ARTICLE ABSTRACTMatrix metalloproteinases (MMP) have been implicated in multiple stages of cancer metastasis. Tissue inhibitor of metalloproteinase-2 (TIMP-2) plays an important role in regulating MMP-2 activity. By forming a ternary complex with pro-MMP-2 and its activator MMP-14 on the cell surface, TIMP-2 can either initiate or restrain the cleavage and subsequent activation of MMP-2. Our recent work has shown that breast cancer cell adhesion to vascular endothelial cells activates endothelial MMP-2, promoting tumor cell transendothelial migration (TEME). However, the mechanism of MMP-2 regulation during TEME remains unclear. In the current study, we present evidence that MMP-14 is expressed in both invasive breast cancer cells (MDA-MB-231 and MDA-MB-436) and lung microvascular endothelial cells (HBMVEC-L), whereas TIMP-2 is exclusively expressed and released from the cancer cells. The tumor cell–derived TIMP-2 was further identified as a major determinant of endothelial MMP-2 activity during tumor cell transmigration in the presence of MMP-14. This response was associated with endothelial barrier dysfunction because coculture of MDA-MB-231 or MDA-MB-436 with HBMVEC-L caused a significant decrease in transendothelial electrical resistance concomitantly with endothelial cell-cell junction disruption and tumor cell transmigration. Knockdown of TIMP-2 or inhibition of TIMP-2/MMP-14 attenuated MMP-2–dependent transendothelial electrical resistance response and TEME. These findings suggest a novel interactive role of breast cancer cells and vascular endothelial cells in regulating the TIMP-2/MMP-14/MMP-2 pathway during tumor metastasis. Mol Cancer Res; 8(7); 939–51. ©2010 AACR.