Calvin Kuo - US grants

DESCRIPTION (provided by applicant): Anti-angiogenic therapy represents an emerging and promising modality for the treatment of malignancies, although controlled comparisons between putative angiogenesis inhibitors has been lacking. Gene therapy approaches are well suited for the experimental delivery of diverse anti-angiogenic proteins, given the ease of vector construction versus recombinant protein, and the convenience of single injection viral administration. We have exploited an adenoviral approach to create an array of adenoviruses expressing anti-angiogenic proteins such as soluble ectodomains of the Vascular Endothelial Growth Factor (VEGF) receptors Flk 1, Flt 1 and neuropilin, as well as the anti-angiogenic factors endostatin and angiostatin. Using these viruses, we have performed the first comparison between VEGF-based and non-VEGF-based anti-angiogenic proteins, and have found superior antiangiogenic and anti-tumor activity with soluble Flk 1 and Flt 1 treatment over tumor-derived proteins such as endostatin and angiostatin. The soluble FIk 1 and Flt 1 adenoviruses exhibit broad-spectrum suppression of numerous human and murine tumors in both subcutaneous and orthotopic models. The current proposal extends upon these findings in several respects. First, we will explore the hypothesis that VEGF blockade can be combined with blockade of distinct angiogenic pathways to achieve additive to synergistic inhibition of angiogenesis and tumor growth. This will be accomplished using several new adenoviruses designed to antagonize the TIE/angiopoietin and EphB4/ephrin-B2 endothelial receptor tyrosine kinase pathways. Second, we will test the hypothesis that the local and systemic efficacy of oncolytic viruses can be improved by simultaneous treatment with soluble Flk 1 and Flt 1 adenoviruses, or by actually modifying the oncolytic viruses to themselves express anti-angiogenic proteins. Finally, based upon the strong and broad-spectrum activity of the murine FIk 1 and FIt 1 soluble VEGF receptors, we will examine the hypothesis that humanized versions of these proteins will effectively suppress tumor growth and tumor angiogenesis. Moreover, these humanized receptors will be expressed from regulated adenoviruses to confer an additional degree of safety as a prelude to eventual clinical use.

DESCRIPTION (provided by applicant): Our work has recently defined an essential role for Wnt signaling in the maintenance of the extensive proliferation that allows complete regeneration of the intestinal epithelium every 5-7 days. Previously, the exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. This has been circumvented by adenoviral expression of Dickkopf-1 (Dkk1), a secreted Wnt antagonist which interacts with Wnt co-receptors of the LRP family. Using adenoviral expression of Dkk1, stringent, conditional and reversible Wnt inhibition in adult animals has been achieved, resulting in repression of expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, and marked inhibition of proliferation in small intestine and colon, accompanied by progressive architectural degeneration with loss of crypts, villi and glandular structure by 7 days. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms, and implicate Wnt proteins as essential growth factors for crypt proliferation in small intestine and colon. Building upon these results, the current proposal will examine the role of Wnts in intestinal regeneration and repair, with the eventual goal of manipulating this pathway for therapy of mucosal disorders such as inflammatory bowel diseases. In the first aim, a systematic quantitative and spatial analysis of Wnt expression patterns in the resting and regenerating intestine will be performed to identify Wnts upregulated during regeneration after radiation or DSS treatment, Dkk1-mediated Wnt blockade, or in IL-10 knockout mice. The functional requirement of Wnt signaling during regeneration will be established by Ad Dkk1 treatment in these animal models. In the second aim, the therapeutic potential of Wnt agonists will be evaluated in the radiation, DSS and IL-10 injury models. Multiple strategies for Wnt stimulation will be utilized, including local/luminal and systemic administration of Wnt proteins, Wnt adenoviruses and small molecule agonists of the Wnt pathway embodied by GSK-3 inhibtors and lithium. In the third aim, the potential functional role of Dkk proteins as endogenous negative regulators of intestinal proliferation will be examined through adenoviral expression of soluble Kremen receptors to sequester and inactivate Dkk, and the examination of Dkk1 effects on Musashil1-expressing intestinal precursor cells.

[unreadable] DESCRIPTION (provided by applicant): The neurovascular unit (NVU) consists of the brain endothelium, along with associated glia, pericytes and neurons. The molecular definition of mediators of cell-cell interaction between these cell types should yield significant insight into vascular function during physiologic and pathologic processes in the CNS. We have constructed a mouse knockout of GPR124, an orphan G-protein coupled receptor originally identified by its overexpression in tumor endothelium. Inactivation of GPR124 by replacement with a lacZ reporter gene results in embryonic lethality with hemorrhage which is strikingly restricted to the brain. Initial analysis of the mutant CNS microvasculature has revealed profound defects of angiogenic migration and sprouting, while expression analysis of GPR124 indicates expression is highly selective for CNS vasculature. The GPR124 phenotype is highly suggestive of defective paracrine signaling within cells of the neurovascular unit, resulting in improper angiogenesis. In the current proposal, the functions of GPR124 will be explored taking advantage of GPR124 knockout mice which we have created. First, the structure of aberrant vasculature in GPR124-/- mice will be studied with respect to qualitative and quantitative deficiencies of with other cells of the NVU, cellular junctions and extracellular matrix. Second, genetic epistatic relationships will be sought between GPR124 and other genes whose knockouts have similar phenotypes, such as integrins and Id transcriptional repressers. Third, cell types of the neurovascular unit, such as glia, pericytes and neurons, will be systematically tested for the ability to elicit GPR124-dependent responses in tissue culture. Finally, conditional approaches to GPR124 inactivation will be pursued using adenoviral and conditional knockout strategies, to facilitate the eventual study of GPR124 function in fully adult organisms. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Erythropolesis, the production of red blood cells (RBCs), is regulated by an intricate network of signals involving the hormone erythropoietin (Epo), and sensing of hypoxia through inhibition of both HIF-1alpha proline hydroxylation and its subsequent interaction with the Von Hippel-Lindau (VHL) protein. Either insufficient or excess erythropoiesis produces disease in humans, with too few RBCs resulting in anemia, and too many RBCs resulting in polycythemia vera. The treatment of anemia alone consumes significant economic resources, with the necessity for either blood transfusions or Epo treatment. We have developed adenoviruses which express soluble ectodomains of the Flkl and Fltl receptors for Vascular Endothelial Growth Factor (VEGF). Single injections in mice of these adenoviruses produce high levels of circulating soluble Flk1 and Flt1 ectodomains which persist for >3-4 weeks, potently suppressing tumor growth and angiogenesis, and producing stringent conditional inactivation of-VEGF in adult animals. Surprisingly, adenoviral expression of Flk1 or Flt1 ectodomains (soluble VEGF receptors, or sVEGFRs) in normal mice strongly stimulates erythropoiesis, with increase in hematocrit increasing from baseline 45% to new levels of 55-75 %. Circulating erythropoietin levels are elevated in parallel; however, the Epo originates from the liver as in embryonic development, not the usual site of synthesis in the kidney. This data implicates VEGF as an unsuspected repressor of hepatic Epo synthesis, and this grant application is focused on determining the underlying moIecular mechanisms. In Aim 1, experiments will expand upon preliminary data implicating perturbation of hepatocyte--endothelial cross-talk in the sVEGFR induction of hepatic Epo. Adenoviral and transgenic expression of Cre recombinase will be used to test the effects of specific inhibition of hepatocyte-produced VEGF on hepatocyte Epo expression. Conversely, co-culture experiments manipulating VEGF will be used to test the effects of endothelial cells on hepatocyte Epo production. Aim 2 will define the specific factors mediating the transcriptional regulation of Epo by VEGF and VEGF blockade. Candidate factors (HIF proteins, HNF4, RXRalpha, GATA factors) will be directly tested by chromatin immunoprecipitation assays and functionally by adenoviral and transgenic expression of Cre to delete floxed alleles of relevant candidates. Additionally, novel factors will be sought by unbiased approaches of DNAse1 footprinting and hypersensitivity assays. The therapeutic implications of the ability to reactivate hepatic Epo synthesis are substantial. These studies should also yield significant insight into the action of VEGF as an unsuspected upstream regulator of RBC homeostasis, into the regulation of hepatic Epo production which is strongly repressed post-natally, and indicate the potential utility of alterations in erythropoiesis as surrogate markers for or a desirable consequence of stringent VEGF blockade.

