Chi-Hung Siu

Role of cadherins in the transendothelial migration of melanoma cells in culture.

Transmigration of cancer cells through the vascular endothelium (diapedesis) is a key event in tumor metastasis. To investigate mechanisms involved in diapedesis, we used laser scanning confocal microscopy to examine the distribution of cadherins of WM239 melanoma cells as they migrated through a monolayer of activated human umbilical vein endothelial cells (EC) cultured on matrigel. Cadherins, including VE-cadherin, but not N-cadherin, were enriched in contacts between EC, whereas N-cadherin, but not VE-cadherin, was found in contacts between melanoma cells. During the early stages of diapedesis, EC located below the attached melanoma cells decreased in height and VE-cadherin disappeared from the EC contact located underneath the melanoma cell. Transendothelial migration began with small melanoma cell processes penetrating the VE-cadherin-negative regions between the EC. Subsequently, melanoma cells became intercalated between EC. Despite the absence of both VE-cadherin and N-cadherin, other members of the cadherin family were present in the heterotypic contacts between EC and melanoma cells. EC surrounding the intercalated melanoma cell subsequently extended processes and spread over the melanoma cell to re-form the endothelial monolayer. Interestingly, the leading margins of these EC processes contained high levels of N-cadherin, but not VE-cadherin. VE-cadherin-rich cell-cell contacts, however, reformed between advancing endothelial processes when they met above the melanoma cell. As the melanoma cells came into contact with the underlying matrigel, they spread out and adopted a fibroblast-like morphology. Addition of anti-N-cadherin antibodies to the assay resulted in a delay in the transendothelial migration of melanoma cells. Together, these results suggest that EC actively participate in diapedesis by disassembling and reassembling VE-cadherin-rich adherens junctions, and that N-cadherin plays an important role in the transmigration of melanoma cells and the reclosure of the endothelium.

Cell–cell interactions during transendothelial migration of tumor cells

A key event in cancer metastasis is the transendothelial migration of tumor cells. This process involves multiple adhesive interactions between tumor cells and the endothelium. After adhering to the surface of endothelial cells, tumor cells must penetrate the endothelial junction, which contains high concentrations of the cell adhesion molecules VE‐cadherin and PECAM‐1. Studies using an in vitro model system, consisting of melanoma cells which are seeded onto a monolayer of endothelial cells cultured on Matrigel, have revealed reorganization of the cytoskeleton and dynamic changes in the cell shape of both tumor and endothelial cells. The initial stages of transmigration are characterized by numerous membrane blebs protruding from the basolateral surfaces of the melanoma cells. Contact regions also show an abundance of microfilaments arising from the underlying endothelial cells. These adhesive interactions lead to the redistribution of both VE‐cadherin and PECAM‐1 and, consequently, a localized dissolution of the endothelial junction. The penetration of the endothelial junction is initiated by melanoma pseudopods. Despite the disappearance of VE‐cadherin from the retracting endothelial junction, heterotypic contacts between the tumor cell and its surrounding endothelial cells show a high concentration of pan‐cadherin staining, suggesting that transmigration of melanoma cells might yet be facilitated by interactions with another member of the cadherin family. Upon adhesion to the Matrigel, melanoma cells begin to spread and invade the matrix material, while the endothelial cells extend processes over the melanoma cells to reform the monolayer. Interestingly, the leading margins of these endothelial processes contain a high concentration of N‐cadherin. VE‐cadherin and PECAM‐1 reappear only when the advancing endothelial processes meet to reform the endothelial junction. Together, these observations suggest that endothelial cells actively participate in the transmigration of tumor cells and specific cadherins are involved in different steps of this complex process. Microsc. Res. Tech. 43:265–275, 1998.

Interleukin-8 secreted by endothelial cells induces chemotaxis of melanoma cells through the chemokine receptor CXCR1

There is increasing evidence that both cell adhesion molecules and soluble factors are involved in tumor metastasis. We have found that endothelial cells secrete chemoattractants that can induce melanoma cell chemotaxis. Protein separation on an ion‐exchange column shows the association of IL‐8 with fractions that contain the chemoattractant activity. This activity is completely lost from the conditioned medium after immunoprecipitation with anti‐IL‐8 antibodies, indicating that IL‐8 is the major melanoma chemoattractant secreted by endothelial cells. IL‐877, the predominant endothelial IL‐8 isoform that contains 77 amino acids, is found to be twice as potent as the more common 72‐amino acid isoform IL‐872. Antibody inhibition studies indicate that the chemotactic response of melanoma cells is mediated by the CXC‐chemokine receptor CXCR1 and not by the more promiscuous CXCR2. When stimulated by tumor necrosis factor α, the nonresponsive WM35 melanoma cells synthesize a higher level of CXCR1 and become chemotactic toward interleukin (IL)‐8. Pretreatment of cells with pertussis toxin nullifies their chemotactic response, suggesting the involvement of G proteins. Antibodies against either IL‐8 or CXCR1 inhibit melanoma transendothelial migration in a coculture assay by 30%. These results are consistent with a role for IL‐8‐induced chemotaxis in the transendothelial migration of melanoma cells.

