(397u) Development of a Novel in-Vitro Tumor Cell Extravasation Model

Cancer is the second leading cause of death in the United States, with metastatic lesions resulting from circulating tumor cells (CTCs) most often causing the mortality.  The basic steps of the metastatic cascade, from initial tumor formation to growth at a secondary site, are a linear sequence of steps composed of a network of mechanisms that have not been fully elucidated.  The goal of this work was to develop a novel nanoarray microfluidic platform allowing for the examination of the steps in the metastatic cascade, particularly extravasations, at the single-molecule level.  It is hypothesized that this platform may also be used as both a screening tool for drug discovery efforts as well as to further basic scientific research in identifying inhibitors of metastasis.  Single-molecule assessments offer several distinct advantages to conventional bulk scale analyses.  In addition to being highly sensitive and requiring small amounts of reagents, single-molecule evaluations allow for interactions to be measured one a time, eliminating the potential for lost distribution information in ensemble averaging. Additionally, structural and functional information can be directly probed and these molecules can be evaluated at native concentrations.  In this work, the optical and spatial resolution attainable using a nanoarray was exploited to study receptor-ligand interactions between cell surface adhesion molecules which are significant in each step of metastatic progression. It has recently been demonstrated that single molecules on the surface of living cells can be examined singularly and with high parallelism using these arrays since filipodia-like extensions on the plasma membrane are readily drawn into the array holes.  In these nanoarrays, single-molecule events such as nanoscale binding, conformation and stoichiometry may be both observed and quantified.  The focus of this work was to develop the nanoarray microfluidic device and complete proof of concept binding studies examining vascular endothelial growth factor (VEGF) receptors (VEGFRs) to observe dimerization of the receptors upon binding on VEGF which has not yet been examined on the single molecule scale.  VEGF was selected for study since it has been demonstrated to modulate extravasations and VEGFR antagonists have already been approved for cancer treatment.  The extension of this nanoarray platform to include a microfluidic channel beneath the array allowed for selective stimulation of the basal side to determine the effects of various stimulants on binding properties.  A discussion of device fabrication and verification as well as preliminary results from extravasation and binding studies will be presented.