Ben Z. Stanger - US grants

DESCRIPTION (provided by applicant): Most organs contain a variety differentiated cell types that are derived from a pool of uncommitted progenitor cells. How the correct number of different cell types are specified - the process of cell fate determination - is a central question in organogenesis. This problem applies to the pancreas, which contains exocrine cells, which produce digestive enzymes, and endocrine cells, which make pancreatic hormones, including insulin. The goal of this project is to understand the mechanism by which pancreatic progenitor cells are instructed to become exocrine or endocrine cells, through the process of cell fate determination. Previous work has suggested that Notch - a complex receptor signaling pathway that is widely used during embryogenesis to control differentiation - plays an important role determining pancreatic cell fate. This proposal aims to answer three questions. First, can manipulation of the Notch signal lead to changes in the proportion of exocrine and endocrine cells that form? This will be addressed by mis-expressing activators and inhibitors of the Notch pathway in transgenic mice to determine the effect on cell fate. Second, how are Notch signals regulated in the pancreas? This will be addressed by characterizing the expression of elements of the Notch pathway when known mediators of endocrine differentiation are expressed in chick embryos. Third, what is the function of the mouse Sel-1l protein, a negative regulator of Notch in C. elegans which is expressed in the mammalian pancreas. This will be addressed by inactivating the Sel-1l gene. Clarifying the mechanism by which cell fate decisions are made in the pancreas will enhance the general understanding of the process of organogenesis. Many degenerative diseases, including pancreatic insufficiency and type I diabetes, result from the loss important differentiated cell types. Practically, an understanding of the mechanisms by which the organism specifies such cell types may confer the ability to experimentally manipulate tissues for therapy.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The liver is a complex and essential tissue. Its multiple functions are facilitated by an intricate three- dimensional organization of the two major "parenchymal" cell types of the liver - hepatocytes and cholangiocytes - relative to blood vessels. Cirrhosis disrupts the normal architectural arrangement of these cells, thereby interfering with the normal flow of blood and bile and preventing productive regeneration of the liver. The clinical consequences of cirrhosis - liver failure and liver cancer - result in thousands of deaths and billions of dollars in health care costs within the United States annually. The long-term objective of this work is to understand how liver differentiation and morphogenesis is normally controlled during development, and to apply this knowledge to innovative new strategies for liver regeneration, both in vivo and ex vivo. [unreadable] Previous work has suggested that Notch - a highly conserved receptor signaling pathway that is widely used during embryogenesis to control differentiation - plays an important role in liver development. Specifically, both human patients and mutant mice with deficiencies in Notch signaling exhibit abnormalities in bile ducts. It is unknown whether such abnormalities in differentiation or morphogenesis. The goal of this proposal is to determine the mechanism of Notch action during liver development and to reveal whether Notch signaling plays a role in the adult liver. [unreadable] These objectives will be achieved through the use of genetically engineered mice and cultured cell lines through three specific aims. First, the normal lineage relationship of cells within the developing liver will be determined. Second, the cellular effects of Notch activation within various compartments of the embryonic and adult liver will be determined. Third, cell culture methods for studying Notch's role in differentiation will be developed. These experiments should provide greater understanding of the mechanism of Notch activity in liver development. Moreover, they will provide a framework for future translational studies aimed at augmenting liver regeneration and morphogenesis in vivo and recapitulating liver development in vitro. [unreadable] Outline for lay audience: The goal of regenerative medicine is to replace a failing tissue with a functioning one by introducing necessary cells into an appropriate environment. The ultimate goal of this research proposal is to devise rational approaches to liver regeneration for use in therapy. As a first step, Notch signaling will be studied to understand how this essential signaling pathway functions during liver development and in the normal function of the liver. [unreadable] [unreadable]

Abstract For patients with type 1 diabetes mellitus (T1DM), the greatest prospect for reduced morbidity and insulin independence is the replacement of [unreadable]-cells coupled with suppression of the underlying autoimmune process. Since the initially dramatic successes of islet transplantation in achieving this goal with donor islets, significant efforts have been directed at identifying and expanding alternate sources of [unreadable]-cells. These efforts have been hampered by several challenges: adult [unreadable]-cells are mainly derived by the replication of existing [unreadable]-cells, non-[unreadable]-cells give rise to [unreadable]-cells with low efficiency, and the [unreadable]-cell phenotype is difficult to achieve or maintain in vitro. In tissues that maintain mass by replication of existing cells, certain forms of injury result in the emergence of a normally quiescent progenitor cell population ("facultative stem cells"). Previous studies have pointed to the presence of such a population in the adult pancreas, but findings have been inconsistent and the precursor population has been elusive. This proposal builds on convincing recent evidence that facultative stem cells reside within the pancreatic ducts and can give rise to large numbers of [unreadable]-cells following a specific form of injury: obstruction of the pancreatic duct. This proposal seeks to identify the ductal cells that exhibit this potential, and to develop practical methods for the efficient initiation of duct-to-islet conversion in vitro or in vivo. Specifically, our goal is to stimulate duct- to-islet conversion in rodents with established diabetes as a proof-of-principle for this approach. The proposed studies have the potential for a major impact on the treatment of T1DM by promoting [unreadable]-cells neogenesis through the introduction of peptides or small molecules into the pancreatic ductal system.

