University of Florida Department of Chemical Engineering 1 ECM Substrate Effects on Cell Motility Jose I. Varillas, Shen-Hsiu Hung, Qian Peng, Stephen H. Arce, Jun Yin, and Yiider Tseng Department of Chemical Engineering, University of Florida, Gainesville, FL ABSTRACT Cell intrinsic motility and morphology are highly affected by its surrounding environmental conditions. Extracellular proteins have been thoroughly studied along with their effects on Rho GTPases, which been closely linked with cellular movement. Therefore, we investigated the contributing effects two ECM proteins, fibronectin and collagen, have on NIH-3T3 fibroblast motility. In this study, cell motility is characterized through a novel biophysical assay that uses the correlations of the cellular and nuclear centroid minutely displacements to precisely explain the subcellular activity of 3T3 fibroblasts on ECM and also quantify their migration capacity. The results suggest that a fibronectin-rich environment positively affects effective cell displacement and migration potential, compared to a collagen substrate which induced stagnant behavior associated with loss of cell polarity and increased cell sampling, or membrane ruffling. The student t-test was applied to indicate the statistical difference (p < 0.001). This provides us with an insight of the ECM effects on subcellular activity and on the cell-ECM interaction in general. Knowledge gained from these experiments could prove useful in cancer prognosis, diagnosis, or treatment. INTRODUCTION Coordinated cellular activity characterized by tightly regulated cycles of polarization, adhesion formation, cytoplasmic protrusion, and rear retraction is referred to as cellular migration and it is of paramount importance in the development and maintenance of multicellular organisms (Ananthakrishnan and Ehrlicher, 2007). Cell migration can be described in three stages detailed by key events. First, cells form lamellipodia and filopodia to perform substrate probing in order to establish, or reinforce, adhesions. The cells then polarize through nuclear relocation in order to help align the cytoskeletal components necessary for movement and support. Finally, the cells release their rear end anchorage by detaching adhesions, allowing for forward movement to occur (Horwitz and Webb, 2003). The migration of cells plays a key role in physiological events such as wound healing (Martin, 1997), morphogenesis (Juliano and Haskill, 1993), and pathological events; in the progression of diseases such as cancer (Bernstein and Liotta, 1994), atherosclerosis (Kraemer, 2000), and arthritis (Ingegnoli et al., 2002). Cell migration includes a cell’s ability to respond to external signals, in the form of chemical and mechanical stimuli, as well as its intrinsic motility. Failure in key migration steps could lead to defects in cell functions that depend on them and result in immunosuppression, autoimmune diseases, defective wound repair, or tumor dissemination. The Extracellular Matrix The extracellular matrix (ECM) is the non-cellular component of all tissues that provides structural support to the cells and it’s highly involved in various important functions such as cell migration (Kuntz and Saltzman, 1997). It also initiates essential biochemical and biomechanical cues required for cell differentiation and homeostasis. The ECM is composed mostly of fibrous proteins, proteoglycans, and polysaccharides, secreted by the cells, which are assembled into an organized network in association with the surface of the cell that produced them. Most normal vertebrate cells can’t survive unless they’re attached to the ECM. This anchorage dependence is lost, however, when cells become cancerous; avoiding anoikis is a key characteristic of metastatic cancer cells. Cell adhesion to the ECM occurs through actin and intermediate filaments. Integrins are transmembrane