Morphological analysis of tumor cell/endothelial cell interactions under shear flow

Abstract

In the process of hematogenous cancer metastasis, tumor cells (TCs) must shed into the blood stream, survive in the blood circulation, migrate through the vascular endothelium (extravasation) and proliferate in the target organs. However, the precise mechanisms by which TCs penetrate the endothelial cell (EC) junctions remain one of the least understood aspects of TC extravasation. This question has generally been addressed under static conditions, despite the important role of flow induced mechanical stress on the circulating cell–endothelium interactions. Moreover, flow studies were generally focused on transient or firm adhesion steps of TC–EC interactions and did not consider TCs spreading or extravasation. In this paper, we used a parallel-plate flow chamber to investigate TC–EC interactions under flow conditions. An EC monolayer was cultured on the lower plate of the flow chamber to model the endothelial barrier. Circulating TCs were introduced into the flow channel under a well-defined flow field and TC cell shape changes on the EC monolayer were followed in vitro with live phase contrast and fluorescence microscopy. Two spreading patterns were observed: radial spreading which corresponds to TC extravasation, and axial spreading where TCs formed a mosaic TC–EC monolayer. By investigating the changes in area and minor/major aspect ratio, we have established a simple quantitative basis for comparing spreading modes under various shear stresses. Contrary to radial spreading, the extent of axial spreading was increased by shear stress.

Introduction

The major cause of death from cancer is metastatic dissemination (Tait et al., 2004), where tumor cells (TCs) locally invade and disseminate through the lymphatic system or blood circulation (Pantel and Brakenhoff, 2004). For hematogenous dissemination, TCs in the circulation must survive destruction by hemodynamic forces and host immune defenses, and then migrate out of the vessel through normal vascular endothelium (extravasation) in order to eventually proliferate in their target organs. TC extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which TCs cross the endothelial cell (EC) junctions and how the presence of fluid shear forces modulates cellular responses remain poorly understood.

Leukocyte interactions with ECs have been extensively studied either under static conditions (Hashimoto et al., 2004) or under flow conditions (Carman and Springer, 2004; Cinamon et al., 2004; Kaplanski et al., 1998; Sheikh et al., 2005) and can serve as a model for interactions of circulating TCs with the vasculature (Takada et al., 1993). It is well known that shear flow has an effect on endothelial alignment and elongation (Dewey et al., 1981; Bruder et al., 2001), on cell cytoskeleton (Galbraith et al., 1998) and therefore on its local mechanical properties (Sato et al., 2000) and finally on mechanotransduction pathways (Li et al., 2005). However, TC–EC interactions have been generally addressed under static conditions in Boyden chambers (Hart et al., 2005; Li and Zhu, 1999; Roche et al., 2003) or on glass slides coated with Matrigel (Voura et al., 2001) or collagen (Longo et al., 2001).

Using a parallel-plate flow chamber, several authors have investigated the role of cell-surface molecules that mediate TC adhesion under flow conditions (Tözeren et al., 1995; Haier et al., 1999; Haier and Nicolson, 2000; Kitayama et al., 2000b; Burdick et al., 2003). However, these studies were focused on transient or firm adhesion steps of TC–EC interactions and did not consider TCs spreading or extravasation. Other studies showed the importance of actin and microtubule fibers reorganization in tumor cell adhesion (Korb et al., 2004), as well as mechanotransduction effects during metastatic adhesion of carcinoma cells, with a particular emphasis on the role of focal adhesion kinase (FAK) (Von Sengbusch et al., 2005).

More recently, Slattery et al. (2005) looked at leukocyte-facilitated TC extravasation and concluded that shear flow significantly reduced melanoma cell extravasation. In a companion paper (Dong et al., 2005), they also showed the role of Mac-1 and ICAM-1-mediated cell interactions with Polymorphonuclears (PMNs) in human melanoma cell transmigration. However, monitoring the whole process of extravasation with live microscopy was not possible with their flow-incorporated Boyden chamber.

In this study, we used a parallel-plate flow chamber to investigate TC–EC interactions under well-defined flow conditions. An EC monolayer was cultured on the lower plate of the flow chamber to model the endothelial barrier. Circulating TCs were introduced into the chamber and TC adhesion to ECs was followed in vitro with live phase contrast and fluorescence microscopy. TCs were distinguished from the EC monolayer by means of fluorescence labeling and their shape changes were quantified. The influence of various shear levels on TC spreading patterns was investigated.