Three-dimensional Scaffold-based Systems for Primary Human Intestinal Culture Abstract The roughly 8 meters of intestine in the adult human plays essential roles in physiologic homeostasis including digestion, nutrient absorption, secretion of hormones, and immune functions. Commensurate with these essential roles, diseases of the intestine are a considerable source of human morbidity and mortality, including but not limited to inflammatory bowel diseases, infection, malabsorptive states and neoplasia. Surprisingly, despite the wealth of investigations into intestinal physiology and pathophysiology, human primary intestinal culture is essentially not utilized at all as an experimental tool. Indeed, many primary tissue types lack appropriate in vitro culture methods, and we hypothesize the dearth of appropriate three-dimensional (3D) culture technologies is the critical limitation. In response, the current proposal seeks to address this notable deficiency through the development of 3D scaffold-based systems for primary human intestinal culture within designed bioreactors. Our innovative approach combines expertise in intestinal biology with the engineering of cellular microenvironments. Through a combination of original scaffolds with tunable properties, discovery and novel application of intestinal mitogens, and theoretical and experimental design of bioreactors with controlled transport properties, we will create well-defined 3D culture systems that enable rapid, high-throughput, iterative optimization of the culture microenvironment. These systems will derive an enabling technology with transformative scientific and therapeutic applications, finally enabling in vitro studies that have never before been possible in primary culture and have instead required transformed cell lines of questionable relevance or in vivo approaches. Such novel downstream applications would include in vitro screening of pharmacologic agents modulating intestinal physiology (intestinal transport, nutrient hydrolysis, or absorption), and intestinal toxicology (chemotherapeutic agents or systemically administered therapeutics). The potential for modeling mucosal immunity (innate versus cellular/humoral) and inflammatory bowel diseases in primary human culture is present, as well as the potential modeling of neoplasia upon introduction of single exogenously introduced cancer stem cells, introduction of oncogenes, or shRNA suppression of tumor suppressors. The ex vivo expansion of intestinal tissue and/or intestinal stem cells for transplantation would be enabled by the proposed studies. Finally, the principles established by these 3D culture systems will provide a paradigmshift in the approaches available to culture diverse organs. The ability to reproducibly culture 3D tissues outside of the body will enable scientists to test new hypotheses and models of disease, to develop high-throughput screens of pharmaceutical targets, and to enable the expansion of cells for regenerative medicine therapies.

DESCRIPTION (provided by applicant): Intestinal homeostasis is maintained by the robust activity of intestinal stem cells (ISC) which allow complete regeneration of the intestinal epithelium every 5-7 days. Recent functional studies have established the existence of two types of ISC, a crypt base columnar (CBC) cell expressing LGR5 and prominin-1, and a distinct ISC population expressing Bmi1, located higher in the crypt at approximately the +4 position and restricted to the small intestine. Despite potent stem cell attributes of the Bmi1+ cells in lineage tracing studies, their regulation, relationship to LGR5+ cells and transcriptome have remained poorly defined. The overall goal of this application is the analysis of the Bmi1+ lineage in vivo and in vitro, using recently developed Bmi1-CreER knock-in mice, our robust methodology for small intestinal culture, and R-Spondin1 and Dkk1 adenoviruses allowing gain- and loss-of-function Wnt manipulation in vivo. Accordingly, Aim 1 will investigate the regulation and functional relevance of the Bmi1+ lineage during intestinal regeneration. The number and fate of Bmi1+ cells will be examined during regeneration in response to radiation or R-spondin1, both in vivo and in vivo using the Bmi1-CreER mouse or cultures derived thereof. Importantly, the functional contribution of the Bmi1+ ISC to intestinal regeneration after radiation or R-spondin treatment will be assessed by diphtheria toxin-mediated lineage ablation. Aim 2 will address the important question of relationships between the Bmi1+ and LGR5+ ISC lineages. Fate mapping of the Bmi1 lineage will be performed in vivo and in vitro to formally demonstrate if Bmi1+ cells or their progeny can express LGR5. Culture of isolated Bmi1+ cells from Bmi1-CreER mice will be performed both within and without an ISC niche to explore if Bmi1+ cells can give rise to LGR5+ cells in vitro. In Aim 3, transcriptional profiling of Bmi1+ cells will be performed and compared to the published LGR5+ transcriptome and target validation performed exploiting in vitro intestinal culture. Finally, Aim 4 will explore the ex vivo expansion and transplantation of Bmi1+ ISC. Our ISC niche-dependent intestinal culture system, as well as niche-free systems will be used to expand Bmi1+ cells ex vivo, followed by single cell or population transplantation in vivo. Questions of plasticity will also be addressed with introduction of small intestine Bmi1+ cells into the colon both in vitro and in vivo. PUBLIC HEALTH RELEVANCE: The intestine possesses highly active stem cell populations with therapeutic relevance to diverse conditions including inflammatory bowel diseases, metabolic disorders and cancer. Here, the Bmi1+ intestinal stem cell population will be investigated with regards to regenerative potential both in vivo and in vitro.

PI: Kuo, Calvin J. Project Summary/Abstract The normal functioning of the adult brain is critically dependent upon an adequate blood supply. The current project will investigate the role of the orphan G-protein coupled receptor GPR124 during adult angiogenesis in the CNS. We have found that GPR124 knockout mice exhibit embryonic lethality at E15.5 from a profound block and highly CNS-specific block in developmental angiogenesis, with formation of forebrain glomeruloid vascular malformations and hemorrhage. We have now generated Tie2- GPR124 transgenic mice in which GPR124 is overexpressed in CNS endothelium under the control of the Tie2 promoter/enhancer. Despite concomitant ectopic GPR124 expression in non-CNS vasculature, the Tie2-GPR124 mice remarkably develop CNS- specific vascular malformations with enlarged, dilated, hyperproliferative vessels. These further reinforce the CNS vascular tropism of this receptor through an independent, complementary gain-of-function approach. The current project will explore the role of GPR124 in adult angiogenesis in the CNS. Aim 1 will exploit our newly developed GPR124 conditional knockout mice to examine the role of this receptor during adult CNS angiogenesis induced by VEGF, or by ischemia in a MCAO ligation model. In Aim 2, the nature of the CNS vascular malformations in Tie2-GPR124 transgenic mice will be further investigated at the level of temporal onset and arteriovenous characteristics. Finally, Aim 3 will model GPR124 signaling in vitro using GPR124-deficient endothelium and putative ligand sources. These studies overall should provide further insight into the role of GPR124 in adult angiogenesis, with particular emphasis on pathophysiologic settings such as stroke and CNS vascular malformations.

