Abstract The acquired ability for tumor cells to migrate and invade through the extracellular matrix within the tumor microenvironment has long been accepted as a hallmark of metastatic potential. Numerous signal transduction pathways and a wide variety of protein classes have been implicated as important players in cell migration and/or invasion. For example, matrix-metalloproteinases (MMPs) have been implicated in the degradation of the basal lamina that is required for the movement of cells through the matrix. Likewise, the MEK/ERK pathway has been implicated in upregulated expression of MMPs in tumor cells. Furthermore, recent evidence has indicated that myosin II also plays a major role in tumor cell invasion. However, the relative contribution of myosin II in cell migration compared to cell invasion remains unclear. In this study we use the myosin II inhibitor, blebbistatin, to concurrently investigate the relative contribution of myosin II to both 2D migration and 3D invasion of human fibrosarcoma derived HT 1080 cells through collagen I and Matrigel matrices. To accomplish this, we utilized a novel assay strategy using a live-cell imaging system, the IncuCyte-FLR, to simultaneously measure migration and invasion of non-labeled cells in a 96-well plate format. Following assay initiation, images of all 96-wells were obtained every three hours until assay completion. Each image was automatically analyzed using phase contrast image based algorithms. Our results indicate that blebbistatin is approximately one order of magnitude more potent in its ability to inhibit cell invasion through both collagen I and Matrigel matrices compared to its ability to inhibit 2D cell migration (e.g. IC50 value for invasion is 5μM compared to the IC50 value for migration, >90μM). Similar, less pronounced results were also observed when human breast adenocarcinoma derived MDA-MB-231 cells were identically evaluated. These results suggest that the myosin II contractile machinery is more heavily taxed during the process of invasion compared to migration in two distinct metastatic cell types. In addition to the role of myosin II, we extended our study to investigate the relative contribution of both MMPs and the MEK/ERK pathway to cell migration and invasion using the inhibitors GM6001 and U0126, respectively. Interestingly, with both inhibitors, we observed minimal effects on cell migration, but significant inhibition of cell invasion through collagen I. In addition, whereas inhibition of myosin II had a significant, immediate effect on invasion, broad inhibition of several MMPs and inhibition of the MEK/ERK pathway did not significantly show an effect until 5, or 10 hours after assay initiation, respectively. These time points are often well past the end point of other commonly used approaches to measure cell invasion. We also found that the results of this assay were dependent both on cell density as well as matrix concentration illustrating the importance of assay optimization. Using this novel, kinetic approach, the results of this study further clarify a more significant role for myosin II in cell invasion when compared to cell migration. We also provide additional evidence that MMPs and MAPK signaling pathways are significantly involved in the process of cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C1.