Determining β2-Integrin and Intercellular Adhesion Molecule 1 Binding Kinetics in Tumor Cell Adhesion to Leukocytes and Endothelial Cells by a Gas-driven Micropipette Assay

Changliang Fu,Chunfang Tong,Manliu Wang,Yuxin Gao,Yan Zhang,Shouqin Lü,Shile Liang,Cheng Dong,Mian Long

doi:10.1074/jbc.m111.281642

Abstract

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β(2)-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β(2)-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.

Highlights

Tumor metastasis requires the detachment of malignant cells from primary tumor, invasion through blood/lymph vessels, transmigration across the endothelium, and adhesion to the host cells [1]
It is critical from the viewpoint of two-dimensional kinetics of ␤2-integrin-intercellular adhesion molecule 1 (ICAM-1) interactions to compare the adhesions between polymorphonuclear neutrophils (PMNs)-endothelial cells (ECs) and PMN-tumor cells (TCs) pairs and to further the understanding of leukocyte-facilitated tumor cell adhesion
The PMN displacement exhibited four distinct patterns of cell retraction: 1) it moved at a high velocity, indicating that no interactions were detected when the two cells were separated; 2) it moved at a relatively low velocity over a distance before its free movement, suggesting that the adhesion presumably occurred with a membrane tether [27]; 3) it stayed still or yielded a very low velocity prior to its free movement, implying that the adhesion appeared likely without a tether extraction; and 4) it did not separate from another cell during entire retraction phase, illustrating that a firm adhesion was formed

Summary

Introduction

Tumor metastasis requires the detachment of malignant cells from primary tumor, invasion through blood/lymph vessels, transmigration across the endothelium, and adhesion to the host cells [1]. Whereas the other techniques are frequently applied to quantify the cell rolling/tethering or aggregation dynamics and the forced rupture of receptor-ligand bond, the adhesion frequency assay with MAT or biomembrane force probe technique is widely used to determine the kinetic rates and binding affinity of surface-bound molecules [21,22,23,24,25,26] In this regard, a red blood cell (RBC) bearing the purified receptors or ligands serves as a force transducer to visualize the occurrence of an adhesive event by taking the advantage of deflection of RBC membrane via a precise micromanipulation [21]. Kinetic rates and binding affinity of interacting molecules were compared between PMN-TC and PMN-EC pairs, and the regulation of ICAM-1 expression on binding kinetics by cytokine TNF-␣ was quantified

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