556 Modeling tumor promoting extracellular matrix dynamics in 3-D suspension culture

Abstract

Increased tissue stiffness, excessive extracellular matrix (ECM) densification and accumulation of collagen fibers and crosslinks are all associated with tumor cell invasion, immune evasion and tumor progression. We have developed a novel assay that recapitulates collagen fiber formation and tumor fibroblast collagen remodeling in vitro. Our method relies on tissue engineered from primary fibroblasts in suspension, and thus avoids the artificial mechanical stress associated with 2D culture on rigid substrates. Using this assay we measure collagen content and crosslinking via colorimetric analysis of total versus immature collagen fibers, relying upon collagen-specific Sirius red staining and analyses of birefringent collagen fibrillar deposits with the use of a polarizing microscope. Quantification of areas occupied by red-colored and green-colored fibrils is therefore a measure of collagen maturation and crosslinking, with red birefringence an indication of mature collagen fibrils and green birefringence an indication of immature collagen fibrils, as demonstrated by the effects of TGFb1 ligand stimulation and TGFb receptor type I inhibition. An increase of immature collagen fibrils over time is indicative of increased remodeling of collagen-rich ECM. Using multiple patient samples, we determined that collagen assemblies produced by squamous cell carcinoma (SCC) fibroblasts (n=4) are continually remodeled in contrast to those from isogenic normal fibroblasts (n=3), and that these differences correlate with invasion when fibroblast matrices are co-cultured with SCC keratinocytes. We are further investigating the parameters of collagen remodeling and tumor cell invasion with the use of b-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, recombinant lysyl oxidase and the lysine analogue trans-4,5-dehydrolysine.