Computerized analysis of tumor cell interactions with extracellular matrix proteins, peptides, and endothelial cells under laminar flow

Arrest and formation of stable adhesive interactions between circulating cells and the endothelium or exposed subendothelial matrix are important processes in many biological situations. We have developed a highly sensitive hydrodynamic assay that utilizes a parallel-plate flow chamber, video microscopy, and digital image processing to separate and measure the primary arrest and adhesion stabilization of flowing cells. Our data indicate that primary cell contact triggers secondary adhesion stabilization, and the secondary events are likely to be critical to metastasis formation. To study the relationship between tumor cell adhesion stabilization and organ-specific blood-borne metastasis, we investigated the adhesion stabilization of metastatic murine RAW117 large cell lymphoma cells to the extracellular matrix proteins fibronectin and vitronectin, several Arg-Gly-Asp (RGD) containing peptides, and microvascular endothelial cells from the liver or lung. The highly liver metastatic RAW117-H10 subline showed the fastest stabilization to fibronectin, vitronectin, and RGD peptides. Poorly metastatic RAW117-P cells had stabilization times 3-10 times longer than for RAW117-H10 cells, while the lung- and liver-metastatic RAW117-L17 subline failed to stabilize at all. The adhesion stabilization of the RAW117-H10 cells to the extracellular matrix proteins and RGD peptides was inhibited by anti-β₃ integrin monoclonal antibodies and RGD peptides. In contrast, the RAW117-L17 subline had the shortest stabilization time to unstimulated microvascular endothelial cells of the lung and hepatic sinusoids, followed by RAW117-H10 cells and RAW117-P cells. Monoclonal antibodies against the β₃ integrin subunit and RGD peptides did not inhibit adhesion stabilization of RAW117-H10 cells to endothelial cells, suggesting that different metastatic variants of large-cell lymphoma cells use differing mechanisms to adhere to organ specific endothelial cells.