DESCRIPTION (provided by applicant): Colorectal carcinoma (CRC) arises from multiple mutations and genomic aberrations in distinct driver cancer genes that that in concert to spur neoplastic development and phenotype. This inherent genetic complexity greatly complicates both personalized diagnosis and treatment. Previously published studies have confined that large numbers of genes will be mutated or subject to genomic aberrations in CRC. A significant and emerging challenge for the post-genomic era is to identify which of these mutated genes are driver loci that functionally drive colon cancer development, versus passenger loci without functional relevance. Finally, it is of the highest priority that one forges these genetic observations with correlations of prognosis and clinical outcome. This can only be done if we better understand the unified biological ramifications of the combined and diverse multigenic driver background which act synergistically to promote CRC tumorigenesis. This proposal details an integrated analysis that will rely on the CRC genomic data generated by the Cancer Genome Atlas Project (TCGA) to discover novel candidate CRC genes and study multigenic CRC driver gene co-mutated / dysregulated modules within the genetic context of other drivers and provide biological validation in a powerful in vitro primary culture CRC model which can be engineered for multiple genetic events. To accomplish these goals, we will develop and implement novel statistical methodologies for the integrative analysis of multiple TCGA genomic and clinical data sets. The goal is to identify and prioritize novel CRC genes either singly or as co-mutated modules in combination with other known driver CRC genes. We will use the rich TCGA data set to conduct an integrated CRC genomic analysis of point mutations, gene expression, copy number aberrations and methylation data. We will prioritize the discovery of mutations and other genomic aberrations of these novel CRC genes that are associated with specific clinical stages of disease and other clinical parameters. These statistical and computational studies will then be directly coupled to rapid and robust functional target validation of candidate loci using our rigorously characterized in vitro primary intestinal culture methodology (Gotani et al, Nature Medicine, 2009), in which we have recently established the transforming activity of established CRC loci such as APC, KRAS and TP53. Genetic deletion and retroviral expression of shRNA, cDNA or mutants thereof will be utilized to evaluate putative individual driver loci, as well as combinatorial oncogene modules. This proposal directly addresses fundamental problems in the exploration and translation of novel colorectal cancer gene discovery in the context of clinical data which is available from TCGA. RELEVANCE (See instructions): Colorectal cancer (CRC) represents the third most commonly diagnosed cancer in the United States. This proposal utilizes a fusion of genomic analysis of a large population of patients, mathematical modeling and culture of intestinal fragments to functionally identify genes that are critical for colon cancer development. These studies have implications for generation of novel diagnostic and therapeutic strategies for colon cancer.

DESCRIPTION (provided by applicant): MicroRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides length which down-regulate gene expression by annealing with the 3' UTR of target mRNAs, repressing translation and inducing mRNA degradation and de-adenylation. The imprecise miRNA recognition of their mRNA targets allows post-transcriptional regulation over hundreds of target mRNA during homeostasis or in response to stimuli. The microRNA miR-126 represents the most abundant endothelial miRNA upon expression profiling, and is expressed in a pan-endothelial fashion during embryogenesis. We have created knockout mice lacking miR-126 which exhibit 50% embryonic lethality associated with edema, hemorrhage, and angiogenic delay. In surviving miR-126 ko mice, adult angiogenesis is delayed for instance in corneal micropocket assays. Interestingly, miR-126 is present in intron 7 of a host gene, Egfl7. The miR-126 ko phenotype actually recapitulates previously described Egfl7 ko phenotypes, and previously described Egfl7 ko mice are now understood to have inadvertently disrupted miR-126 expression. An essential role for a miRNA during tumor angiogenesis has not been previously demonstrated. The current application explores the role of miR-126 in tumor angiogenesis based upon strong miR-126/Egfl7 expression in tumor endothelium and preliminary data in the MMTV- PyMT transgenic model of breast cancer. Aim 1 investigates whether constitutive genetic deletion of miR-126 inhibits tumor progression and angiogenesis and extends survival in the MMTV-PyMT model. Aim 2 evaluates the therapeutic potential of miR-126 inhibition through temporally conditional miR-126 ko with our floxed mouse allele in pre-established MMTV-PyMT tumors. Conditional miR-126 ko will serve as a reference standard for pharmacologic miR-126 inhibition in the MMTV-PyMT model via both antagomirs, as well as a novel 2'-F/methoxyester anti-miR chemistry. Further, miR-126 inhibition by either genetic deletion or antagomir/anti-mir treatment will be compared and combined with VEGF inhibition. Finally, Aim 3 investigates mechanisms of miR-126 action during breast tumorigenesis through compartment-specific deletion in endothelium, in vitro characterization of miR-126 ko endothelium, and endothelial tip- and stalk cell phenotypes. Overall, these investigations utilize complementary approaches of rigorously characterized miR-126 genetic knockout mice and novel antagomir and 2'-F/methoxyester anti-miR therapeutic strategies to explore the first functional linkages between an endothelial microRNA and tumor angiogenesis. The demonstration of a functional requirement for miR-126 during tumor angiogenesis and progression would have significant implications for design of future anti-angiogenic therapies. PUBLIC HEALTH RELEVANCE: The inhibition of tumor blood vessels, termed anti-angiogenic therapy, is a promising new modality for cancer therapy. The majority of current anti-angiogenic therapies target a blood vessel growth factor termed Vascular Endothelial Growth Factor. Here, we explore the relevance of a RNA molecule, miR-126, as a novel factor regulating tumor blood vessels, with implications for development of new anti-angiogenic therapies.

DESCRIPTION (provided by applicant): Cancer arises from the acquisition and concerted action of multiple mutations and genomic aberrations in discrete combinations of tumor suppressors and oncogenes, known as drivers. Large cancer genome-scale sequencing studies such as TCGA, are now operative and the Cancer Target Discovery and Development (CTDD) Network seeks to bridge the gap between the enormous volumes of data generated... and the ability to use these data for the development of human cancer therapeutics. A secondary goal for the CTDD Initiative is that in five years, the entire CTDD Network is expected to identify and characterize targets for approximately 25 or more (if possible) cancer types. and for applicants to have or build the capacity for in depth analyses and experimental approaches utilizing datasets for many cancer types. A broad coverage is the paradigm for this initiative. Here, the wealth of TCGA data will be directly coupled to robust in vitro functinal validation of candidate cancer driver modules using primary mouse 3D organoid cultures of diverse tissues arrayed in high-throughput format. In Aim 1, the Hanlee Ji and Sylvia Plevritis groups will identify co-segregating mutational modules from TCGA datasets from multiple solid tumor types, using complementary methods of supervised Bayesian analysis and Unsupervised Module Network Analysis for Master Regulators. In Aim 2, these prioritized mutational modules, stratified for clinical significance, will undergo direct functional validation in a broadly applicble, multiplexable, in vitro 3D primary organoid system developed by the Calvin Kuo group, which is amenable to combinatorial gene engineering. In collaboration with Bill Hahn, this will utilize high throughput lentiviral introduction of cDNA or shRNA to systematically interrogate the genes within amplicons and deletions, contextually modeled in the TCGA mutational background in which these copy number variations occur. Additionally, co-segregating mutational modules from diverse tissues will undergo systematic deletion in organoid cultures to define minimal module composition, and we will pursue process development to extend the range of tissues from which organoids can be modeled. Overall, these studies describe bioinformatic and in vitro modeling approaches that are robustly portable across a variety of organ systems for functional interrogation of diverse TCGA datasets, as highly responsive to RFA-CA-12-006 and the CTDD, and with attendant implications for cancer biology, diagnosis and therapy.

