Decoupling the phenomenological effects of collagen fiber density and matrix stiffness on A549 tumor spheroid

The tumor microenvironment consists of a phys- ical gradient field that guides tumor invasion. Collagen, a major component of the extracellular matrix (ECM), forms a fibrous network with flexible stiffness. Increased ECM stiffness enhances collective tumor migration, an indicator of metastasis. However, tumor growth can also deform and remodel the collagen matrix into a dense-fiber environment, which may limit tumor migration. Therefore, the mecha- nisms underlying tumor behavior in response to individual matrix properties must be clarified. In this study, we devel - oped a collagen-coating technique to delineate the effects of fiber density and matrix stiffness on tumor growth. Low and high collagen concentrations represented low- and high- fiber-density substrates, respectively, which were applied to glass or polyacrylamide gels, representing stiff and soft matrices. The stiff matrix supported A549 tumor spheroid expansion, whereas the collagen fiber density slightly coun- teracted stiffness-guided cancer migration. Specifically, the degree of collagen fiber density decelerated tumor spheroid expansion on the stiff matrix but not on a soft matrix. Our study provides a novel engineering approach for decipher - ing the effects of both matrix stiffness and fiber density. These results suggest that fiber density exerts its inhibitory effects on stiff matrices.