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Date of Award
Doctor of Philosophy (PhD)
Epithelial cells form multi-layered tissue scaffolding that makes up every organ in the body. Along with epithelial cells, the basement membrane (BM) and connective tissue are composed of various proteins that sculpt the organs and protect them from foreign macromolecules. Epithelial cells respond to various cues, both chemical and mechanical, from their surrounding matrices to aid in maintenance and repair of these layers through degradation and deposition of extracellular matrix (ECM) proteins. In cancer progression, epithelial cells lose their normal function of supporting tissue structure and instead adopt more aggressive behaviors through an epithelial-to-mesenchymal transition (EMT) of their cellular traits. In some cases, these cells may break away from the primary tumor and begin to develop a secondary tumor elsewhere, a process known as metastasis. In these processes, cells are constantly interacting with highly heterogeneous and layered matrices that can influence cell behavior. This dissertation investigates how mechanical properties of both intact and defective layered matrices influence epithelial cell response. Chapter 1 details an introduction to epithelium structure and health, as well as how the mechanical properties of the matrix supporting the epithelium structure regulate epithelial cell behavior. Chapter 2 investigates the role of basement membrane (BM) integrity in maintaining epithelial monolayer health. A common outcome of EMT is degradation of surrounding ECM as cells become more mechanically active and migratory. Conversely, it is not known whether a break or defect in the BM could, in turn, induce EMT. We address this question in two parts. First, we layer BM-mimicking collagen-IV coatings onto tunable polyacrylamide (PA) hydrogels, induce a defect into the layered hydrogel, and then culture normal epithelial cell networks on the defect-laden substrates. Second, we layer the BM-like matrix atop a collagen matrix to investigate the impact of the BM defect on cell invasion. Our results show that defects in BM-mimicking substrates induce EMT in normal epithelial cells by promoting degradation of the BM itself, triggering mechanical activation of normal epithelial cells in 2D and further progressing EMT induction. We also demonstrate that BM defects can trigger epithelial cell invasion into the surrounding ECM through the same BM degradation mechanism. In chapter 3, we address the ability of normal and cancerous epithelial cells to sense distant matrix stiffness. Epithelial cells have been shown to be highly sensitive to substrate stiffness, particularly the stiffness of their immediate surroundings. However, epithelial cells reside in layered environments in which distinct layers stiffness and fiber structure can vary greatly. Yet, little is known about the ability for epithelial cells to sense these distant layers. Here, we investigate the extent to which distant matrix stiffness can affect cell migration by fabricating collagen-I matrices of varying thickness onto PA hydrogels of tunable stiffness. We have found that while normal epithelial cells are only able to sense the stiffness of their immediate surroundings, cancer cells show sensitivity to distant matrix stiffness through fibrous collagen matrices. Cancerous epithelial cells are able to sense distant matrix stiffness through greater actin fiber assembly, cell force generation, and by utilizing the fibrous intermediate collagen matrix to deform the distant PA substrate. By inhibiting the force-generating cellular machinery or by crosslinking the collagen network, we eliminate the ability of cancer cells to sense distant matrix stiffness. Our findings show that cancer cells may be affected by more than just their immediate surroundings, and, in turn, may influence the behavior of distant epithelial cells.
Lori Setton, Gretchen Meyer, Elliot Elson, Robert Mecham,
Available for download on Sunday, August 29, 2021