Crossing the endothelium: E-selectin regulates tumor cell migration under flow conditions

Diapedesis is a vital part of tumor metastasis, whereby tumor cells attach to and cross the endothelium to enter the circulation. Specific adhesion molecules, expressed by both the tumor and endothelial cells, mediate this process. This review summarizes recent findings regarding the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions mediated by E-selectin. Using a laminar flow chamber and a tissue engineered human blood vessel, E-selectin was found to regulate initial attachment and rolling of colon cancer cells and also the subsequent diapedesis through the endothelium. Three different mechanisms of diapedesis were reported to be regulated by E-selectin; the formation of a mosaic chimeric layer of tissue, paracellular diapedesis between endothelial cells and transcellular diapedesis, in which tumor cells were transported via large vacuoles within the endothelial cells. Moreover activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase by E-selectin was further required for paracellular diapedesis. This study is the first to report these observations under dynamic and shear stress conditions.Key words: E-selectin, endothelium, tumor cells, diapedesis, site-specific metastasisDuring metastasis, tumor cells must complete a number of stages to successfully migrate from one site in the body to another. These include detachment of cells from the primary tumor, degradation and migration through the surrounding extra cellular matrix, extravasation and survival in the circulation and arrest in distant capillaries.¹ Once at the secondary site, they must undergo the reverse process before initiating growth and angiogenesis to support their development. This is a highly inefficient process, with few cells completing all stages to establish metastases.²The process of extravasation involves the diapedesis of tumor cells across the endothelium (‘dia’ meaning through and ‘pèdan’ meaning to leap). Similar to neutrophil extravasation at sites of inflammation, tumor cells initially adhere to the endothelium forming loose interactions via adhesion molecules before rolling and finally forming firm attachments, prior to diapedesis. Site-specific metastasis has been described for a number of tumors, with specific interactions between the tumor and endothelial cells potentially playing a pivotal role. Expression of E-selectin by endothelial cells following activation by inflammatory cytokines for example, is reported to be involved with the homing of colon cancer cells to the liver.^3–5 This review summarizes recent findings by Tremblay et al. (2008), published in Cancer Research, in which three distinct mechanisms of transendothelial migration by colon cancer cells are described, ultilizing E-selectin adhesion molecules on the endothelium (Fig. 1): (1) paracellular diapedesis at endothelial junctions, (2) formation of mosaic chimeric tissue of tumor and endothelial cells and (3) transcellular diapedesis.⁶Open in a separate windowFigure 1Schematic representation of the three mechanisms that colon cancer cells use during diapedesis of the endothelium. Under flow conditions, tumor cells initially form loose attachments with the endothelium, prior to rolling and forming firm attachments, mediated by specific adhesion molecules such as E-selectin. Following firm adhesion, tumor cells initiate diapedesis by (1) migrating between the junctions of endothelial cells (paracellular diapedesis), (2) forming a mosaic chimeric layer of both cell types (although this may not lead to complete diapedesis) and (3) passaging through the endothelial cells in large vacuoles (transcellular diapedesis).To demonstrate the importance of E-selectin in the adhesion of colon cancer cells to the endothelium, a laminar flow chamber was initially used. Human umbilical vein endothelial cells (HUVECs), expressing WT or a truncated form of E-selectin (with a cytoplasmic domain deleted) (ES-ΔICD) grown as a monolayer on glass slides, were treated for four hours with IL-1β to induce E-selectin expression. Cultures were then placed in a laminar flow chamber with culture medium and shear stress applied. HT29 colon cancer cells were then injected into the flow chamber, video sequences were recorded after 10 and 30 minutes and the subsequent HT29 cells that had adhered to the HUVECs was counted. The results demonstrated that colon cancer cells adhered to the endothelium under flow conditions using E-selectin, but only following treatment with IL-β. Blocking E-selectin and mutating the intracellular domain of E-selectin both prevented tumor cell adhesion. Similar results were also generated using a tissue-engineered human blood vessel, to more closely mimic the in vivo situation.⁷ Using these systems, the authors verified that E-selectin activation by circulating colon cancer cells is involved in p38 and ERK activation, via the intracellular domain.Using HT29 cells labeled with Vybrant Di1, the authors next used time-lapse microscopy over a 24-hour period to investigate the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions. With endothelial cells expressing WT and truncated E-selectin, E-selectin was found to mediate initial attachment and rolling of the HT29 cells on the endothelial layer, in support of their previous results. Interestingly, most adhered tumor cells penetrated the endothelium, with 75% inserting themselves within the endothelial cell layer, creating a chimeric mosaic (Fig. 1). Many of these cells however did not complete migration through the endothelium and this was especially the case when using endothelial cells expressing E-selectin with the truncated intracellular domain, suggesting that this region was required for tumor cells to complete diapedesis.Of the cells that penetrated the endothelium, only 25% of the colon cancer cells completed diapedesis. This process was observed by two different mechanisms (Fig. 1). Firstly, at the junction of three endothelial cells, HT29 cells migrated between the cells (i.e., paracellular diapedesis); this required E-selectin signaling to ERK. Secondly, transcellular migration was also observed, in which tumor cells attached to the endothelial cells (not at the cell junctions) and induced endothelial cell retraction and blebbing. As a result tumor cells were engulfed within large vacuoles in the endothelial cells and transported across the cell. This was accompanied by loss of endothelial contact with the extracellular matrix. Interestingly this process was not fatal for all endothelial cells and some remained in the culture medium.In conclusion, this is the first paper to demonstrate three different mechanisms by which colon cancer cells migrate through the endothelium under conditions of shear stress, in an E-selectin-dependent fashion. Although a large proportion of tumor cells remained within the endothelium as a mosaic layer and did not complete diapedesis, the authors concluded that such cells form a subpopulation of tumor cells that are capable of initiating, but not completing extravasation, potentially due to nature of the tight junctions between the endothelial cells.^8,9 If proliferation is then stimulated, such cells may further be responsible for the development of local metastases within the vessel. A small percentage of tumor cells however completed diapedesis. This occurred either at the junction of endothelial cells (paracellular) or of particular interest whereby tumor cells crossed through the endothelial cells (transcellular). Similar data have been described previously for neutrophils,¹⁰ but this is the first study to demonstrate this phenomenon with tumor cells via E-selectin. This work provides new insights in understanding the mechanisms by which tumor cells cross blood vessel walls and the metastatic process.