DESCRIPTION (provided by applicant): The tremendous regenerative capacity of the intestine, with epithelial turnover every 5-7 days, is mediated by the carefully orchestrated activity of intestinal stem cells (ISC). The recent identification of molecular markers for ISCs, combined with in vivo lineage tracing and in vitro primary organoid culture methodologies has revolutionized ISC biology. The resultant wealth of findings have showcased ISCs as a robust general paradigm for stem cell behavior and have elicited a next generation of studies exploring the ISC niche, interrelationships and interconversion of actively cycling versus quiescent ISC populations, and translational applications of ISC transplantation. Accordingly, the current application is a continuation of our prior 5 year project for the NIDDK Intestinal Stem Cell Consortium (ISCC), and is responsive to RFA-DK-13-012, Intestinal Stem Cell Consortium Research Projects (U01), seeking to define conditions controlling proliferation and differentiation of intestinal stem cells, determine the developmental lineage of characterized populations of stem cells in the small intestine and methods for expanding and grafting stem cells back into the intestine. Here, we build upon our preliminary data from the prior cycle with novel reagents and methodologies. Aim 1 explores R-spondins as regulators of Lgr5+ ISC symmetric cell division combining adenovirus-mediated gain- and loss-of-function approaches with in vivo lineage tracing, and pursuing mechanistic and niche localization studies. In Aim 2, interrelationships between diverse cycling and quiescent ISC populations will be investigated by RNA-Seq, and novel lineage relationships of Bmi1+ ISC will be explored. Aims 3 and 4 explore translational aspects of ISC biology, with Aim 3 utilizing organoid culture to evaluating the effects of R- spondins on colon cancer stem cells, and Aim 4 attempting the phenotypic correction of a Mendelian intestinal epithelial defect by ISC transplantation. Overall, these studies describe an integrated approach to ISC biology and translation that is highly responsive to RFA-DK-13-012, Intestinal Stem Cell Consortium Research Projects (U01), with implications for stem cell biology, and intestinal disease pathogenesis and therapy.

? DESCRIPTION OF THE OVERALL U19 APPLICATION (provided by applicant): The human gastrointestinal tract exhibits diverse and essential roles in processes as widespread as digestion, absorption, secretion and immunity. However, these same crucial functions represent a significant vulnerability by which bacterial and viral gastrointestinal pathogens induce substantial morbidity and mortality in humans worldwide. While animal models are invaluable to the study of basic mechanisms of infectious disease, the interactions between some of the most prominent gastrointestinal pathogens with host tissues are species specific and host restricted, particularly for infections that have co-evolved with humans. Thus, the study of many pathogens has heretofore required animal-adapted strains that do not accurately recapitulate the human disease pathophysiology and host range restriction in mice, or alternatively oncogenically transformed human cell lines that do not model normal tissue characteristics. Recent advances in culture of primary tissues as 3-dimensional organoids that reproduce multilineage differentiation and organ architecture represent a promising technology to modeling the interaction of human enteric pathogens with human gastrointestinal epithelium in vitro. The overall goal of this application is to create alternative model systems for a variety of human enteric diseases and pathogen-associated specific immune responses using optimized 3D human gastrointestinal organoid cultures via the co-culture of (1) primary gastric and intestinal organoids with (2) bacterial/viral pathogens and/or (3) immune effector cells. We have thus assembled a multidisciplinary, collaborative, and synergistic team at Stanford University including Calvin Kuo (gastrointestinal organoid culture), Manuel Amieva (Helicobacter pylori), Harry Greenberg (Rotavirus), Sarah Heilshorn (microenvironment bioengineering), Sean Bendall (CyTOF), Elizabeth Mellins (immunology), and Denise Monack (Salmonella Typhi). Together, these PIs form the Stanford Novel, Alternative Models for Enteric Diseases Cooperative Research Center (Stanford NAMSED CRC) Program. The Stanford NAMSED CRC is composed of Cores A-C (Administration, Organoid Production, and Advanced Co-Culture Engineering and Single Cell Statistics of Gut Immunology ACCESS-GI), and Projects 1-2, which study bacterial and viral pathogen interactions with human GI organoids, respectively. Towards these goals, we employ human organoid technologies incorporating epithelial-only or epithelial/mesenchymal components to create tissue-specific models of bacterial (Helicobacter pylori, Salmonella Typhi, Salmonella Typhimurium) or viral (rotavirus) infections, with or without immune effector cells or stimuli. We deploy innovative supporting technologies including CyTOF mass cytometry, bioengineered extracellular matrices, microfluidics and CRISPR-based gene editing. Overall, the Stanford NAMSED represents a synergistic group devoted to the integrated modeling of enteric pathogens as an epithelial-immune organoid unit.

? DESCRIPTION (provided by applicant): Recent in depth molecular analyses of human malignancies, through projects such as The Cancer Genome Atlas (TCGA), have revealed numerous frequently identified mutations; however, only a subset of these actually contribute to the development of particular cancers. The malignant phenotype is often the result of synthetic genetic interactions between multiple genomic and epigenomic aberrations. As such, subsets of tumors have specific co-occurring mutations or genomic alterations that cooperate in a co-dependent manner. The goal of this application is to identify the critical co-dependent molecular pathways that cooperate with known driver genomic alterations in oral squamous cell carcinoma (OSCC), one of the most frequent malignancies of the head and neck. The insight gained will, in turn, provide a platform for novel drug discovery and/or rationale for the investigation of novel combinations of existing drugs. Aim 1 will use sophisticated new bioinformatics algorithms developed by the Gevaert lab to integrate mutation and copy number alteration data with DNA methylation and gene expression data in OSCC TCGA data sets. These algorithms will be used to predict, with high probability, candidate genetic interactions among heterogeneous OSCC tumors and to identify master regulators of gene modules that are related to particular biologic processes, such as metastasis. In Aim 2, candidate gene interactions and master regulators will be validated by the Sunwoo lab using next generation in vivo synthetic lethality assays, using patient-derived xenografts to more closely reflect the primary tumor. Candidate master regulators of metastasis will also be evaluated using in vivo assays. In Aim 3, the Kuo lab has adapted their experience in culture and oncogenic transformation of gastrointestinal 3D air-liquid interface primary organoid cultures to OSCC. Accordingly, our validated wild-type oral mucosal organoid protocols will be used to introduce co-occurring mutations and gene alterations into wild-type human and mouse oral mucosa tissue to functionally validate the oncogenic activity and multigenic transforming synergy of putative OSCC genes from Aims 1 and 2. In Aim 3, the 3D organoid culture approach will also be used to grow primary human OSCC tumor organoids directly from surgical samples, for in vitro chemosensitivity testing, correlation against exome sequencing mutational status and shRNA/sgRNA-based gene validation. This bi-directional strategy of (1) targeting co-occurring mutations in patient-derived xenografts and primary tumors and (2) introducing co-occurring mutations into normal oral mucosa will provide important insight into our understanding of the synthetic genetic interactions in OSCC. Further, the functional and genetic data from Aims 2 and 3 will be channeled back to Aim 1 to continuously update the bioinformatics models.

