(772e) Assessment of Microgroove-Aligned Endothelial Cells for in Vitro Simulations of Cancer Cell Adhesion

A thin layer of endothelial cells line the entirety of circulatory vessel walls.  Adhesion of cancer cells to this endothelial cell lining is an important step in the metastatic cascade and researchers are currently using in vitro techniques to investigate these interactions.  Under static culture conditions, endothelial cells grow in a characteristic cobblestone pattern rather than growing in straight lines due to the absence of shear stresses that would normally be present.  It has recently been suggested that such changes in cell morphology may also affect surface expression profiles, which can alter how frequently or strongly cancer cells bind to endothelial cells.  While flow adapting endothelial cells is important prior to studying cancer cell binding in vitro, traditional methods can be cumbersome due to the fact that the cells have to be exposed to flow for an extended period of time under controlled environmental conditions.   In this study, we investigated the efficacy of a microgroove-aligned method for acquiring a flow adapted phenotype.  Human Umbilical Vein Endothelial Cells (HUVECs) were cultured on glass slides that were fabricated using UV lithography which employed SU-8 2000.5, an epoxy-based negative photoresist, such that microscale grooves were present on the slides. The portions of the SU-8 photoresist exposed to UV light polymerized and remained on the slide while the unexposed areas were removed with developer, leaving only the hardened grooves. The resulting microgrooves were approximately 0.5 microns in both width and depth and were separated by 0.5 micron spaces.  Actin staining and fluorescent microscopy were used to compare the morphology of human umbilical vein endothelial cells (HUVECs) grown under static conditions on unmodified glass slides versus micropatterned glass slides.  Results demonstrated that desirable elongation and unidirectional growth was achieved using the microgroove-aligned system. Surface expression analyses of the HUVECs were also conducted to evaluate expression of four common cellular adhesion molecules involved in cancer metastasis (ICAM-1, VCAM-1, E-Selectin and P-Selectin) for HUVECs cultured on unmodified glass slides versus micropatterned slides. HUVECs grown on unmodified glass slides and stimulated with TNF-alpha (tumor necrosis factor alpha) were also employed as a positive control in these analyses, as TNF-alpha is commonly used in adhesion assays to stimulate adhesion molecule expression.  Fluorescent imaging was used to obtain histograms of image pixel intensities corresponding to adhesion molecule expression as well as spectrophotometer readings for further comparisons. Preliminary results indicate upregulation of VCAM-1 on HUVECs cultured on microgrooves as compared to unmodified controls, more reminiscent of in vivo expression.  Also presented will be a proof of concept cancer adhesion study in a parallel plate flow system employing unmodified and microgroove-aligned statically grown HUVECs versus HUVECs grown under flow conditions.