(62bc) Understanding Cancer Cell Motility and Interactions with the Extracellular Matrix

Cancer research focuses primarily on biochemical signaling in the tumor microenvironment. While this is one crucial aspect of understanding cancer, the effects of mechanical signals, such as stiffness and dimensionality, are proving to be important as well. However, most experiments performed in this area so far have been done on 2-dimensional (2D), planar substrates, which do not accurately represent the physiological environment in vivo. Prior research shows that cells embedded in 3-dimensional (3D) extracellular matrices display drastically different motility characteristics and, as they do not form focal adhesions in 3D matrices, the differences might be due to the altered role of the focal adhesion proteins. Nonetheless, it has proven difficult to isolate the reason for these differences, as altering the 3D extracellular matrix leads to changes not only in stiffness but also in pore size and ligand density.

The first objective of my research is to develop a method that will provide controlled stiffening of a 3D collagen gel, while maintaining constant pore size and ligand density. For the first time, this will allow for quantitative determination of the effect of stiffness alone on cell motility. There are many methods of altering the stiffness of a 3D collagen gel. Here, we chose to compare the effect of increasing collagen concentration?which alters stiffness, pore size, and ligand density--against using Transglutaminase II enzyme (TGII) as a cross-linker. We show that the pore size and ligand density of Transglutaminase II crosslinked gels are unchanged compared to the uncrosslinked gel. Cell viability and effect of stiffness on 3D motility were assayed by embedding HT1080 Human Fibrosarcoma cells in the matrix and using time-lapse microscopy along with other novel techniques including intracellular microrheology and a 3D matrix traction assay. The results showed wild type cell velocity decreased as collagen concentrations increased as well as when the matrix was stiffened with TGII.

Since focal adhesion proteins are thought to be important in the mechanotrasduction of external signals to the inside of the cell, we next embedded HT1080 cells with various focal adhesion proteins knocked down in uncrosslinked and crosslinked gels. We measured motility characteristics, such as velocity and number of protrusions, of the knockdown cells in a crosslinked matrix and compared them to those measured in uncrosslinked gels and also to wild type cells in both conditions. This allowed us to determine which focal adhesion proteins are important to the cell in sensing stiffness in 3D matrices.

Our findings indicate that stiffness of the extracellular matrix indeed plays a critical role in cell motility by altering the velocity and reducing the number of protrusions. The development of this novel and accessible technique of stiffening 3D matrices using TGII allows for exclusive analysis of an important variable to cancer cell motility?stiffness. Further study will lead to a greater understanding of the molecular mechanisms driving cell migration and may reveal a possible treatment or prevention method for cancer. Isolating and understanding the mechanical transduction pathway in a cancer cells will provide greater insight into the mechanism of metastatic tumors and the role that the surrounding extracellular matrix plays.