PROJECT SUMMARY The overall objective and goals of the Stanford NAMSED CRC Administrative Core A are to manage, coordinate, and supervise all Center activities to effectively and optimally support the Program's goals to develop alternative model systems for enteric diseases. The Administrative Core A will be led by Dr. Calvin Kuo and Dr. Manuel Amieva as Co-Core Leaders and will involve the other Project and Core Leaders on a Center Steering Committee to monitor progress toward the Specific Aims, which are to: 1) Implement administrative & leadership mechanisms via a management plan that will facilitate communication and cooperation among the Stanford project and core leaders and with the greater NAMSED Consortium and investigators at other institutions to ensure a maximally productive research effort; 2) Monitor the progress of each of the Research and pilot projects and their interactions with the scientific cores, and to promote effective communications among them; 3) Provide an efficient, centralized unit for the fiscal and administrative operation of the NAMSED CRC activities; 4) Solicit and evaluate proposals on an annual basis in the Stanford research community for Pilot Developmental Research Projects (DRPs), and to monitor their progress. 5) Provide infrastructure support for Stanford NAMSED CRC investigators to develop collaborative studies with other members of the NAMSED Consortium.

? DESCRIPTION (provided by applicant): Mutations in the KRAS locus are amongst the most pervasive genetic alterations in human cancer. In colorectal cancer (CRC), KRAS mutations are not only prevalent (~42%) but crucially impact therapeutic response to EGFR inhibitors. Despite the attractiveness of KRAS as a therapeutic target, direct pharmacologic inhibition of constitutively active mutant KRAS or its downstream effector pathways has proven challenging, considerable effort and recent promising progress notwithstanding. Since KRAS mutant cancer cells may possess additional dependencies on either downstream pathways or acquire secondary mutations, such synthetic lethal vulnerabilities represent valuable therapeutic opportunities. Unfortunately, prior KRAS synthetic lethal screens have suffered from complications of reproducibility and off-target effects, compounded by potential context-dependent effects from the complex and heterogeneous 2D cancer cell lines employed. As explored in the current application, primary 3D colon organoid cultures represent a novel approach to identification of KRAS synthetic lethal interactions in CRC. Such organoids are particularly well characterized for colon and combine native differentiation and tissue architecture with experimental tractability. Importantly, they provide an unusual opportunity for mutant KRAS to be studied in the setting of primary human colon tissue with a limited subset of deliberately engineered oncogenic mutations opposed to transformed cell lines with potentially large numbers of confounding genetic alterations. Our goal is to explore KRAS synthetic lethality in colorectal cancer, via a synergistic effort from Multi- PIs Calvin Kuo and Kevan Shokat, the Co-Investigators Haian Fu, Michael McManus and Olivier Gevaert and collaborators Stanley Qi and Michael Bassik. In Aim 1, we generate engineered primary human colon organoids with CRISPR APC mutation or more complex genotypes ± KRAS mutation (G12D, G12C, G12V, etc.), as well as colorectal cancer patient-derived organoids from surgical specimens (CRC-PDO) with wild- type or mutant KRAS status. Aim 2 pursues activity-based proteomic characterization of kinases that are altered by mutant KRAS in engineered colon or CRC-PDO organoids ± mutant KRAS and functionally evaluates these kinases as synthetic lethal targets. In parallel, Aim 3 robustly multiplexes organoids into multiwell format to identify small molecule synthetic lethal compounds by high-throughput screening. Lastly, Aim 4 utilizes a complementary genetic approach to CRC KRAS synthetic lethality based on shRNA and CRISPRi-mediated gene silencing, both for orthogonal functional validation of synthetic lethal candidates arising in Aims 2 and 3, but also for unbiased druggable genome screens to identify novel synthetic lethal interactions. Overall, we present a highly integrated approach to KRAS synthetic lethality in CRC where novel organoid methods are directly coupled to proteomic, small molecule and genomic screening technologies and bound by multi-center cross-validation of candidate loci and small molecules.

SUMMARY Non-small cell lung cancer (NSCLC) is an aggressive malignancy in which limited treatment options are further compromised by treatment resistance. Immunotherapy, particularly against the PD-1/PD-L1 immune checkpoint, has transformed NSCLC treatment with durable responses and comparatively minimal side effects in both second-line treatment of metastatic disease and, recently, first line therapy. Despite these impressive responses there are equally impressive but poorly defined intrinsic and/or acquired resistance mechanisms. Across solid tumor types, the response rate targeting the PD-1 axis in unselected patients is only ~20-30%. In previously untreated NSCLC, the overall response rate (ORR) to the anti-PD-1 antibody pembrolizumab is only ~45% even with patient pre-selection for >50% IHC PD-L1 tumor positivity, PD-L1 negative patients also exhibit anti-tumor response and the active search for alternative biomarkers of response in NSCLC has been unfulfilled. Thus, the significant resistance mechanisms impairing response to PD-1-targeted agents in NSCLC and other diverse solid tumors have remained intractable to both biomarker discovery and accompanying mechanistic definition. Project 2 of this U54 application thus directly addresses the pressing issue of intrinsic and acquired resistance to PD-1-targeted immunotherapy in NSCLC through analysis of an invaluable cohort of on-treatment longitudinal biopsies. Aim 1 pursues deep single-cell RNA-seq profiling of the immune component of NSCLC anti-PD-1 on-treatment biopsies using a highly efficient, microfluidic bead-based protocol allowing unsupervised discovery of cell clusters, T cell activation or exhaustion states and transcriptomic insights into immunotherapy resistance. Aim 2 exploits our 3D Patient-Derived Tumor Organoid (PDO) cultures that represent the first in vitro functional recapitulation of the PD-1-dependent immune checkpoint and tumor infiltrating lymphocytes (TILs) within clinical NSCLC biopsies. Here, we create functional organoid culture models of NSCLC immunotherapy resistance from longitudinal biopsies, measuring TIL activation upon in vitro anti-PD-1 organoid treatment and correlating against patient response. Lastly, Aim 3 performs prospective liquid biopsy and exome sequencing to determine mutational signatures of anti-PD-1 resistance that functionally regulate immune checkpoints. These Aims utilize synergistic expertise from the Stanford site of the U54 with Calvin Kuo (Project 2 PI; organoid culture, single cell RNA-seq), Ron Levy (tumor immunotherapy) and Heather Wakelee and Suki Padda (NSCLC immunotherapy trials), all in close coordination with Project 1 clinical biopsies, liquid biopsies and whole exome sequencing from Trever Bivona and Sourav Bandyopadhyay of the UCSF site. Overall, we present a comprehensive approach to intrinsic and acquired resistance to PD-1 inhibition in NSCLC via complementary single cell, organoid and sequencing analysis of longitudinal on-treatment biopsies.

This application investigates regulation of central nervous system (CNS) angiogenesis and BBB integrity by the membrane protein Reck, a glycophosphatidylinositol (GPI)- anchored protein with large extracellular domain (ECD). In a fascinating convergence of data from many groups including ourselves, substantial overlap exists between the knockout mouse phenotypes for Reck, the G protein-coupled receptor family member Gpr124 and the Wnt7a/7b ligands. These phenotypes are all unified by marked deficits in developmental CNS angiogenesis with glomeruloid vascular malformations and forebrain-specific embryonic lethal hemorrhage. Further, Reck and Gpr124 synergistically promote canonical Wnt signaling with pronounced specificity for Wnt7a/7b and not other Wnt family members. Here, we perform mechanistic exploration of the contribution of Reck to Gpr124- and Wnt7a/7b-mediated signaling and blood-brain barrier (BBB) function. Here we investigate regulation of BBB integrity by the RECK/GPR124/Wnt pathway in vitro and in vivo. Aim 1 biochemically characterizes RECK/GPR124- regulated Wnt7a/7b signaling by defining domains of Reck and Gpr124 that underlie their physical interaction and functional promotion of Wnt7a/7b signaling. Aim 1 also investigates potential complex formation with Wnt7a/7b and the established Wnt receptors Frizzled (Fzd) and LRP. Aim 2 creates an in vitro culture model of the RECK/GPR124/Wnt pathway using primary brain endothelial cells (ECs) isolated from genetically modified mice and analyzes effects of pathway modulation on BBB marker expression and function. Aim 3 investigates the role of the RECK/GPR124/Wnt pathway in BBB integrity post-stroke using endothelial-specific and inducible knockout of Reck genetically modified mice and the tMCAO stroke model, and superimposed effects of Gpr124 knockout or Wnt pathway manipulation. Overall, these studies utilize complementary biochemical, cell biological, and genetic strategies to explore RECK/GPR124/Wnt7 regulation of Wnt/?-catenin signaling and BBB function.

