(772a) Traction Stress As a Modulator for Protease Dependent Invasion of Cancer Cells
AIChE Annual Meeting
2013
2013 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Cell Biomechanics I
Friday, November 8, 2013 - 8:30am to 8:48am
Introduction: The ability of cancer cells to invade into and migrate within the extracellular matrix (ECM) is central to metastatic dissemination. Conventionally, this process was thought to occur via proteolytic degradation of the pericellular ECM network. However, studies demonstrating that cell invasion and migration can be only partially attenuated by protease inhibitors suggest the existence of protease independent mode of migration as a compensation mechanism. Although recent findings have indicated the role of physicochemical cues of the ECM in dictating the particular mode of migration, the intracellular signal by which a particular mode of migration occurs remains elusive. In this study, we demonstrate that cell generated traction stress modulates the transition from protease independent to dependent mode during invasion of cancer cells into 3D matrices through the activation of a regulatory Golgi transport pathway known as CARTS.
Materials and Methods: Single cell invasion assay into Matrigel was utilized to probe the transition from protease independent to dependent mode of invasion and the role of ECM properties on this. To this end, Matrigel, embedded with fluorescent particles, were spin coated onto glass surfaces to obtain thicknesses of 10, 20, and 30 µm. Cancer cells, MDA-MB-231, were plated onto these matrices to initiate the invasion process. As a function of invasion depth, individual cells were analyzed for the traction stress utilized to deform the matrix and whether these forces result in any permanent deformation of the Matrigel due to proteolysis. Furthermore, we examined the location of a key transmembrane matrix metalloproteinase (MT1-MMP) as well as its colocalization with the Trans-Golgi Network Marker 46 (TGN46) and a secretory protein involved in the CARTS pathway, Pancreatic Adenocarcinoma Up-regulated Factor (PAUF).
Results and Discussion: By decreasing the thicknesses of the Matrigel on the length scale of a single cell, we increased the mechanical resistance encountered by the invading cells while keeping other factors such as ligand density and other interfacial properties unaltered. We observed that the protease induced permanent deformation of the Matrigel occurs at traction stresses of approximately 120 Pa irrespective of Matrigel thickness. We also applied mechanical stresses up to 200 Pa via glass bead modified probes using atomic force microscopy and failed to observe the mechanical yielding of Matrigel. Furthermore, at stresses below 120 Pa, we observed that MT1-MMP remains within the cytoplasm as the cell utilize bleb formation to penetrate into the Matrigel. At larger forces, we observed the translocation of MT1-MMP to the cellular periphery as well as actin and cortactin rich protrusions at the leading edge of the cell indicating the formation of invadopodia. We next examined whether the translocation of the MT1-MMP occurs through CARTS pathway during this process. We observed that the depletion of the TGN46 through inhibition of Protein Kinase D abrogated the invasion processes. In addition, we observed the colocalization of TGN46 and PAUF with MT1-MMP within cells generating traction stresses larger than 120 Pa.
Conclusions: Traction stresses generated by the cells during their invasion triggers the transition from protease independent to dependent mode of migration. During protease indepedent mode, cells utilize bleb formation devoid of MT1-MMP on the cellular periphery to deform the matrix while during protease dependent mode, cells utilize invadopodia-like structures with MT1-MMP localized to the cellular periphery to degarde the matrix. We aslo shows that the transport of MT1-MMP during this process occurs through a regulatory Golgi transport pathway known as CARTS.