Celeste M. Nelson - US grants

[unreadable] DESCRIPTION (provided by applicant): Malignant transformation of the breast and other organs is associated with dramatic changes in the microenvironment surrounding neoplastic cells, including a reactive fibrotic stroma characterized by increased production of inflammatory cytokines, excessive accumulation of extracellular matrix (ECM), and an increase in tissue stiffness. Contractile myofibroblasts are key mediators of the biochemical and biophysical properties of the fibrotic tumor microenvironment. Additionally, the transdifferentiation of myofibroblasts from tissue cells and their subsequent activation is controlled by a combination of soluble factors and contractile tension. The increased tissue stiffness associated with fibrosis may thereby generate a positive feedback loop to facilitate tumor progression and metastatic invasion; delineating the microenvironmental effects and effectors will require sophisticated, tractable model systems. Here we describe the development of an experimental model that can define how alterations of biochemical and biophysical cellular microenvironment can stimulate myofibroblast development and activation, and how formation and activation of myofibroblasts in tissue structures affects progression to malignancy. In Specific Aim 1, we will determine the biochemical and biomechanical requirements of the substratum microenvironment for the transdifferentiation process. In Specific Aim 2, we will use a novel three- dimensional microlithography-based organotypic culture mimetic of the mammary epithelial ductal network to determine how myofibroblast transdifferentiation affects the microenvironment of the duct at the biochemical, mechanical, and cell population levels. Given that the presence of fibrotic foci in breast tumors correlates with metastasis and negative prognosis, and might hinder the efficacy of tumor therapies, the new physiologically relevant models developed in this work will have significant impact for discovery and evaluation of novel therapeutic targets to combat fibrosis genesis and tumor progression. PROJECT RELEVANCE: Repair of tissue damage involves the generation of specialized fibrous tissues that assist in tissue remodeling; deregulation or inappropriate activation of these repair processes can lead to fibrosis. Increasing evidence suggests that tissue fibrosis is a significant risk factor for development of cancer of the breast, lung, and many other organs. We present here a three-dimensional, microlithography-based model that can be used to break down the key steps involved in the earliest stages of fibrosis genesis. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The tree-like architecture of the mammary gland is generated by branching morphogenesis, a reiterative process of branch site initiation and tubule invasion from a pre-existing epithelial structure. Branching is controlled by the interplay between positive and negative regulators, defects in either of which can give rise to aberrancies ranging from hyperplasia to malignant growth. Our long term goal is to delineate how these positive and negative signals are integrated spatially within the tissue to determine which cells branch, and thereby define the branching pattern. We have developed a lithography-based three-dimensional organotypic culture model that recapitulates the architecture of mammary epithelial ducts, enables micrometer-resolution control of tissue geometry and microenvironment, and provides quantitative data in a physiologically relevant context. The engineered ducts execute a complete series of morphogenetic events that can be predicted computationally. Using this culture model, we have shown that the position of branching is determined in part by the concentration profile of transforming growth factor (TGF)-21, an autocrine inhibitory morphogen. Furthermore, we have found that cells located in positions that branch up- regulate the expression of mesenchymal markers during morphogenesis. Based on these preliminary and published data, we propose: 1- To investigate the features of the TGF21 concentration profile perceived and transduced by mammary epithelial ducts. 2- To determine the mesenchymal markers differentially expressed during morphogenesis, and whether these are necessary and/or sufficient to define position of branching. We will further test whether the pattern of mesenchymal gene expression is regulated by the TGF21 inhibitory profile. 3- To begin to dissect how branching is regulated by the physical properties of the microenvironment, by determining whether the extracellular matrix alters branching pattern, TGF21 inhibitory concentration profile, or neo-expression of mesenchymal markers. These studies will provide insight into the local cues and gene expression changes that govern position of branching. [unreadable] [unreadable] PROJECT RELEVANCE: Cells integrate information from stimulatory and inhibitory signals during branching morphogenesis to develop into the tree-like structure of the mammary gland; disruption or misregulation of these signals can lead to neoplastic growths and eventual development of frank tumors. Here we present studies aimed at understanding how mammary epithelial cells perceive inhibitory signals and translate them into patterned differences in gene expression during branching morphogenesis. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY The vertebrate lung develops via a process known as branching morphogenesis, wherein subgroups of epithelial cells are instructed reiteratively to form clefts or buds and thereby generate a space-filling tree with a sufficient surface area for gas exchange to support breathing after birth. An aberrant mechanical environment within the thoracic cavity can disrupt branching and cause fetal pulmonary hypoplasia, a major cause of respiratory insufficiency of the newborn. It is unclear how mechanical stresses control or disrupt the branching program. Here, we describe experiments combining tissue engineering approaches with investigations of intact embryonic lungs to define how mechanical stresses are transduced into gene expression changes that drive branching morphogenesis. Engineered lung tissues and computational models will be used to predict the role of mechanical stresses in branch site initiation. In Specific Aim 1, we will determine whether and how mechanical stresses regulate branching morphogenesis of engineered embryonic mouse lung tissues and intact chick and mouse embryonic lungs. In Specific Aim 2, we will define the mechanically induced gene expression changes that drive lung branching. To our knowledge, this work will represent the first comprehensive analysis of mechanically responsive genes in branching morphogenesis in culture or in vivo. We expect that the gene expression patterns revealed will uncover new avenues to explore for medical treatment of mechanically-induced diseases such as fetal pulmonary hypoplasia.

