Rapid fabrication of collagen bundles mimicking tumor-associated collagen architectures

Abstract

Stromal collagen is upregulated surrounding a solid tumor and presents as dense, thick, linearized, and aligned bundles. The collagen bundles are continually remodeled during tumor progression, and their orientation with respect to the tumor boundary has been correlated with invasive state. Currently, reconstituted-collagen gels are the standard in vitro tumor cell-extracellular matrix interaction model. The reticular, dense, and isotropic nanofiber (~900 nm-diameter, on average) gels do not, however, recapitulate the in vivo structural features of collagen bundling and alignment. Here, we present a rapid and simple method to fabricate bundles of collagen type I, whose average thickness may be varied between about 4 μm and 9 μm dependent upon diluent temperature and ionic strength. The durability and versatility of the collagen bundles was demonstrated with their incorporation into two in vitro models where the thickness and alignment of the collagen bundles resembled various in vivo arrangements. First, collagen bundles aligned by a microfluidic device elicited cancer cell contact guidance and enhanced their directional migration. Second, the presence of the collagen bundles in a bio-inert agarose hydrogel was shown to provide a route for cancer cell outgrowth. The unique structural features of the collagen bundles advance the physiological relevance of in vitro collagen-based tumor models for accurately capturing tumor cell-extracellular matrix interactions. STATEMENT OF SIGNIFICANCE: Collagen in the tumor microenvironment is upregulated and remodeled into dense, thick, and aligned bundles that are associated with invasive state. Current collagen-based in vitro models are based on reticular, isotropic nanofiber gels that do not fully recapitulate in vivo tumor stromal collagen. We present a simple and robust method of rapidly fabricating cell-scale collagen bundles that better mimic the remodeled collagen surrounding a tumor. Interacting with the bundles, cancer cells exhibited drastically different phenotypic behaviors, compared to nanofiber scaffolds. This work reveals the importance of microscale architecture of in vitro tumor models. The collagen bundles provide physiologically relevant collagen morphologies that may be easily incorporated into existing models of tumor cell-extracellular matrix interactions.

Keywords: 3D culture; Collagen; Hydrogels; Invasion; Microfluidics; Tumor microenvironment.