Indiana University-Purdue University Indianapolis (IUPUI) / Stiffening and softening of extracellular matrix (ECM) are critical processes governing many aspects of biological processes. The most common practice used to investigate these processes is seeding cells on two-dimensional (2D) surfaces of varying stiffness. In recent years, cell-laden three-dimensional (3D) scaffolds with controllable properties are also increasingly used. However, current 2D and 3D culture platforms do not permit spatiotemporal controls over material properties that could influence tissue processes. To address this issue, four-dimensional (4D) hydrogels (i.e., 3D materials permitting time-dependent control of matrix properties) are proposed to recapitulate dynamic changes of ECM properties. The goal of this thesis was to exploit orthogonal enzymatic reactions for on-demand stiffening and/or softening of cell-laden hydrogels. The first objective was to establish cytocompatible hydrogels permitting enzymatic crosslinking and stiffening using enzymes with orthogonal reactivity. Sortase A (SrtA) and mushroom tyrosinase (MT) were used sequentially to achieve initial gelation and on-demand stiffening. In addition, hydrogels permitting reversible stiffening through SrtA-mediated peptide ligation were established. Specifically, poly(ethylene glycol) (PEG)-peptide hydrogels were fabricated with peptide linkers containing pendent SrtA substrates. The hydrogels were stiffened through incubation with SrtA, whereas gel softening was achieved subsequently via addition of SrtA and soluble glycine substrate. The second objective was to investigate the role of dynamic matrix stiffening on pancreatic cancer cell survival, spheroid formation, and drug responsiveness. The crosslinking of PEG-peptide hydrogels was dynamically tuned to evaluate the effect of matrix stiffness on cell viability and function. Specifically, dynamic matrix stiffening inhibited cell proliferation and spheroid formation, while softening the cell-laden hydrogels led to significant increase in spheroid sizes. Matrix stiffness also altered the expression of chemoresistance markers and responsiveness of cancer cells to gemcitabine treatment. markers and responsiveness of cancer cells to gemcitabine treatment.

<div>Pancreatic cancer is the present third leading cause of all cancer-associated deaths with a under 9% 5-year survival rate. Aggressive tumor progression and lack of early detection technique lead to the fact that most patients are diagnosed at terminal stage - pancreatic ductal adenocarcinoma (PDAC). Despite that numerous therapeutic approaches have been introduced, most options cannot advance to or fail at the clinical trials. It has been suggested that previous failure is due to insufficient understanding of PDAC tumor microenvironment (TME). Human PDAC is composed of severely fibrotic tissue (i.e., desmoplasia) that harbors a variety of malignant cells (e.g., pancreatic stellate cells, cancer-associated fibroblasts, macrophages, etc.), excessive extracellular matrices (ECM), as well as abnormal expression of growth factors, cytokines, and chemokines. Multiple cell-cell and cell-ECM interactions jointly result in a stiffened, hypoxic, and fluid pressure-elevated PDAC tissue. The resulting pancreatic TME not only physically hinders penetration of therapeutics, but also dynamically interacts with the residing cells, regulating their behaviors.</div><div><br></div><div>Increasing tumor tissue stiffness in PDAC is not only a passive outcome from desmoplasia, but an active environmental factor that promotes tumor survival, growth, and invasion. However, traditional in vitro cell culture systems such as two-dimensional (2D) culture plate and animal models are not ideal for mechanistic understanding of specific cell-matrix interactions. Therefore, dynamic hydrogels have been introduced as a category of advanced biomaterials that exhibit biomimetic, adaptable, and modularly tunable physiochemical property. Dynamic hydrogels can be precisely engineered to recapitulate a variety of aspects in TME, from which to investigate the role of dynamic tumor-stroma interaction in PDAC progression. The goal of this dissertation was to exploit synthetic polymers (i.e., poly(ethylene glycol) (PEG)) or natural ECM (i.e., gelatin and hyaluronic acid (HA)) as precursors to prepare the dynamic cancer-cell laden gels. The design utilized the orthogonal thiol-norbornene photopolymerization to prepare the primary homogenous xxvi</div><div><br></div><div>gel network. Next, through further functionalizing gel precursors with phenolic derivatives, enzymatic reaction (i.e., tyrosinase) or flavin mononucleotide (FMN)-mediated photochemistry could be harnessed to manipulate the dynamic changes of substrate mechanics. Experimentally, a computational model and the associated validation were presented to investigate the process of gel stiffening. Finally, these techniques were integrated to prepare cell-laden gels with spatial-temporally tunable properties that were instrumental in exploring the synergistic effects of dynamical matrix stiffening and presence of HA in promoting epithelial-mesenchymal transition (EMT) in PDAC cancer and stromal cells.</div>