PROJECT SUMMARY The airway epithelium of the mammalian lung develops in the presence of exogenous fluid forces exerted from fetal breathing movements and peristaltic contraction of the surrounding smooth muscle. Defects in the mechanical environment of the thoracic cavity, including those due to congenital diaphragmatic hernia or oligohydramnios, can lead to pulmonary hypoplasia and respiratory failure after birth. Although several major biochemical signals, including fibroblast growth factor 10 (FGF10), have been identified in the control of airway branching morphogenesis, the signaling defects resulting from mechanical perturbations are unclear. Here, we propose to use microfluidic approaches to replicate the mechanical environment of the fetal chest cavity and explore effects from fluid pressure, volume, and flow on development of embryonic mouse lung explants. We will combine these microfluidic approaches with timelapse imaging of lungs explanted from transgenic reporter mice, particle imaging velocimetry analysis of the fluid flow within the airways, and molecular analysis of mechanotransductive signaling in the regulation of the FGF10 signaling axis. In Specific Aim 1, we will determine how static transmural pressure and luminal fluid volume regulate branching of the airway epithelium, development of the mesenchyme, and expression of FGF10 and its known regulators. We will also quantify mechanical regulation of proliferation, apoptosis, and cell shape changes in the epithelium, mesenchyme, and mesothelium. In Specific Aim 2, we will mimic the pressure changes that result from fetal breathing movements and quantify the effects of these dynamic changes on morphogenesis, gene expression, and fluid transport within the developing lung. This work will isolate the effects of pressure, volume, and flow and define precisely how each contributes to morphogenesis of the airways and their surrounding mesenchyme at both the cellular and molecular levels. We expect that this model system will open new avenues of investigation for identifying medical treatments to combat pressure-induced diseases such as fetal pulmonary hypoplasia.

DESCRIPTION (provided by applicant): The airway epithelial tree is sculpted in the embryo via branching morphogenesis, a process in which new daughter branches sprout laterally off a main stem (domain branching) or split from the tip of a parent branch (planar or orthogonal bifurcations). These branching events are physical by nature and occur within a dynamic mechanical environment which includes the contractility of the epithelium itself, static and phasic contractions of the surrounding airway smooth muscle, and distending transmural pressures from the presence of fluid within the lumen of the tree. Although abnormal development of the embryonic lung is frequently observed in fetuses with defects that cause mechanical alterations in these tissue compartments, the physical contributions of each that are responsible for driving the branching process are unknown. Signaling downstream of Wnts regulates airway branching and smooth muscle differentiation, and is likely responsive to mechanical alterations in the developing lung. Here, we hypothesize that the mechanical behavior of airway smooth muscle plays a central role in driving branching morphogenesis, and that both epithelial contraction and luminal fluid pressure regulate branching in part by altering signaling pathways that control smooth muscle differentiation and contractility. We will combine transgenic reporter mice with high-resolution real-time spinning disk confocal microscopy, microfluidic devices, three-dimensional traction force microscopy, and computational modeling to define how the mechanical behaviors of the airway epithelium, smooth muscle, and luminal fluid collaborate to direct branching morphogenesis. In Specific Aim 1, we will use transgenic reporter mice to determine how airway smooth muscle differentiation and contraction affect domain branching and terminal (planar and orthogonal) bifurcations of the airway epithelium. In Specific Aim 2, we will use microfluidic devices to control the transmural pressure across embryonic lung explants and define the role of transmural pressure in airway smooth muscle differentiation, epithelial branching, and mechanical signaling. In Specific Aim 3, we will characterize the contractility of the airway epithelium, quantify the forces exerted by the epithelium during branching, and determine how epithelial contractility directs mechanical signaling in the surrounding mesenchyme. This work will provide a complete mechanical portrait of the tissue compartments during morphogenesis, and define how each component contributes to the physical changes required to sculpt a new branch. We expect that the mechanical behaviors and signaling pathways revealed by this work will uncover new therapeutic options to treat fetuses and neonates who present with abnormalities in lung development.