DESCRIPTION (provided by applicant): This proposal is focused on morphogenesis of the biliary system, the conduit for transport of bile from hepatocytes to the intestine. Dysfunction of the bile ducts at any point along its intricate branched structure can be a source of morbidity and mortality, and re-establishment of normal biliary integrity is essential for recovery from injury. The experiments described in this proposal will provide insight into how connectivity is established along the entirety of the biliary tree, delineate the mechanisms by which Notch - a pathway whose deficiency causes bile duct deficiency in humans - regulates liver development, and determine the role of this pathway in the function of adult bile ducts. The long-term objective of this research is to understand how the liver achieves and maintains its three dimensional architecture. The proposed experiments will employ Cre/lox technology and real-time imaging to examine several parameters of biliary morphogenesis in vivo: formation of the extrahepatic-intrahepatic biliary junction, formation of the ductal-canalicular junction, and the establishment of biliary cell polarity and tubule formation. A central goal of the proposal is to test the hypothesis that Notch signaling controls bile duct development by initiating a cellular "tubulogenesis program," and specific candidate mediators of this process will be tested for their role in tubulogenesis in vitro. In addition, studies will be performed to examine how Notch signals are coordinated with other known regulators of bile duct development (e.g. HNF1b, HNF6, TGFb). Finally, experiments are proposed that will test the hypothesis that Notch signaling continues to exert an effect on liver biology throughout life, by maintaining ductal cell integrity and spatially organizing the response to injury. PUBLIC HEALTH RELEVANCE: At present, the only effective treatment for patients with end-stage liver failure is transplantation. A major challenge to the development of alternative approaches is the inability to recapitulate normal liver cell organization (morphogenesis) and function outside of the body. The ultimate goal of this research is to delineate the events that guide differentiation and morphogenesis of the liver normally, and thereby devise rational approaches to liver regeneration and replacement.

DESCRIPTION (provided by applicant): It is widely accepted that the immune system can exert either positive or negative influences on tumor growth. In many instances, tumors create an immunosuppressive microenvironment which contributes to tumor escape from immune destruction, particularly in pancreatic ductal adenocarcinoma (PDA). We have previously demonstrated that CD40 agonists can alter the tumor microenvironment and trigger a macrophage-dependent destruction of PDA in both humans and genetically engineered mice. Moreover, using a novel genetic lineage-tracing methodology, we now have preliminary data that invasive behavior and other elements of the metastatic cascade occur at the earliest stages of disease and are critically regulated by inflammation. Thus, we hypothesize that targeting immunosuppressive mechanisms in PDA will provide novel approaches to therapy for this otherwise treatment-resistant disease. In particular, we hypothesize that CD40 activation can re-educate both macrophages and T cells to trigger regression of a primary PDA tumor and impede metastatic spread in concert with other elements of the immune system. Our ultimate goal is to devise new therapies for PDA based on an understanding of immune regulatory networks in the tumor microenvironment. This proposal uses the multi-PI mechanism and will incorporate both mouse models and human patients. Specific Aims are to: (1) Understand the immunological mechanism(s) underlying the anti-tumor effect of agonist CD40 mAb, (2) Understand how inflammation and CD40 activation influence the metastatic cascade, 3) Determine the clinical and immunological impact of CD40 mAb in a clinical trial of patients with resectable pancreatic carcinoma.

? DESCRIPTION: The goals of our proposal are to bring together an expert team of bioengineers and stem cell/developmental biologists to create a Human Islet Biomimetic that will facilitate (i) long term culture and manipulation of human islets and (ii) maturation of stem-cell derived or reprogrammed islets. Specifically, we will combine our expertise in cell and developmental biology with our experience molding three dimensional vascularized scaffolds in which cellular inputs, matrix composition, and microscale organization (including flow) can be varied with precision. Although much is known about islet function under homeostatic conditions in vivo, current methods for studying islet physiology and pathophysiology are severely limited. Studies that rely on model organisms - particularly mice - are hampered by cellular and molecular discrepancies between human and rodent islets. The use of human islets for studies of islet physiology is also problematic, as limited availability and exposure to non-physiological conditions during isolation impede the use of this cellular source. Most importantly, there is no system currently available which supports the full function of islets or b-cells for more than a fe days in culture. Thus, our understanding of islet function and dysfunction - particularly as it relates to type 1 diabetes (T1D) - has been constrained by the lack of tools for maintaining and studying human islets in vitro. Into this gap, we will take cadaveric human islets, pancreatic progenitors from human ES and iPS cells, and endocrine cells that are trans-differentiated from intestinal stem cells as starting material, and incorporate them into innovative scaffold devices that provide control over local structure, cellular content, and fluid dynamics to stabilize b-cell function. Overall, we plan to reconstitute human islet biomimetics that recapitulate the diverse cellular types and their organization within the natural human islet. In addition, we will use the system to explore the reasons why islets are prone to lose function when placed ex vivo and to model human islet diseases. This system will be critical for the success of other HIRN consortia, as well for the b-cell biology community at large by providing an accessory system for studying b-cell survival, immune interactions, and alternate sources of b-cells. Our Aims are as follows: Aim 1: To establish a human islet biomimetic for sustained islet viability and function in vitro. Aim 2: To optimize human islet biomimetic function with respect to glucose sensing, insulin release, and stable maintenance of islet phenotypes. Our study is designed to provide a deeper understanding of the molecular and cellular events that lead to islet dysfunction in T1D and related islet disorders and to help develop strategies to restore normal islet function in these disorders.

