for training a virtual biopsy GAN would be difficult or impossible to achieve (e.g., the retina).

NIH Research Projects · FY 2025 · 2021-09

Abstract/Project Summary Metastatic breast cancer and relapse following therapy are dependent on (1) development of intrinsic resistance to targeted and endocrine therapies, and (2) resistance to recognition and destruction of cancer cells by the immune system. The Stanford Breast Metastasis Center (SBMC) is focused on (1) quantifying the timing of metastatic dissemination in breast cancer (2) functionally delineating the contribution of cellular and microenvironmental crosstalk on metastatic proclivity, and (3) characterizing the mechanisms of responses by metastatic cells to therapies. In order to achieve these goals, mechanistic computational models that capture dynamic and emergent tumor cell intrinsic and extrinsic properties are needed as are clinically annotated longitudinal tissue cohorts and experimental models that capture disease heterogeneity. The SBMC addresses each of these outstanding challenges. First, we have established an unparalleled collection of clinically annotated breast cancer cohorts sampled through treatment and metastasis, including both prospective and retrospective longitudinal cohorts, with multiple metastatic sites. We leverage a living biobank of breast cancer patient- derived organoids (PDOs) from primary tumors and metastases that recapitulate the heterogeneity of disease, high-risk of relapse subgroups and tumor-immune interactions and greatly facilitating the proposed functional studies. We characterize these vast tissue resources and model systems using state-of-the-art molecular profiling technologies to probe tumor tissue in situ at single cell and subcellular resolution. Specifically, with Multiplexed Ion Beam Imaging by Time of Flight (MIBI-TOF) and matrix-assisted laser desorption ionization imaging (MALDI) we simultaneously visualize the composition, lineage, function and spatial distribution of tumor and stromal cell populations and perform co-registered analysis of the glycome. We integrate these data within the genomic landscape of metastatic disease and analyze these data within robust machine learning and computational frameworks to uncover disease dynamics and features associated with clinical outcomes. Lastly, we conduct genome-scale CRISPR screens in 3D breast cancer models to systematically define oncogenic dependencies, therapeutic vulnerabilities and macrophage-tumor cell interactions. This integrated systems biology and functional genomics approach will contribute to a quantitative and mechanistic understanding of metastatic breast cancer and the dynamic relationship between tumor cells and the host, with implications for therapeutic targeting.

NIH Research Projects · FY 2025 · 2021-09

Abstract Neuropeptides have a critical role in modulating sleep and wakefulness and offer unique opportunities to treat sleep disorders. Among them, Neuropeptide S shows outstanding features: i) Administration of NPS increases wakefulness and reduces anxiety; ii) Neuropeptide S knockout mice show display increased NREM and anxiety; iii) Mutations of the NPS receptor that give rise to overactive signaling result in short sleep in humans and mice; iv) Expression of NPS is restricted to a few thousand neurons distributed across five main clusters in the basomedial amygdala, dorsomedial thalamus, Kolliker-Fuse/parabrachial area, pericoerulear region and nucleus incertus. These regions have been directly or indirectly associated with arousal and anxiety, but the detailed mechanisms as to how the modulate sleep architecture are unknown. We have recently generated a new line of mice expressing cre recombinase under the control of the endogenous NPS gene promoter (NPS-IRES-cre mice). Here we propose to use these mice and a combination of circuit mapping tools to decipher the mechanisms by which NPS modulates sleep/wake cycle. First, we will use viral-mediated tracing to determine if the five clusters of NPS+ neurons are interconnected, and their anatomical relationship with known arousal circuits. In a second aim, we will use fiber photometry to determine the activity profiles of the five clusters of NPS cells across the sleep/wake cycle and in response to stress and positive emotional stimuli. We will also determine which arousal circuits are activated by optogenetic stimulation of NPS, and which circuits activate NPS neurons. We will also assess whether NPS stimulation affects locomotor activity, anxiety, core body temperature and other physiological variables that may confound the arousal effect. In aim 3, we will test whether individual clusters of NPS neurons are necessary for NPS’s effects on sleep by using opto and chemogenetic inhibition. Finally, we will use a CRISPR-based approach to introduce NPS gene mutations in individual NPS+ cell clusters and determine whether NPS release in these brain regions is essential to control sleep/wake architecture and anxiety behaviors. The results from these experiments will shed new light into the function of NPS and NPS+ neurons, as well as the interconnection between them and arousal circuits. These data may lead to improved treatments of neuropsychiatric disorders associated with imbalances in arousal systems.

NIH Research Projects · FY 2025 · 2021-09

Celiac disease (CeD) is a gluten-induced, HLA-DQ2 or -DQ8 dependent inflammatory disorder of the small intestine for which no non-dietary therapy is available. Transglutaminase 2 (TG2) is the target of CeD-specific autoantibodies and is also involved in disease pathogenesis. In a CeD patient TG2 catalyzes the formation of deamidated gluten peptides that bind to HLA-DQ2/8 with high affinity and are recognized as epitopes by disease- specific CD4+ T cells. However, the location where TG2 exerts its pathogenic action is unknown. The overarching hypothesis of our proposal is that TG2 derived from enterocytes shed into the intestinal lumen is the source of pathogenically relevant enzyme in CeD. This luminal TG2 reacts with gluten peptides to form covalent complexes recognized by TG2-specific B cells in Peyer’s patches. In turn, these B cells present gluten antigens to disease-specific T cells, while also deriving help from these T cells. This hypothesis can explain how TG2 autoantibodies are formed in CeD. Three Aims involving in vitro and in vivo studies are designed to test our hypothesis, while taking advantage of the complementary capabilities of the three collaborating laboratories. Specific aim 1: Two key features of the above hypothesis will be tested at a biochemical level. First, the ability of antigenic gluten peptides to form metastable covalent intermediates at the TG2 active site will be probed. Second, a novel isotope labeling assay will be developed to verify that luminal TG2 in the mouse intestine can recognize and deamidate dietary gluten. Under this Aim we will also engineer a gut-impermeable TG2 inhibitor, which will be used in Aim 3 to validate the pathogenic role of luminal TG2. Specific aim 2: Using shed enterocytes collected from the human jejunal lumen as well as human organoid cultures, we will demonstrate that intestinal epithelial cells harbor abundant catalytically competent TG2 at the time of shedding. The ability of human enterocyte-derived TG2 to form covalent adducts with antigenic gluten peptides will also be verified. Our organoid cultures will also be used to identify disease-relevant environmental factors that are most effective at increasing steady-state TG2 activity in the small intestinal lumen. Specific aim 3: This Aim will test the pathogenic role of enterocyte-derived luminal TG2 in two mouse models of CeD. In one model of established CeD, we will test whether TG2-gluten peptide covalent complexes in the gut lumen can stimulate collaboration between TG2-specific B cells and gluten-specific T cells. In another model where CeD can be induced by feeding gluten, the requirement for TG2 expression in enterocytes or alternately in the myeloid compartment will be tested. First-generation TG2 inhibitors are already undergoing human clinical trials. If the overarching hypothesis of our proposal is correct, it will establish that TG2 inhibition in the intestinal lumen is sufficient to protect against gluten- induced villous atrophy in CeD. Not only will this motivate the design of a safer and more efficacious therapies, but our gut-impermeable inhibitors may also serve as next-generation drug prototypes for this lifelong disorder.