Morphogenesis is the process by which simple tissues fold into the complex shapes of mature organs, such as the branching airways of the lung. This tissue folding is caused by patterns of mechanical forces (pushing and pulling the tissue) that are induced by the expression of different genes throughout the tissue. The final shapes of the lungs of mammals, birds, and reptiles are vastly different, suggesting that the mechanical forces and genes expressed are different as well. This award supports fundamental research to define the gene expression changes and mechanical forces that fold the airways of the lungs in three different species (mouse, chicken, and alligator). Understanding what is unique and what is conserved between lungs of different species will provide insight into how different organs evolved over time. Additionally, being able to manipulate the expression of genes to control organ development is critical for tissue engineering applications for the biomedical and healthcare industries. Results from this research will therefore have economic and societal benefits for the United States. This research combines techniques and insights from several disciplines, including developmental biology, mechanobiology, and biomedical engineering. This interdisciplinary approach will help broaden the participation of underrepresented minority groups (at the undergraduate and high school levels) and enhance outreach interactions with the lay population.

Mammals, birds, and reptiles have evolved different anatomical strategies for the conduction and diffusion of air through the lungs, which must result from differences in airway morphogenesis in the embryo. The objective of this project is to define and quantify these evolutionary morphogenetic differences. The research team will use three-dimensional traction force microscopy, real time confocal imaging, and continuum mechanical modeling to quantify the forces exerted during morphogenesis of the distinct airway architectures of embryonic mice, chicks, and alligators. In parallel, high-throughput gene expression analysis will be conducted for each morphogenetic movement in each species. These analyses will reveal the distinct gene expression changes that control mechanical forces within the epithelium as it folds lateral branches (mammals and birds), bifurcating branches (mammals and reptiles), and anastomosing airways (birds and reptiles). This research adopts for the first time the principles of mechanobiology within the framework of evolutionary developmental biology to create a comprehensive physical description of tissue morphogenesis, and will uncover the basic mechanical differences underlying lung development in different species.

? DESCRIPTION (provided by applicant): Breast cancer development is regulated by extracellular biophysical and biochemical cues. Among signaling mediators, members of the Rho family of small GTPases have been identified as controlling the growth, motility, invasion, and metastasis of breast cancer cells. We have previously implicated the splice isoform Rac1b as a key player in activation of epithelial-mesenchymal transition (EMT) and cellular invasiveness in breast cancer cells. Our recently published and preliminary data demonstrate that the key step in Rac1b control of cellular phenotype is through direct interaction with and activation of NADPH oxidase and consequent production of reactive oxygen species (ROS), and that the assembly of the Rac1b-NADPH oxidase complex is controlled by the extracellular matrix (ECM) and tissue tension. We hypothesize that Rac1b acts as a key signaling nexus to integrate mechanical and chemical signaling inputs from the surrounding ECM and tissue to control development of the malignant phenotype. We propose to test this hypothesis through the use of micropatterning, molecular biology, and numerical modeling with cultured cells, transgenic animals, and human breast tissue biopsies. In Specific Aim 1, we will combine engineered substrata with molecular biology approaches to define the molecular mechanisms through which the substratum microenvironment promotes Rac1b membrane localization, NADPH oxidase assembly, and ROS-mediated EMT. We will focus specifically on signaling via integrin-linked kinase (ILK). In Specific Aim 2, we will combine sophisticated three-dimensional engineered and organotypic culture models with experiments using transgenic mice to define how the microenvironment of the normal host epithelium affects Rac1b membrane localization, integrin signaling, EMT, and motility of the resident tumor cells. In Specific Aim 3, we will use breast tissue samples from women who have been found to have benign breast disease to define how age-associated changes in lobular structure and composition affect ILK, Rac1b signaling, and breast cancer risk. The proposed work will significantly advance understanding of how biochemical and biomechanical signals are integrated in the control of Rac1b- and ROS- associated tumor progression and how activation of EMT is controlled by microenvironmental signals. These advances will have direct relevance to our understanding of normal tissue development and progression to malignancy.