? DESCRIPTION (provided by applicant): The overall goal of this project is to define the epigenetic and transcriptional mechanisms underlying cellular reprogramming, a process by which cells switch from a hepatocyte program to a biliary program in vivo. In the liver, correct spatial organization is essential for function, and disruption of normal architecture (by fibrosis) underlies most cases of severe chronic liver disease. The configuration of the biliary tree is of particular importance, and bile duct dysfunction (from obstruction, destruction, or congenital malformation) is a significant cause of liver-associated morbidity and mortality. A clearer picture of how bile ducts are made and remodeled during injury and regeneration will thus have a major impact on our understanding of disease pathogenesis, informing novel strategies for regenerative therapies. In the last grant period, we made several important discoveries related to biliary development, adult cell plasticity, and liver regeneration. Among these was the finding that the Notch pathway - a conserved signaling module known to be important in bile duct development - acts by coordinating biliary differentiation with morphogenesis through a novel tubulogenesis mechanism. Furthermore, we found that newborn and adult hepatocytes retain significant cellular plasticity, exhibiting the ability to undergo in vivo reprogramming (also termed metaplasia or trans-differentiation) to biliary epithelial cells (BECs). Such reprogramming occurs as part of the liver's physiological response to injury and requires Notch signaling. Moreover, Notch itself can drive reprogramming by pushing cells through a stepwise cascade of transcriptional, morphological and functional changes. These discoveries lay the groundwork for the current proposal, with the objective of defining the mechanisms underlying cellular reprogramming in vivo and exploiting these insights for the treatment of cholestatic disease in humans. These objectives will be achieved through the following two interrelated Specific Aims: Specific Aim 1: Determine the molecular mechanisms of hepatocyte-to-biliary reprogramming in vivo. Specific Aim 2: Examine the function and therapeutic potential of biliary reprogramming in vivo. These efforts will provide an unprecedented window into the transcriptional, functional, and epigenetic changes that occur during a change in cell identity in vivo. Importantly, because biliary reprogramming is part of the liver's normal injury response, these insights will have direc physiological relevance. Finally, by using both established mouse models and an innovative human transplantation system in which reprogramming occurs, these studies may provide mechanistic insights that can be used to tip the balance between hepatocytes and BECs, leading to novel treatments for liver disease.

PROJECT SUMMARY Inter-tumoral heterogeneity ? the fact that every tumor has distinct genetic, epigenetic, and stromal features ? poses a challenge for precision cancer therapy. While genetic variants that influence a tumor?s response to targeted therapy (i.e. ?actionable mutations?) have received great attention, comparatively less is known about the causes of variation in the tumor microenvironment (TME). Nevertheless, such variation in the TME is likely to be of critical importance, as prior work has shown that the response to immunotherapy correlates with the abundance of T cells and other immune populations within a tumor [1,2]. We and others have shown, in the context of pancreatic ductal adenocarcinoma (PDA), that tumor-derived factors shape the microenvironment in vivo, dictating the relative abundance of different stromal populations. For example, tumor-derived GM-CSF recruits myeloid cells to the tumor, fostering an immunosuppressive environment [3,4], while tumor-derived Sonic Hedgehog causes an accumulation of myofibroblasts and other changes in the TME [5,6]. However, additional cancer cell-derived factors that act in similar fashion remain to be identified, representing an unexploited source of novel targets. In our preliminary work, we found that human PDA exhibits a wide spectrum of immune activity. Surprisingly, and in contrast to other tumor types, the presence or absence of an active immune signature was unrelated to the neo-antigen burden of a given tumor. We therefore hypothesize that cancer cell-intrinsic factors critically shape the immune microenvironment and drive immune heterogeneity. We further hypothesize that (i) a tumor?s immune makeup determines its response to immunotherapy and (ii) anti-tumor responses can be improved by modulating the immune infiltrate. Here, we propose an innovative approach to delineate the biology of immune heterogeneity in PDA, including novel implantable and genetically engineered mouse models studied in parallel with samples from an extensive tumor bank and prospectively collected from two clinical immunotherapy trials. Cellular, molecular, and clinical consequences of inter-tumoral heterogeneity will be assessed in the context of response to immunotherapy. Our ultimate goal is to understand and manipulate the immune microenvironment in PDA for therapeutic benefit, and we will approach this goal through the following three interrelated Specific Aims: Aim 1. Identify the molecular mechanism(s) underlying heterogeneity of immune infiltration Aim 2. Assess the impact of immune heterogeneity on the response to immunotherapy Aim 3. Elucidate the causes and consequences of immune heterogeneity in human PDA