Cell migration and invasion are critical steps in many physiological processes (e.g., embryogenesis, tissue homeostasis, wound healing) and disease states (e.g., cancer, atherosclerosis). A key component to these motility related cellular functions is proteolysis of extracellular matrix (ECM) proteins to remove physical barriers as well as modulate key signaling components. While it is well accepted that proteolysis is important during cellular migration, it has been difficult to directly examine and quantify the spatiotemporal regulation and coordination of proteolytic matrix remodeling.
We aim to develop a materials based fluorescent reporter system to characterize protease activity in three- dimensional environments and study how changes in the extracellular environment influence this activity during melanoma progression. Specifically, the proposed research plans aims to: 1) Develop a tunable 3D culture platform to investigate how extracellular microenvironment regulates melanoma cell proteolytic activity and 2) Examine how stromal cells influence melanoma cell proteolysis and migration. A progression of melanoma cell lines will be cultured in 3D hydrogels with fluorescent enzyme sensitive peptides incorporated as pendant functional groups to measure proteolysis of three enzyme classes: matrix metalloproteinases, cathepsins, and uPA. Regulation of proteolysis by the microenvironment will be investigated by culturing cells singly or in clusters to understand the role of homotypic cell-cell interactions and by varying the elasticity of the hydrogel to elucidate the role of cell-matrix interactions on local and global protease activity. Finally, usin primary fibroblasts isolated from healthy tissue or tumor associated fibroblasts, we will investigate the effect of stromal cell co- culture on melanoma cell migration and proteolysis. Collectively, this characterization should advance the basic understanding of the coordination of matrix remodeling and cell migration, provide a new method that allows spatial characterization of local protease activity, and lead to new insights into which proteases to target for more effective cancer therapies.
During cancer progression, tumor cells degrade the surrounding microenvironment, enabling the growth of new blood vessels to supply the primary tumor and allowing tumor cells to migrate to secondary sites, i.e. metastasis. To develop new therapies to inhibit this degradation, there is a need for a better understanding of the specific enzymes involved in degradation and how they are regulated. Towards this goal, this application develops new techniques to measure and visualize specific enzymes involved in degradation during cell migration and then investigates how degradation is regulated by cell-cell and cell-matrix interactions.
|Tokuda, Emi Y; Jones, Caitlin E; Anseth, Kristi S (2017) PEG-peptide hydrogels reveal differential effects of matrix microenvironmental cues on melanoma drug sensitivity. Integr Biol (Camb) 9:76-87|
|Leight, Jennifer L; Tokuda, Emi Y; Jones, Caitlin E et al. (2015) Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition. Proc Natl Acad Sci U S A 112:5366-71|
|Tokuda, Emi Y; Leight, Jennifer L; Anseth, Kristi S (2014) Modulation of matrix elasticity with PEG hydrogels to study melanoma drug responsiveness. Biomaterials 35:4310-8|
|Leight, Jennifer L; Alge, Daniel L; Maier, Andrew J et al. (2013) Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate. Biomaterials 34:7344-52|