1531871(Arnold)

Addressing today's grand challenges in energy, environment, health, and security relies on the development of novel materials with extraordinary properties and the understanding of natural materials tasked with unprecedented performance. The properties and performance of a material depend on its structure across different size scales and although much information can be gained by looking at the surface, to truly understand the design and use of materials it is necessary to characterize their internal three dimensional structure without destroying the systems or devices in which they are used. This project seeks to achieve this goal by acquiring an X-Ray tomographic microscope (XRM) capable of visualizing the three dimensional structure of materials with submicron spatial resolution while controlling such environmental variables as tension-compression, temperature and fluid flow and without destroying the sample in the process. In doing so, Princeton will be able to complement its existing expertise in two dimensional characterization and create a user facility that will open the door to new kinds of characterization aimed at developing a deeper understanding of materials ranging across areas such as biological materials for tissue engineering, geological materials for carbon sequestration, or electrochemical materials for energy storage. These and numerous other frontier research efforts will enable Princeton and neighboring researchers to meaningfully and substantially contribute to the solution of pressing problems confronting the nation and the world. The XRM's ability to visualize the internal structures of materials will be incorporated to education and outreach programs that seek to inspire and excite young people about science and engineering and to instill confidence and motivation for academic achievement.

The goal of this project is to create a user facility that enables non-destructive, time resolved, 3-D structural and chemical characterization of materials to probe aspects of structure-property-processing-performance relations, formation and evolution of defects, compositional heterogeneity, and the spatial organization of phases for broad classes of materials. Through this funding we will acquire an X-Ray tomographic microscope (XRM) capable of visualizing the 3D structure of materials with submicron spatial resolution and the added ability to control such environmental variables as tension-compression, temperature and fluid flow. The x-ray microscope has a unique source-sample-detector design that provides unprecedented sensitivity to contrast imaging along with multiple length scale imaging of the same sample, spanning the nanometer scale (100 nm voxel dimension) up to the device-scale (25 micron voxel). Users will come from across the sciences and engineering, and specimens of immediate interest will include batteries, fuel cells, electronic devices, polymers, composites, biofilms, living tissues, cements, rock cores, sediments, vegetation and soil cores. This XRM's large working distances provide space for in situ cells that will be developed to enable time-dependent, in situ, nondestructive studies of materials under their real-world operating conditions.

PROJECT SUMMARY Current in vivo and in vitro models of human cancer remain limited in their ability to replicate progression to invasive disease in an easily accessible and physiologically relevant format. Tissue-engineered tumors may provide a more powerful system by enabling modular control over key aspects of a tumor and its microenvironment, such as vascular density or interstitial pressure. This collaborative study seeks to develop and apply new methods of engineering vascularized tumors in vitro, in which the cellular, physical, and genetic composition of the tumor and its microenvironment can be controlled with high spatial and temporal resolution. The collaborative team consists of experts in biomaterials and tissue engineering (Tien), quantitative developmental and tumor biology (Nelson), mechanics (Ekinci), and clinical tumor biology (Radisky) and pathology (Nassar). The core enabling technology, which we have been developing over the past fifteen years, is the use of three-dimensional (3D) micropatterned extracellular matrix hydrogels as scaffolds for directing the 3D organization of engineered tissues. Specifically, the proposed work will create microscale human breast tumors that contain perfused capillaries and draining lymphatics, which provide routes for tumor cell escape and enable the capture of those cells for downstream expression profiling. Interstitial stresses and biochemical composition will be analyzed by non-invasive imaging and repeated sampling of interstitial fluid, respectively, to provide longitudinal data for correlation with tumor cell behavior. This work will also create vascularized collagenous stroma that can accept human breast tumor biopsies as in vitro patient-derived xenografts, for the discovery of candidate mutations that favor tumor invasion and escape; these mutations will then be tested in hypothesis-driven analyses using the engineered breast tumors. More broadly, this work will disseminate these microscale tissue engineering technologies to cancer research laboratories for adaptation to other types of cancers and tumor cell behaviors.

