Tuning Gel Stiffness with Surface Chemistry to Study Cancer Cell Behavior

Cancer metastasis occurs when cancer cells migrate away from the source of the tumor to other tissues in the body. Treatment options become limited once the cancer has metastasized, which motivates an understanding of what factors impact cancer cell migration. Metastasis is driven by extracellular matrix (ECM) cues such as chemical gradients, orientation of ECM fibers, or stiffness of the ECM that direct cell migration. In this study, we examined how cancer cells respond to changes to the stiffness of a collagen gel in a 3-D environment. Previous studies have used gel density or cross-linking in polymer networks to control the stiffness of various gels. However, changing gel density or cross-linking can result in a system with different properties, such as number of cell-ECM attachment sites, which also impacts cell behavior. It is known that attaching a soft gel to a stiff substrate and adjusting the thickness of the soft gel can be used to control local stiffness in a material. We used this approach in addition to chemical linkage between a surface and a soft gel to control the local gel stiffness. A study showed that cells had a larger area when located closer to the interface between a soft gel and hard material. Therefore, it was predicted that cells located near a chemically-modified glass surface would have larger areas due to the larger local stiffness at the surface. To test this hypothesis, MDA-MB-231 breast cancer cells were embedded in collagen gels between two glass coverslips of a specific surface chemistry (clean glass or glutaraldehyde-treated) and gel thickness. The cells were stained for F-actin, and morphological properties of the cell were measured using ImageJ. The cells were found to have larger areas when located near a glutaraldehyde-treated glass surface compared to the cells near the surface of clean glass regardless of soft gel thickness. In addition, cells found further from the glutaraldehyde-treated surface had smaller areas than those found near the surface. On the other hand, when cells were embedded in collagen between two clean glass coverslips, the measured cell area was the same regardless of distance from the surface or collagen gel thickness. This indicates that there exists some distance from the surface in which cells can no longer sense the chemical linkage between the collagen fibers and the glutaraldehyde-treated glass surface. This result is significant because it demonstrates that cells respond differently in the ECM when located near stiffer tissue within the body. This finding could be used in tissue engineering applications where surface linkage might control cell function or in fundamental studies where researchers are trying to understand responses to stiffness.