Protocol

In Vitro Assays for the Extracellular Matrix Protein-Regulated Extravasation Process

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA

¹Corresponding author (wang0011{at}mc.duke.edu)

INTRODUCTION

Extravasation, the process by which circulating tumor cells pass through the blood vessel wall, is a critical step of metastasis. Extracellular matrix (ECM) proteins secreted by the cancer cells are likely to play an interactive role in the dynamic interaction between cancer cells and endothelial cells during the extravasation process. This protocol describes two in vitro assays, the transendothelial cell migration (TEM) assay and the vascular permeability assay, which are used to demonstrate the involvement of ECM proteins in cancer cell extravasation. Both assays employ primary human umbilical vein endothelial cells (HUVEC) to reconstitute a vessel wall (HUVEC monolayer) on a porous filter membrane within a Transwell chamber. The TEM assay examines the efficiency of cancer cells to migrate through the vessel by co-culturing the cancer cells with the endothelial monolayer. The vascular permeability assay allows the study of the impact of secreted ECM proteins on the permeability of the vessel wall by applying conditioned medium from cancer cells to the endothelial monolayer.