PROJECT SUMMARY The branched architecture of the airways of the lungs permit the transfer of approximately six liters of air per minute between the external surroundings and the alveoli. The airway epithelial tree accomplishes gas exchange, mucus production, and pathogen clearance and blocks the entry of water, particulates, and microbes. To accomplish these diverse biological functions, the airway epithelium is comprised of several distinct cell types that differentiate from common progenitors during embryonic development, the first of which is the pulmonary neuroendocrine cell. Disrupting the differentiation of the specialized epithelial cell types negatively affects airway morphogenesis, and abnormally high numbers of pulmonary neuroendocrine cells are found in several congenital and acquired diseases of the lung. As it differentiates, the epithelium secretes ions and water across its apical surface, causing fluid to fill the lumen of the airways with a transmural pressure high enough to inflate the lungs. Defects that cause a decrease in transmural pressure are associated with both underdeveloped lungs and an increase in pulmonary neuroendocrine cells, but the specific role of pressure and the molecular signaling downstream of this mechanical cue are unknown. By combining time- lapse confocal imaging with an innovative microfluidic culture system, we found that transmural pressure controls the rate of lung development and the expression of markers of neuroendocrine cells. Using next- generation sequencing analysis, we found that low transmural pressure decreases the expression of targets of Notch, the master regulator of pulmonary neuroendocrine differentiation, and YAP, a known mechanosensor. Here, we hypothesize that transmural pressure coordinates the growth and differentiation of the different cell types within the epithelium by signaling through Notch and YAP. We will combine microfluidic devices with engineered mice, high-resolution time-lapse spinning disk confocal microscopy, and next-generation sequencing analysis to define the relative roles of pressure, Notch, and YAP in the regulation of pulmonary neuroendocrine progenitor fate decisions. In Specific Aim 1, we will use microfluidic chest cavities, engineered mice, time-lapse imaging, and single cell RNA-sequencing to define physically how transmural pressure regulates the pulmonary neuroendocrine population in the developing lung. In Specific Aim 2, we will use microfluidic chest cavities, reporter mice, and chromatin immunoprecipitation approaches to define whether and how transmural pressure regulates Notch signaling in the embryonic airway epithelium. In Specific Aim 3, we will determine whether pressure signals through YAP to affect pulmonary neuroendocrine differentiation and the Notch pathway. This work will define how mechanical signals from the microenvironment are transmitted to the first progenitor fate decision in the developing airway epithelium. We expect that our results will reveal novel insights into mechanical control of progenitor differentiation during tissue development and suggest new therapeutic targets for defects in lung development.

Cells can be coaxed into forming organ-like structures outside of the body. These “organoids” would help scientists study organ development, function, and disease. Organoids often fail to form in a reproducible manner, currently limiting their utility. This project will invent new approaches to reliably build organoids that mimic the lung. The investigators will track and control organoid formation using light and mechanical forces. The project will introduce high school and college students from underrepresented communities to scientific research. The project will also share its approaches by building an international symposium.

The minimal functional unit of the lung is the alveolus, which is comprised of alveolar epithelial type I (AT1) cells interspersed with type II (AT2) cells, surrounded by a meshwork of myofibroblasts that helps maintain 3D structure. The ability to reproducibly generate organoids that mimic the alveoli of the lung would have immense promise for studies aimed at understanding tissue function, the fundamental processes of respiratory infection, and the biomechanics of tissue structures during health and disease. Unfortunately, current protocols to generate alveolar organoids fail to reproduce native tissue structure. This RECODE project will uncover the rules necessary to differentiate alveolar progenitor cells into precise ratios of AT1:AT2 cells, and the contractile signaling that permits myofibroblasts to fold the epithelium into an alveolus. This transformational goal will be accomplished via a highly innovative combination of expertise from quantitative developmental biology, mechanobiology, biomaterials, computational modeling, and synthetic biology, which will be used to identify the spatiotemporal dynamics that governs alveolar differentiation and morphogenesis. The proposed research is divided into three main conceptual advances. Aim 1 focuses on using real-time fluorescent reporters, mathematical modeling, and optogenetics approaches to define the biochemical signaling dynamics necessary for specifying bipotent progenitors into AT2 cells. Aim 2 takes advantage of synthetic materials, real-time reporters, and optogenetics to uncover the mechanical signaling necessary for specifying bipotent progenitors into AT1 cells. Aim 3 combines computational modeling, 3D printing, and optogenetics to uncover and reproduce the patterns of contraction used by myofibroblasts to fold the epithelium into the alveolus. Altogether, this work will identify the design rules required to construct organoids that reproducibly differentiate into tissue structures that mimic alveoli within the lung.

This RECODE award is co-funded by the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences, the Developmental Systems Cluster in the Division of Integrative Organismal Systems, and the Engineering Biology and Health Cluster in the Division of Chemical, Bioengineering, Environmental, and Transport Systems.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.