PROJECT SUMMARY/ABSTRACT The deluge of multi-scale ?omics? data from The Cancer Genome Atlas Project (TCGA) and other cancer profiling projects has revealed remarkable genetic and epigenetic complexity and tremendous intrapatient variation. Accordingly, a particularly acute need exists for accurate, scalable human cancer models that can functionally interrogate these extensive datasets, identify driver oncogenic events from benign passengers and characterize their relevance to treatment response. For the last four years the Stanford Cancer Target Discovery and Development (CTD2) Center has pursued human ?organoid? culture methods for cancer modeling and driver oncogene discovery. Primary 3D organoid cultures afford the unusual opportunity to initiate cancer de novo within the epi/genetic ?tabula rasa? of cultured primary human wild-type tissue, versus the corresponding and often poorly-defined complexity of long- passaged 2D cancer cell lines. This creates a highly defined baseline for cancer modeling and functional driver oncogene validation that is leveraged throughout. Our overall approach applies state-of-the-art systems biology and robust computational resources to large-scale cancer profiling datasets, thus nominating candidate drivers that undergo direct functional evaluation in human organoid culture. This experimental scope leverages a highly synergistic team of Calvin Kuo (reporting PI, organoids), Hanlee Ji (multi-PI, cancer ITH, genomics), Christina Curtis (multi-PI, tumor evolution, cancer systems biology), Olivier Gevaert (cancer systems biology, epigenetics) and Michael Bassik (high-throughput functional genomics). Accordingly, Aims 1 and 2 couple bioinformatic prioritization of TCGA copy number alteration (CNA) and methylation data for driver discovery via organoid-based barcoded lentiviral screens and orthogonal cDNA, shRNA and CRISPR approaches. Aim 3 exploits the ability to longitudinally observe de novo genomic and epigenomic evolution in oncogene- engineered wild-type organoids to nominate networks of cooperating oncogenes that undergo iterative organoid functional validation. Lastly, Aim 4 explores the utility of organoids to model de novo treatment resistance, using archetypal targeted and chemotherapy perturbagens as proof-of-principle and employing single cell RNA- seq/intratumoral heterogeneity and exome sequencing endpoints.

ABSTRACT The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has threatened global health. The severity of disease and rising number of deaths from SARS-CoV-2 have raised an urgent need for effective therapies. Besides respiratory symptoms, 20-50% of patients exhibit gastrointestinal symptoms such as diarrhea and emesis. In addition, clinical evidence shows that viral RNA can be found in rectal swabs, indicating that the intestine may be a critical target of SARS-CoV-2 infection. In this proposal, we engineer novel high-affinity blocking agents for known entry receptors of SARS-CoV-2 to prevent infection of human intestinal cells and pursue a longer-term goal of structure-based discovery of novel receptor targets. Aim 1 designs blocking agents that target the known interaction of SARS-CoV-2 S protein with its primary entry receptor ACE2 (angiotensin-converting enzyme 2), as well as with a novel co-receptor, CD147 (accessory protein for monocarboxylate transporters), both of which are expressed in human small intestinal and colon epithelial cells. In Aim 1 we will engineer an ACE2/CD147 bi-specific agent that can simultaneously target both SARS-CoV-2 S protein receptors to improve the efficiency and specificity of viral blockade. We utilize in vitro protein evolution by yeast cell surface display to generate high-affinity ACE2 and CD147 ECDs with improved affinity for SARS-CoV-2 S protein versus the wild- type ECDs These will be combined into a single bispecific agent containing both ACE2 and CD147 affinity-matured ECDs and assayed in human intestinal organoids. In particular, we deploy intestinal organoids with a ?flipped polarity? where the apical ACE2-expressing aspect faces outwards towards the surrounding ECM/media instead of towards the interior lumen to better model physiologic viral infection. In Aim 2, we will screen a CRISPRa activating library for additional human SARS-CoV-2 secretome targets. The SARS-CoV-2 secretome, i.e. virus-encoded secreted or surface-exposed transmembrane proteins, also facilitates infection of host cells and provides novel targets for SARS- CoV-2 therapeutics. This proposal leverages expertise of Chris Garcia (Multi-PI of the parental R01) in protein engineering, immunotherapeutics, and structural biology with Calvin Kuo (Multi-PI of the parental R01) expertise in organoid generation and disease modelling to design targeted therapeutics for SARS-CoV-2. We also utilize collaboration from the Manuel Amieva and Catherine Blish groups in organoid apical-basal polarity inversion and BSL3 SARS-CoV-2 infection, respectively.

Project Summary/Abstract The intestine is endowed with a remarkable ability to continually regenerate its epithelial layer, both to maintain tissue homeostasis and to heal injury, mediated by Lgr5-expressing active and injury/reserve intestinal stem cells (ISCs) and complex cellular and humoral niche. Disruption of these regenerative properties results in absorptive or barrier dysfunction or frank overgrowth in malabsorptive, infectious, inflammatory and neoplastic intestinal disorders. An improved understanding of ISCs and their niche is therefore crucial to the clinical translation of this biology as is the mission of the NIH Intestinal Stem Cell Consortium (ISCC). As a current ISCC member laboratory, we have extensively collaborated to discern the biology of homeostatic and injury/reserve ISCs relative to their niche. Here, we continue these studies in three aims. In Aim 1, we continue our prior studies on the Lgr5+ ISC niche to explore crosstalk between Lgr5+ ISC and its closely associated PDGFRA+ mesenchyme via the concept that PDGFRA-expressing cells sense and respond to Lgr5+ ISC damage, while Lgr5+ ISC conversely elaborate stimulatory factors for PDGFRA+ cells. This extends preliminary data where primary manipulation of Lgr5+ ISC by radiation or R-spondin results in secondary effects on PDGFRA+ cell number, proliferation and expression of niche factors including R-spondins. This Lgr5+ ISC/PDGFRA+ crosstalk will be studied by deletion of R-spondins in PDGFRA+ populations and by genetic and pharmacologic manipulation of PDGF signaling. This is complemented by enteroid reconstitution studies and unbiased tandem single cell RNA-seq and ATAC-seq of PDGFRA+ cells. Aim 2 furthers our studies of air-liquid interface (ALI) intestinal organoids containing epithelium and mesenchyme towards pre-clinical translation. First, we will extensively characterize stromal components by IF and perform unbiased single cell RNA-seq, to inform and optimize the ALI culture method and stromal preservation therein. Secondly, we develop in vivo orthotopic transplantation of human and mouse ALI intestinal organoids, leveraging biocompatible ECMs and leveraging second-generation bioengineered Wnt agonists having Fzd-subtype specificity. Thirdly, we apply mouse ALI organoids to achieve the first organoid transplantation correction of a disease phenotype in a Ferroportin (Fpn)-KO model of intestinal iron absorption deficiency anemia. Lastly, Aim 3 exploits our ability to convert bioengineered Wnt agonists into Fzd-subtype specific antagonists for biological probes and therapeutics. Fzd-subtype specific antagonists will be used to probe the requirement of specific Fzds during intestinal homeostasis in vivo, with parallel modeling in enteroid culture. Overall, each of these aims represent highly collaborative studies that would not be possible without the integral participation of fellow ISCC labs and the ISCC Coordinating Center, towards advancing the dual ISCC goals of defining niche biology and therapeutic translation.

