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.

Fig. 1

Radial spreading of TCs under flow (shear stress =1.6 Pa) observed using phase contrast (a–c) and fluorescence (d–f) microscopy at t=0 (a,d), t=20 min (b,e) and t=45 min (c,f) after injection of TCs on the EC monolayer. The fluid flow is oriented from right to left of the images.

Fig. 2

Axial spreading of TCs under flow (shear stress = 1.6 Pa) observed using phase contrast (a–c) and fluorescence (d–f) microscopy at t=0 (a,d), t=20 min (b,e) and t=45 min (c,f) after injection of TCs on the EC monolayer. The fluid flow is oriented from right to left of the images.

Fig. 3

Time course of TCs projected area evolution for radial and axial spreading on an EC monolayer under a shear stress of 1.6 Pa. Results are shown as mean ± SD (n=19 for radial and 22 for axial TCs). Dotted line: radial spreading of TCs on fibronectin-coated glass slide (n=20).

Fig. 4

Time course of TCs minor/major aspect ratio for radial (triangles) and axial (squares) spreading on an EC mono layer under a shear stress of 1.6 Pa. Results are shown as mean ± SD (n=19 for radial and 22 for axial TCs). Dotted line: radial spreading of TCs on fibronectin-coated glass slide (n=20). Inset illustrates the best fitting ellipse defining the major and minor axes for a selected cell. * indicates statistical significance (p<0.005) of axial compared to radial aspect ratio from t=20 to t=70 min.

Fig. 5

Effect of shear flow on the number of TCs: a) % attached TCs on the EC monolayer (relative to the number of TCs remaining on the EC monolayer at the end of the 10-min incubation time, which corresponds to a total number of 309, 362 and 379 TCs observed at 0.2, 0.8 and 1.6 Pa, respectively), b) % TCs undergoing radial or axial spreading (relative to the initial number of attached cells). Results are shown as mean ± SD from 8 random fields observed in two independent experiments for each flow rate. The numbers over each bar correspond to the total number of cells observed in each case. * indicates statistical significance (p<0.005) of % attached TCs at 0.8 and 1.6 Pa as compared to 0.2 Pa.

Fig. 6

Effect of shear flow on the evolution of TCs projected area: (a) radial spreading, no significant difference is found between the different shear levels, (b) axial spreading, the cell area at 0.2 Pa is significantly lower than at 0.8 and 1.6 Pa (p<0.005) from t=20 to t=120 min. Results are shown as mean ± SD for each applied shear stress.

Fig. 7

Minor/major ratio versus area (trajectory) for the two spreading patterns at two different shear stresses, from t=0 to 70 min.

Fig. 8

Confocal series showing typical TCs undergoing: radial spreading (a–c), axial spreading (d–f) on an EC monolayer at 1.6 Pa shear stress for 30 min. Fixed cells were stained for TCs cytokeratin (b,e), actin fibers (c,f) and nuclei (a,d). Individual images are shown in an apical-to-basal direction. Z=0 μm corresponds to the first section as illustrated in the upper sketch. In (a–c), actin filaments of adjacent ECs clearly overlay external borders of the radially spread TC (arrowheads). In (d–f), lateral borders of an axially spread TC are in close contact with adjacent ECs (arrows). N corresponds to TC nuclei.