| The process of tumor transvascular metastasis is a complex biologic events that includes the following steps:1)Tumor cells growth ,and release of neoplastic cells from the primary tumor mass.2)Tumor invasion to the basement membrane, migration and intravasation into blood vessels.3)Adhesion to the vascular endothelium of metastatic sites, infiltrate through the endothelium and extravasation through the vessel wall.4)Invasion of the recipient tissue bed and proliferaton within the tissue. The extravascular migration of tumor cells is the critical steps in transvascular metastasis, it is the course of interaction between tumor cells and endothelial cells.First the tumor cells,entry into the blood circulation, must arrest at the metastatic site and adhere to the luminal surface of the endothelial cells, it is the premise of extravasation through the vessel wall. Mechanisms involved after tumor cells adhesion have received less attention. In particular, signal transduction mechanisms triggered within endothelial cells during extravascular migration of tumor cells are not well understood. It has ever been supposed that tumor cells migration through endothelial cells are by endocytosis. But recently studies provide evidence that the process of tumor metastasis is similar to it of neutrophil migration in inflammation, and migration of the circulating neutrophil from the vascular compartment is through a gap between adjacent endothelial cells into surrounding tissue in acute inflammation. Thus, we speculate that the same mechanism regulates tumor extravascular migration across the endothelial barrier.In the present study, We constructed an in vitro model of tumor cell transmigration across a monolayor of HUVEC, and using it to observe extravascular migration of tumor cells in extravascular migration of tumor cells.We constructed the model of tumor cells extravascular migration. Briefly, first HUVECs were seeded onto the transwell culture chambers containing polycarbonate membrane inserts, which was coated with thicker collagen gel. Since collagen are the essential elements of basement membrane of blood vessels, thus we can construct the monolayer vessel endothelial cell and extracellular matrix in vitro. then, when HUVECs reached confluence, we added tumor cells to the surface of confluent HUVEC monolayer. The critical step during construction of the model is the formation of HUVEC monolayer. We confirmed confluency by Giemsa's stain and measuring electrical resistance. Using this model system, we observe extravascular migration of tumor cells, and found that the tumor cells can migrate into collagen gel through endothelial cells in a time-dependent manner. According to this experiment we selscted the high invasive HT1080 tumor cells to do the next experiment.Since the change of electrical resistance can explain the alteration of vascular endothelial permeability quantitatively, to investigate the relation between tumor cell extravascular migration and vascular endothelial permeability, we measured the electrical resistance of the confluent HUVEC monolayer during HT1080 cells migration. We found that the electrical resistance reduced rapidly during HT1080 migration, this suggest that the extravascular migration of tumor cells can increase vascular endothelial permeabitity, and the increase of vascular endothelial permeabitity is due to the contraction of endothelial cells and opening of gaps between endothelial cells. It also confirm that tumor cells migrate into extravascular tissue through a gap between adjacent endothelial cells.The formation of gaps between adjacent endothelial cells means the alteration of endothelial cell junctions. There are two kinds of endothelial cell junctions described as tight junctions and adherence junctions. It was reported that actin polymerization may directly open these junctions through interaction of actin filament and junction-associated proteins. To assess actin reorganization during tumor cells migration, HUVECs were stained with rhodamine-p...
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