ABSTRACT Primary human organoid models are an increasingly deployed platform for in vitro infectious disease modeling. The COVID-19 pandemic, engendered by the novel coronavirus SARS-CoV-2, represents a grave threat to public health and physiologic in vitro infection models are therefore urgently needed. This supplement request for U19AI116484, Stanford Cooperative Center for Novel, Alternative Model Systems for Enteric Diseases (Stanford NAMSED), requests funding to create new models for SARS-CoV-2 infection using novel human lung organoid technologies in collaboration with Dr. Ralph Baric at UNC, a recognized coronavirus authority. These studies exploit SARS-CoV-2 infection of organoids using a feeder-free, chemically defined human lung organoid system (Calvin Kuo lab), lung organoids with integrated immune components (Calvin Kuo), methods for robust apical-basal inversion of lung organoid polarity (Manuel Amieva), BSL3 single cell RNA-seq (Catherine Blish) and SARS-CoV-2-GFP indicator strains and BSL3 facilities (Ralph Baric). The SARS-CoV-2 infection of lung organoids will occur in BSL3 containment at both UNC and Stanford to compare apical versus basal infection routes, document how epithelial infection initiates secondary immune responses, and overall generate improved 3D physiological models of SARS-CoV-2-GFP infection relevant to therapeutics screening.

ABSTRACT The COVID-19 pandemic, engendered by the novel coronavirus SARS-CoV-2, is a grave threat to public health, with lung infection and respiratory failure. However, the intestine is also targeted by SARS-CoV-2, as many patients present with GI symptoms and the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is abundantly expressed by intestinal epithelium. COVID-19 mortality is strongly associated with systemic inflammation as in ?cytokine storm?, fostering attempts at therapeutic immunomodulation, and raising a critical need for in vitro human systems modeling SARS-CoV-2-induced immune cell interactions with intestinal epithelium. Conventional 3D organoid cultures (?enteroids?) allow intestinal epithelial SARS-CoV-2 infection but unfortunately omit immune cells. Here we generate a holistic intestinal in vitro model of SARS-CoV-2 infection using air-liquid interface (ALI) organoids containing both epithelium and infiltrating immune cells en bloc without artificial reconstitution. The complex intestinal immune system of ALI intestinal organoids contains various innate and adaptive immune cells, highly diverse T cell receptor (TCR) and B cell receptor (BCR) repertoire, and plasma B cell-derived antibody transcripts. Importantly, immune components in these ALI organoids respond efficiently to epithelial damage and ALI intestinal organoids are highly susceptible to bacterial and viral infections. Here, we utilize this unique ALI organoid technology with integrated immune components to explore sequelae of SARS-CoV-2 infection. Aim 1 establishes and optimizes BSL3 SARS-CoV-2 infection of ALI intestinal organoids, exploiting a novel eversion method to relocate the apical aspect of ACE2-expressing cells to the external surface, allowing survey of SARS-CoV-2 infection of different regions of small intestine and colon. The time course of tissue-resident SARS-CoV-2-induced cross-talk between intestinal epithelium and immune cells is unknown, as hindered by lack of human experimental systems. Thus, Aim 2 performs a scRNA- seq and CyTOF study of SARS-CoV-2-induced immune responses within ALI organoids to (1) create a network model of the temporal propagation of immunity and bidirectional communication between epithelium and immune cells and (2) perform therapeutic testing against nodal vulnerabilities, correlating against clinical status (naïve, convalescent, immunized, cross-reactive coronavirus). Current epithelial organoid systems also do not allow study of adaptive immunity. Aim 3 thus recapitulates SARS-CoV-2 adaptive immune responses by co-culturing ALI intestinal organoids with newly developed human lymph node organoids. Within such SARS-CoV-2-infected co-cultures we correlate adaptive immune responses with clinical status including prior cross-reactive coronavirus infection and SARS-CoV-2 naïve, convalescent and ultimately immunized states. Overall, we leverage collaboration from Mark Davis (human LN culture) and Catherine Blish and Scott Boyd (SARS-CoV-2) to create human in vitro systems modeling SARS-CoV-2-induced innate and adaptive immunity, with relevance for pathogenesis investigations and therapeutics testing.

Stanford/UNC Biomimetic U19 Research Center PROJECT SUMMARY/ABSTRACT ? CENTER OVERVIEW Infectious diseases continue to pervasively afflict global health and socioeconomic stability despite substantial prevention and treatment initiatives. Respiratory and gastrointestinal pathogens rank amongst the most intractable infectious diseases, particularly notable for recurrent waves of zoonotic coronaviruses and the recent COVID-19 pandemic, engendered by SARS-CoV-2. Overall, an urgent need exists for improved in vitro experimental models of human disease to study pathogenesis and to validate therapeutics. The central mission of the Stanford/UNC Biomimetic U19 Research Center is thus to deploy novel 3-dimensional organoid culture models to elucidate the biology and therapy of respiratory and gastrointestinal infectious pathogens. Our application is a renewal of our prior Stanford NAMSED U19 Research Center and is comprised of two Cores and three research Projects, leveraging complementary and synergistic expertise of our investigators at Stanford University and the University of North Carolina. The Center continues to be led by the Multi-PIs, Calvin Kuo and Manuel Amieva, who also co-lead Core A (Administrative Core). Core B (Organoid Core) is led by Calvin Kuo and provides novel capabilities for lung and GI organoid culture, gene editing and multiplexed screening. The three Projects extensively utilize organoid biomimetics for exploration of GI and respiratory pathogens. Project 1, (PI, Manuel Amieva) investigates H. pylori and Salmonella colonization, competition and invasion in the GI tract, while Project 2 (PI, Harry Greenberg) investigates rotavirus host range, neutralization, and M cell interactions in enteric biomimetics. Project 3 (PI, Ralph Baric) is a new addition and extensively uses organoids to model SARS-CoV-2, other closely related epidemic and pre-epidemic emerging coronaviruses and 1918 H1N1 influenza to reveal common and unique host networks associated with severe pulmonary outcomes. The activities of the Stanford/UNC Biomimetic U19 Research Center reside within three overarching Aims. In Aim 1, our Center performs organoid modeling of the epithelium-pathogen interface to investigate pathogenesis, susceptibility and host range restriction. This employs robust reverse genetics and CRISPR screens to systematically manipulate host versus viral/bacterial compartments, within novel apical-basal polarity modulated distal lung/alveolar, nasal sinus, stomach and intestinal organoid systems. Aim 2 defines how SARS- CoV-2, pre-epidemic coronaviruses, rotavirus and Salmonella can perturb reciprocal cross-talk between tissue epithelium and resident immune cells. This exploits a unique 3D air-liquid interface organoid method preserving GI and lung epithelium en bloc with diverse endogenous infiltrating immune cell types without artificial reconstitution. Lastly, Aim 3 performs organoid-based evaluation of therapeutic candidates against SARS-CoV- 2, pre-epidemic coronaviruses and rotavirus in medium- to high-throughput formats including epithelium and/or immune cells. Overall, the explorations of the Stanford/UNC Biomimetic U19 Research Center directly apply advanced organoid systems to the investigation and therapy of recalcitrant infectious pathogens.

