Molecular Tracking of Proteolysis During Breast Cancer Cell Extravasation: Blockage by Therapeutic Inhibitors

Elucidating the mechanism of focal proteolysis at the cell-matrix interface during metastasis is an ongoing challenge. We have completed the dissection of components of proteolytic machinery required for breast cancer cells (metastatic MDA- MB231 and non-metastatic MCF-7) transendothelial migration (TEM). Modulation of individual molecules demonstrates the functional cooperation of furin, cell surface adhesion molecules (alpha(sub v)Beta(sub3), CD44), and matrix metalloproteinases (MMP-2, MMP-9 and MTI-MMP) during the process of TEM. Confocal microscopy shows co-localization of molecules and MMPs critical for TEM, and disruption of these molecules reduces TEM of MDA-MB231 cells. MMP-2 and MTI -MMP localization matches the imprint of spatially restricted fluorogenic gelatin digestion. The digestion occurs in a stage-specific manner and becomes most evident during the migratory phase of tumor cell TEM. Of the above breast cancer cell lines, MDA- MB231 is able to undergo efficient TEM, and MTI-MMP emerges to be one of the key molecules involved for this event. The lack of several key components in MCF-7 cells impairs its ability to transmigrate. We have now extended our analyses of protease activity by these cells in vivo to the mammary gland, illustrating their differential capacity to degrade the matrix components in the transplanted plug. Our data reveal how specific molecular interactions result in a cooperative proteolytic interface at the metastatic tumor cell surface. We have shown this in vitro utilizing the breast tumor cell-endothelial cell microenvironment during TEM, and in vivo in the mammary tissue environment.