(278a) Modulating Cell-Matrix Interactions Is an Effective Method to Control Cell-Cell Junctions and Thus Improve the Predictive Capability of the Caco-2 in Vitro Model of Drug Permeability
Oral bioavailability is a key parameter in the development of any therapeutic compound, be it a biologic or a small molecule drug. The traditional preclinical method to screen for the oral bioavailability of a drug in vitro is the Caco-2 assay in which human colorectal adenocarcinoma cells (Caco-2) are cultured as a monolayer on top of collagen I-coated transwell inserts. While the Caco-2 assay has been a very successful in vitro predictor for many drug compounds, it has long been known to be ineffective at evaluating drugs that are transported via the paracellular route. Previous studies have demonstrated that paracellular transport is regulated by the presentation and organization of tight junction proteins at cell-cell contact points. The development and maintenance of tight junctions is influenced by focal adhesions at cell-matrix contact points, and tight junctions and focal adhesions are connected through the actin cytoskeleton. Therefore, we hypothesized that regulation of focal adhesions through matrix engineering could be leveraged to regulate tight junction formation and hence enable significant paracellular transport across Caco-2 monolayers. Protein engineering was used to synthesize a recombinant engineered extracellular matrix (eECM) based on an elastin-like amino acid sequence. Two independent matrix parameters, stiffness and density of integrin-engaging RGD ligands, were used to alter focal adhesion formation. As expected, the number of mature focal adhesions, as quantified by talin and vinculin staining, was significantly reduced on more compliant matrices and on matrices with fewer RGD ligands. Interestingly, the average Caco-2 cell area on the less adhesive eECM substrates was significantly less than that for cells grown on collagen I, and the eECM-cultured Caco-2 cells had similar cell area to primary native intestinal tissue epithelial cells. Furthermore, Caco-2 cells cultured on softer, lower integrin-engaging eECM exhibited a more small intestinal phenotype compared to collagen I-cultured Caco-2 cells, with increased expression of the tight junction marker claudin-2 and the small intestinal markers sucrase-isomaltase and aminopeptidase N. In drug permeability assays, the eECM cultured Caco-2 cells displayed a significant increase in paracellular transport of the model drugs inulin-FITC, ranitidine, famotidine, and nadolol. Notably, the use of eECM in this assay was found to not affect the permeability of dexamethasone and naproxen, drugs that are transported via transcellular routes. Our results demonstrate that the substitution of an eECM material for collagen I would be an easily implementable strategy for improving the physiological accuracy of the Caco-2 assay in preclinical screens for oral absorption of pharmaceutical drugs.