Role of reactive oxygen species (ROS), metalloproteinase-2 (MMP-2) and interleukin-6 (IL-6) in direct interactions between tumour cell spheroids and endothelial cell monolayer

Metastasis is a multistep process involving a variety of direct cell-cell, cell-matrix and paracrine interactions. In the present study, we examined some consequences of direct interaction between tumour cells and endothelial cells in vitro. When multicellular spheroids of two human tumour cell lines (HeLa and Hep-2) were transferred onto a human umbilical vein endothelial cell (HUVEC) monolayer, a peri-spheroidal zone of damaged endothelial cells was observed after 24 h co-culture. To determine the cause of this damage, the production levels of superoxide anion (O2−), interleukin-6 (IL-6) and metalloproteinase-2 (MMP-2) were measured both in co-culture and in monocultures of the tumour cell spheroids and endothelial cells. Attachment of HeLa and Hep-2 cellular spheroids to the HUVEC monolayer resulted in 1.6-fold and 2.1-fold increases in O2− release, respectively. Also, the MMP-2 level was five times greater in the co-culture than in the tumour spheroid monoculture. The increase of IL-6 in the co-culture model, on the other hand, was only slight. However, a 2 h preincubation of endothelial cells with LPS (10 μg/ml) prior to the transfer of spheroids induced a significant increase in the production of this cytokine compared to an appropriate control (an LPS-activated endothelial cell monolayer). These results strongly suggest that both ROS and MMP-2 are involved in endothelial cell injury when tumour cells cross the endothelial barrier. Moreover, IL-6, which participates in the inflammatory response, may also be involved in the extravasation of tumour cells.