Bioactive Poly(ethylene glycol)-based Hydrogels for Characterization of Matrix Influences on a Lung Cancer Metastasis Model
West, Jennifer L.
Doctor of Philosophy
Pathological changes to tumor extracellular matrix (ECM) composition, mechanics, and architecture promote cancer progression and metastasis. Exploration of tumor-ECM interactions using in vitro matrix-mimetic culture systems has largely been restricted to naturally-derived matrix materials that permit limited experimental control. Such study of a novel lung adenocarcinoma model in Matrigel™ (MG) has suggested key matrix cues that mediate epithelial-mesenchymal transition (EMT) and metastasis. In this thesis work, synthetic hydrogel scaffolds based on poly(ethylene glycol) (PEG) featuring high experimental control and modular bioactivity were used to study matrix influences on the EMT-prone model line 344SQ. Encapsulation of 344SQ cells in PEG hydrogels modified for cell adhesivity and cell-mediated enzymatic degradability induced formation of lumenized, polarized spheres mimicking the epithelial phenotype observed in three-dimensional MG. Tuning matrix stiffness, adhesive ligand concentration, and ligand spatial presentation altered epithelial morphogenesis. Exploration of the EMT phenotype of PEG-encapsulated 344SQ cells revealed TGFβ-initiated changes in morphology, polarity, expression levels of EMT marker genes and their epigenetic controller, and the organization of cell-secreted ECM. Notably, a potent role for adhesive ligand was illuminated as matrices with low PEG-RGDS concentration even in the absence of TGFβ induced formation of spheres with a post-EMT phenotype by several of these measures. A matrix-invasive phenotype was also revealed by altering matrix structural parameters and tuned with incorporation of an alternative protease-cleavable sequence. Finally, the influence of cell-cell contacts was explored by covalent incorporation of cadherin proteins into the matrix. Matrix-tethered E- and -N-cadherin affected 344SQ sphere development in otherwise non-cell-adhesive matrices and modulated polarity and the degree of TGFβ response. Further, in 344SQ with a knockdown of the essential polarity-determining protein Scribble, matrix-tethered cadherin influenced the formation of a phenotype with partially normalized epithelial polarity with corresponding differences in membrane localization of cell-expressed E-cadherin. Overall, this thesis demonstrates the utility of the more experimentally controllable PEG system in studying ECM influences on cancer progression with findings providing greater insight into stromal biomechanical, biochemical, and cell-cell factors that mediate lung adenocarcinoma epithelial morphogenesis and EMT. These contributions help advance the state of the field towards a goal of developing new metastasis-targeting cancer therapeutics.