Cell shape changes and cytoskeleton reorganization during transendothelial migration of human melanoma cells

Abstract An in vitro system has been established to study the migration of human melanoma cells through a monolayer of endothelial cells. Endothelial cells were cultured to confluence on Matrigel before the seeding of melanoma cells. Laser scanning confocal microscopy showed that, prior to migration, melanoma cells appeared round and showed cortical F-actin staining. The initial stage of transmigration was characterized by numerous membrane blebs protruding from basolateral surfaces of the melanoma cells, and contact regions showed an abundance of filaments arising in the underlying endothelial cells. Later, pseudopods from the melanoma cells inserted into contact regions between endothelial cells. Eventually, the melanoma cells intercalated with the endothelial cells. At this stage, many endothelial filament bundles terminated at contacts between the endothelial cells and the transmigrating melanoma cell, suggesting active interactions between the two cell types. Upon contact with the Matrigel, melanoma cells began to spread beneath the endothelium, displaying a fibroblastic morphology with prominent stress fibers. To reestablish the monolayer, adjacent endothelial cells extended processes over the melanoma cell. Tumor necrosis factor α did not affect the transmigration of melanoma cells from cell lines isolated from several stages of metastasis. However, tumor necrosis factor did promote the transmigration of melanoma cells derived from a non-metastatic lesion. These results thus define cell attachment and cell penetration of the monolayer as two distinct steps in transmigration and suggest that tumor necrosis factor may enhance the metastatic potential of tumor cells.

Role of NCAM, cadherins, and microfilaments in cell‐cell contact formation in TM4 immature mouse sertoli cells

To determine events that lead to the formation of intercellular contacts, we examined the spatial and temporal distribution of NCAM, cadherins, and F-actin in TM4 cells by immunofluorescence and laser scanning confocal microscopy. TM4 cells exhibited epithelioid characteristics and formed large overlapping lamella-like cell-cell contacts that contained a high concentration of NCAM. NCAM-rich lamellae formed from smaller NCAM patches at the ends of filopodia-like contacts between adjacent cells. Cadherins, as visualized by a pan-cadherin antibody, were present in a pattern distinctly different from that of NCAM. Although in filopodia-like contacts, both cadherins and NCAM were often concentrated at filopodial tips, in the larger lamella-like contacts that developed later, cadherins were located in an irregular punctate pattern only at the distal and more apical margins of the slanted NCAM-rich contact regions. Patterns of NCAM and microfilament (MF) bundle distribution were distinctly different, suggesting that the ends of these MF bundles were not physically linked to NCAM. By contrast, cadherins were concentrated at the ends of MF bundles at all stages of contact formation examined. Interestingly, this association of cadherins with MF bundles was mostly seen at the edge of the overlapping processes. In the lower cell process, MF bundles at the contact site were often arranged in random fashion, indicating an asymmetric distribution of MF in the junctional region. However, N-cadherin was enriched only at sites where MF bundles from both the upper and lower cell processes were aligned and terminated at the junctional membrane. Thus the organization of the actin cytoskeleton at cell-cell contact sites is influenced by the differential localization of different cadherins. These data also suggest that different mechanisms are involved in the accumulation of NCAM and cadherins in cell-cell contact regions.

Platelet-endothelial cell adhesion molecule-1 (CD31) redistributes from the endothelial junction and is not required for the transendothelial migration of melanoma cells

We have examined the role of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) during the transendothelial migration of melanoma cells using a novel in vitro system. Comparable studies have suggested the involvement of PECAM-1 in leukocyte transendothelial migration. Such studies have been confirmed using in vivo models of inflammation. These studies prompted us to examine the role of PECAM-1 in tumor cell transendothelial migration. Anti-PECAM-1 monoclonal antibodies, known to block leukocyte transendothelial migration, were tested in co-cultures of human melanoma cells seeded on a monolayer of human lung microvascular endothelial cells. None of these antibodies inhibited the transmigration of melanoma cells. Moreover, confocal microscopy revealed the dissolution of the PECAM-1 adhesion complexes in the endothelial junctions associated with melanoma cells and the lack of PECAM-1 in heterotypic contacts between transmigrating melanoma cells and adjacent endothelial cells. These data, therefore, indicate that PECAM-1 is not required for the transendothelial migration of melanoma cells.