DISCUSSION

Extravasation is a highly regulated process. It requires a self-activated signaling that increases the adhesion and motility of the cancer cells and a forward signaling that induces breaches in the endothelium layer (Bernstein and Liotta 1994; Chambers et al. 2002; MacDonald et al. 2002; Weis and Cheresh 2005). The TEM and vascular permeability assays are simple and effective methods for demonstrating the functional involvement of molecules in the extravasation process in vitro (Lampugnani et al. 1992; Voura et al. 2001; Tremblay et al. 2006; Gupta et al. 2007; Karnoub et al. 2007; Ma et al. 2008; Welm 2008). The two protocols described here were designed to study the ECM proteins in particular (Ma et al. 2008). In the TEM assay, cancer cells are dissociated from the culture plates using Versene (which contains EDTA but not trypsin) instead of trypsin/EDTA. This modification helps to preserve the transmembrane and membrane-bound proteins on the cell surface, which are important players in the co-culture system. The TEM assay provides direct evidence for protein-regulated extravasation, because it mimics dynamic interaction between cancer cells and endothelial cells. However, counting the number of transmigrated cells is time consuming and sometimes subjective. In the vascular permeability assay, conditioned medium containing the secreted factors from cancer cells is used to treat the endothelium monolayer. Macromolecules (FITC-dextran) are used to examine the permeability of the endothelium monolayer. Although the readout of this assay is not sufficient to draw conclusions, it is relatively objective and high-throughput. The vascular permeability assay is ideal for drug and inhibitor screening (Tremblay et al. 2006; Ma et al. 2008). Combining the results from both assays should provide a comprehensive understanding of the ECM protein-regulated extravasation process.