Abstract P4-06-04: Rho-Family GTPase Activity in Invading Mammary Tumor Cells Visualized by Two-Photon Excitation Laser Scanning Microscopy

Abstract

Abstract Background: Rho-family GTPases play critical roles in the regulation of tumor cell motility and invasion. Despite the vast accumulated knowledge of Rho-family GTPase regulation in two-dimensional culture models, their three-dimensional spatio-temporal regulation in vivo remains poorly understood because of technical difficulties. The ability of tumor cells to migrate in vivo depends on both physical and biochemical characteristics of the tumor microenvironment, i.e., extracellular matrix, stromal cells and myeloid cells as well as tumor cell properties. Therefore, development of methods for directly measuring the activity of Rho-family GTPases in vivo is required for further understanding the mechanism of tumor invasion. Material and Methods: To visualize the activity of Rho-family GTPases in vivo, we developed fluorescence resonance energy transfer (FRET)- based probes comprising of fluorescent proteins. The probes were stably expressed in 4T1 mouse mammary tumor cells by means of retrovirus-mediated gene transfer. 4T1 mouse mammary tumor cells highly expressing FRET probes were selected by fluorescence activated cell sorting (FACS) and implanted subcutaneously in nude mice. The 4T1 cells formed tumors within 5 to 10 days. The activity of Rho-family GTPases was examined by two-photon excitation laser scanning microscopy (TPLSM). The collagen matrix surrounding tumors was visualized by second harmonic signal. TexasRed-dextran was injected into nude mice intravenously before observation for the visualization of blood vessels. Results: We found that 4T1 mouse mammary tumor cells assumed an elongated, mesenchymal phenotype in vivo and in three-dimensional culture models. Cell motility in vivo was significantly slower than in 2D cell culture models. In the in vivo models, the elongated tumor cells demonstrated cell movement along aligned collagen fibers away from the primary tumor mass. Furthermore, we observed that Rac1 activity was higher at the tumor-stroma borders than at the tumor cores, using FRET imaging. Discussion: In this study, we successfully monitored the activity of Rho-family GTPases in vivo by FRET imaging. We also confirmed that 4T1 mouse mammary tumor cells mainly exhibited a mesenchymal phenotype when these tumor cells invaded into the surrounding stromal tissue. Recent studies have shown that some tumor cells exhibit two different modes of individual cell movement in 3D cell culture models: mesenchymal movement is characterized by an elongated cell shape and a dependence upon ECM proteolysis, whereas in amoeboid movement, cells have a rounded morphology, and are less dependent on proteases. We hereby demonstrate the utility of a three-dimensional, in vivo imaging technique toward understanding the mechanisms underlying tumor invasion. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-04.