PROJECT SUMMARY The immune system remarkably distinguishes between self and non-self/self-aberrant antigens, affording exquisite anti-tumor specificity and inhibition of tumorigenesis. However, tumor immunosurveillance is unfortunately opposed by tumor cell evasion of the immune response. Immune checkpoint blockade (ICB) targeting PD-1, PD-L1, CD40 and others, as well as adoptive cell transfer (CAR-T, bulk TILs) favorably modulate this equilibrium for therapeutic benefit. However, response rates are often incomplete, progressive disease is common, and predictive biomarkers are suboptimal. The development of next-generation immunotherapies has been hindered by a lack of in vitro models that functionally recapitulate syngeneic interactions between tumor and infiltrating immune cells. In response, we have developed organoid methods that culture primary human tumor biopsies together with their infiltrating immune components as a cohesive unit without reconstitution. These ?patient-derived tumor organoids? (PDO) preserve tumor cells alongside endogenous T, B, NK cells and macrophages, robustly recapitulate the T cell receptor clonotype repertoire of the original tumor, and crucially, manifest tumor-infiltrating lymphocyte (TIL) expansion, activation and tumor cell killing in response to anti-PD-1/PD-L1 therapeutic antibodies (Cell, 2018). The PDO system thus represents a holistic organoid model of human tumor-immune interactions. Here, we leverage the PDO technique to investigate immunotherapeutic mechanisms and treatments in PD-1-responsive cutaneous squamous cell carcinoma (cSCC) and melanoma, exploiting pre- and post-treatment human biopsies and mouse models. Aim 1 hypothesizes that checkpoint inhibition induces a complex and sequential network response involving immune-tumor and immune-immune crosstalk. Thus, Aim 1 employs the ability to perform serial time-course sampling of PDOs to define a single cell RNA-seq network cellular crosstalk model of the early anti-PD-1-stimulated anti-tumor immune response over multiple acute time points typically inaccessible to clinical biopsies performed after months. Importantly, comparison of this immune propagation in responding versus non-responding mouse and human organoids will define nodal points conferring resistance. Aim 2 improves bulk TIL adoptive transfer immunotherapy by using PDOs as living bioreactors to enrich tumor-reactive mouse and human melanoma TILs by anti-PD-1 checkpoint inhibition, followed by testing of enhanced anti-tumor activity in vitro and in vivo. Lastly, Aim 3 performs a co-treatment trial comparing anti-PD-1 responses of pre-treatment biopsy cSCC PDOs to clinical outcomes. Further, post- treatment biopsy PDOs are re-challenged with anti-PD-1 and a novel agent inactivating PD-1 by dephosphorylation. We thus utilize the holistic PDO model preserving endogenous tumor epithelial and immune components en bloc to investigate and improve cancer immunotherapy via our team of Calvin Kuo (organoids), Mark Davis and Chris Garcia (tumor immunology) and Anne Chang and Dimitri Colevas (cSCC clinicians).

PROJECT SUMMARY (Core B: Organoid Core) Infectious disease research is substantially hampered by lack of experimental models necessary to study interactions between pathogens and target tissues; a glaring deficiency amplified by the recent COVID-19 pandemic. The recent advent of 3D organoid methods embodies a more physiologic in vitro infectious disease platform. Over the past 5 years, the Stanford NAMSED U19 Center has operated an Organoid Core, led by Calvin Kuo, which has distributed approximately 74 organoid lines for modeling infectivity of gastrointestinal pathogens, resulting in numerous collaborative publications. The Organoid Core has also produced new air- liquid interface (ALI) organoids that maintain epithelium and infiltrating immune populations en bloc without reconstitution (Cell, 2018) and created new protocols for long-term, feeder-free, chemically defined human lung distal organoid culture, as well as novel human nasal sinus organoids (Cell Stem Cell, 2020). The overall goal for Core B (Organoid Core) in our renewal application of the Stanford NAMSED U19 for a Stanford/UNC Biomimetic U19 Research Center is to expand upon our previous success and provide novel ex vivo models for studying gastrointestinal and respiratory pathogens. Thus, Core B will serve as a central biohub for our Center by generating and distributing next-generation human gastrointestinal and respiratory organoid cultures to support Projects 1-3. In addition to these core responsibilities, the Kuo lab will optimize innovative organoid-based assays that will provide additional support for Projects 1-3 and resources for the global research community. In Aim 1, human ?epithelial-only? organoids from the stomach, small intestine, distal lung and nasal airways will be generated, characterized, and distributed for Project 1 (H. pylori, S. Typhi, S. Typhimurium), Project 2 (rotavirus), and Project 3 (SARS-CoV-2, MERS, pre-epidemic coronaviruses and 1918 influenza H1N1 virus). There has been a particular lack of organoid methods incorporating endogenous immune populations without reconstitution. Thus, Aim 2 develops innovative ALI organoid methods preserving epithelial and endogenous infiltrating immune cells en bloc for defining cross-talk between epithelial and immune compartments during GI and pulmonary infections. Lastly, Aim 3 optimizes multiplexed organoid platforms for pathogen infection and drug screening to provide novel methods for evaluating infection, therapeutics and immune responses. Overall, Core B of the Stanford/UNC Biomimetic U19 Research Center leverages leading-edge expertise in primary 3D human organoid culture for the study of enteric and respiratory pathogens. Additionally, we capitalize on novel organoid methods preserving both the epitheilal and immune components to provide innovative holistic tools to dissect pathways involved during pathogenic infection, with both basic and translational implications.

PROJECT SUMMARY (Core A: Administrative Core) The overarching goal of the Stanford/UNC Biomimetic U19 Research Center Administrative Core is to provide the administrative support necessary to form a cohesive, integrated, and efficient Center. In doing so, the Administrative Core will optimally support the Center?s goal to create in vitro model systems using optimized 3D human organoid cultures to study human respiratory and enteric infectious diseases. The Administrative Core will serve as a central hub for the management and coordination of all Center activities and will extend the successful practices implemented during the current NAMSED award. We will maximize efficiency, cost- effectiveness, and productivity by centralizing common administrative and operational resources used by each Project and Core. The core will be led by Drs. Calvin Kuo and Manuel Amieva and will involve the Project and Core leaders and NIAID Program Staff on a Center Steering Committee. The Administrative Core will convene weekly subgroup meetings, monthly full meetings, monthly Steering Committee meetings, and an annual retreat to foster collaboration and synergy. Day-to-day operations of the Administrative Core will be overseen by a dedicated Center Manager. The Administrative Core has the following Specific Aims: 1) Provide administrative leadership and integration of projects and cores across the proposed Center via a Management Plan. 2) Convene internal and external advisory board meetings and facilitate meaningful interactions between advisory boards and Center members. 3) Provide an efficient, centralized unit for the fiscal operation of Center activities. 4) Establish practices to monitor project productivity and assess use of core resources. 5) Provide infrastructure support for Stanford/UNC Biomimetic U19 Research Center investigators to develop collaborative studies with other members of the Biomimetics Consortium and with pertinent scientific communities. 6) Coordinate and implement broad results dissemination.