Clin. Exp. Metastasis, 1995, 13, 155 164 A simple fluorometric assay for quantifying the adhesion of tumour cells to endothelial monolayers Elizabeth A. Price, Deirdre R. Coombe* and J. Clifford Murray University of Nottingham Laboratory of Molecular Oncology, CRC Academic Department of Clinical Oncoloyy, City Hospital, Nottingham, UK and *Institute.for Child Health Research, West Perth, Australia (Received 7 November 1994," accepted 17 January 1995) A static adhesion assay employing 6-carboxy-3',6'-diacetylfluorescein (6-CFDA) as a fluorescent marker has been developed to study the interactions of turnout cell lines with endothelial monolayers. This assay allows simple, safe quantification of cell-cell adhesion using living cells. It has been used to demonstrate that the integrin adhesion molecule VLA-4 mediates the attachment of RPMI-7951 melanoma cells to human umbilical vein endothelial cells (HUVEC) which have been activated by TNF~. In addition, MDA-MB-231 breast adenocarcinoma cells display greater adhesion to microvessel endothelial cells than to large vessel endothelial cells. Keywords: adhesion, carcinoma, endothelium, melanoma, metastasis Introduction The primary cause of therapy failure in cancer patients is the inability to prevent the spread of tumour cells from the initial site to distant organs and the subsequent formation of metastatic lesions. Dissemination of tumour cells occurs via the blood or lymphatic circulation and metastases form where these cells are able to escape from the circulatory system and proliferate within the tissues [1]. The evidence indicates that extravasation of tumour cells requires first their adhesion to the vascular endothelium, followed by their transmigration through the endothelial layer and its underlying basement membrane. The interaction of tumour cells with the endothelium is the point in the metastatic cascade which is most accessible to therapeutic intervention. Although much is known about the adhesion molecules mediating the interactions of leukocytes with endothelial cells [2], our knowledge of those mediating tumour cell Address correspondence to: E. A. Price, University of Nottingham Lab. of Molecular Oncology, CRC Academic Dept. of Clinical Oncology, City Hospital, Hucknall Road, Nottingham NG5 IPB, UK. Tel:(+ 44) 602 627927; Fax: (+ 44) 602 627923. interactions with endothelium, and their role in metastasis formation is less clear [3]. It is likely that different adhesion pathways are utilized by cells derived from different tumour types. Similarly, the interactions of tumour cells with large vessel and microvessel endothelia could have different molecular bases. Much of our current knowledge of human endothelial cell biology arises from studies with human umbilical vein endothelial cells (HUVECs). However, increasingly the evidence indicates that endothelial cells derived from different types of vessels are phenotypically and functionally different [4]. In addition most pathological events, including tumour cell extravasation, occur within the microvasculature [5]. Recent studies comparing human dermal microvascular endothelial cells and HUVEC have revealed differences in their expression of adhesion molecules. For example, ICAM-1 expression on unstimulated microvessel endothelial cells is substantially higher than that seen on HUVEC, whereas CD44 is highly expressed on HUVEC but only weakly on dermal microvascular cells [6]. In vitro adhesion assays have been used to examine i 1995 Rapid Communications o[Oxlbrd Ltd Clinical & Experimental Metastasis Vol 13 No 3 155