An accurate understanding of cell migration requires an understanding of not only its chemical and biological determinants, but also its response to the extracellular environment. In vivo, cells are surrounded by an extracellular matrix in all three dimensions, characterized by the simultaneous action of multiple cellular and extracellular variables. In addition to cellular adhesion and traction forces, the dense fibers in 3D matrices can block cell movement in the absence of matrix degrading enzymes (also known as matrix metalloproteinases, or MMPs). Therefore, in order to fully comprehend the underlying mechanisms by which cells migrate in vivo, it is necessary to study motility in an environment that mimics physiologically relevant physical and chemical characteristics. Our current research utilizes peptide functionalized PEG gels formed via thiol-ene polymerizations, and also aims to exploit sequential orthogonal reactions to dynamically and spatially change the cellular microenvironment. Our goal is to undertake a systematic method of identifying how the key factors known to regulate cell motility act synergistically in native-like three-dimensional matrices. We collaborate with Natatlie Ahn’s group in Chemistry and Biochemistry at CU to better understand signaling mechanisms, as well as researchers in the School of Medicine to apply this knowledge towards the screening of drugs for melanoma treatment. Materials are being developed to understand the role of MMPs in cancer progression and how human mesenchymal stem cells migrate to aid in bone repair. Finally, the information gathered from our cell tracking experiments are combined with computational studies from Mohammad Zaman’s group at Boston University to provide further insights into the fundamental aspects of cancer metastasis and tumor cell motility.