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Using fixed microvessel endothelial cell monolayers the molecules involved in the adhesion of liver-preferring murine RAW117 large cell lymphoma cells to murine liver-derived microvessel endothelial cells were identified by affinity isolation. Detergent lysates obtained from poorly (P) or highly (H10) liver-metastatic cells inhibited RAW117-H10 cell adhesion to hepatic sinusoidal endothelial (HSE) cell monolayers. Allowing detergent lysates of cell surface-labeled RAW117 cells to bind to fixed HSE cell monolayers and eluting the bound components indicated that several tumor cell surface molecules (approximately 70, approximately 35, approximately 32, approximately 22, and approximately 14 kDa) might be involved in RAW117 cell-HSE cell adhesion. The approximately 35 kDa component was cation dependent in its binding to target HSE cells. Increasing detergent concentration had no effect on binding of the approximately 35 kDa component to HSE cell monolayers, whereas treatment with 0.5 M NaCl resulted in its selective elution from HSE cells. Incubation of the HSE cell monolayers with detergent lysates from cell surface-labeled RAW117-H10 cells resulted in selective depletion of the approximately 35 kDa component, suggesting that the binding is saturable. This divalent cation-dependent molecule is one of the major tumor cell surface components bound by several types of endothelial cells and murine hepatocytes, whereas there was poor binding of this component to unfixed or fixed human red blood cells. The purified, partially (approximately 40%) sequenced molecule had amino acid sequence identity with murine but not bovine annexin II, indicating that it was not bound from the bovine serum used to grow RAW117 cells. Using antibodies specific for annexin II flow cytometry indicated equivalent amounts of annexin II are expressed on RAW117 cell surfaces in the absence or presence of excess EDTA, whereas annexin I was only found in low amounts on the surfaces of RAW117 cells. Annexin II antibodies inhibited by approximately 40-50% the adhesion of RAW117 tumor cells to live or fixed endothelial cells, and purified tumor cell surface fractions containing the approximately 35 kDa component partially inhibited (approximately 35%) RAW117 cell-HSE cell adhesion. The data indicate that annexin II is expressed on the extracellular surface of RAW117 cells, and cell surface-annexin II mediates a portion of the Ca(2+)-dependent RAW117 cell adhesion to liver microvessel endothelial cells.

Poorly liver metastatic large-cell lymphoma RAW117-P cells were sequentially selected in vitro for increased adhesion to murine hepatic sinusoidal endothelial cells. After three or four sequential selections, the selected sublines showed increased rates of adhesion to target hepatic microvessel endothelial cells and increased formation of experimental metastases in the liver. However, the endothelial cell adhesion-selected RAW117 sublines were generally unstable and gradually lost their enhanced adhesive and metastatic properties during passage in culture. Highly metastatic, liver-selected RAW117-H10 large-cell lymphoma cells were more resistant to the cytostatic effects of interferon-gamma (IFN-gamma) than poorly metastatic unselected parental RAW117-P cells. When tested for their sensitivity to IFN-gamma, the endothelial cell adhesion variants were significantly more resistant than the unselected RAW117-P cells, but after a 72-h treatment with IFN-gamma, the in vitro-selected cells lost their enhanced endothelial cell adhesion characteristics, their potential to colonize the liver, and their ability to grow when injected at subcutaneous or intramuscular sites. In contrast, the metastatic potential of similarly treated RAW117-P cells was unaffected by IFN-gamma during a 72-h treatment. Sequential selection of RAW117-P cells for increased resistance to IFN-gamma in vitro resulted in variant lines that were refractory to the growth-inhibiting effects of IFN-gamma, and these IFN-gamma-selected variants were also less adhesive to liver microvessel endothelial cells. The IFN-gamma-selected variants also lost their experimental metastatic potentials completely and their tumorigenicities at sites of subcutaneous or intramuscular injection. Cytofluorographic analysis indicated reduced cell surface expression of H-2Kd antigen and fibronectin receptor on the selected variant cells but no change in cell surface mu heavy chain immunoglobulin. The unselected and selected RAW117 lines had similar sensitivities to natural killer (NK) cell-mediated cytolysis, indicating that the in vivo differences were probably not due to differences in NK cell-mediated cytolysis. The results suggest that selection for adhesion to organ microvessel endothelial cells or sequential exposure to certain cytokines can affect the adhesive, growth and metastatic properties of RAW117 cells without modifying their responses to NK cells.

Metastatic variant cell lines of the murine RAW117 large cell lymphoma were used to study the cell surface components involved in syngeneic tumor cell/microvessel endothelial cell interactions. Poorly liver-metastatic parental RAW117-P cell line adhered to murine hepatic sinusoidal endothelial cell monolayers at significantly lower rates than the liver-selected, highly liver-metastatic RAW117-H10 line and both cell lines were poorly adherent to lung microvessel and bovine aorta endothelial cells. Viable, 2% 1-butanol-treated RAW117-H10 tumor cells formed fewer liver tumor nodules in experimental metastasis assays than untreated H10 cells and were significantly less adherent to murine hepatic sinusoidal endothelial cell monolayers. When 2% 1-butanol extracts of metabolically labeled or CHAPS detergent lysates of cell surface-labeled tumor cells were analyzed for their ability to bind to fixed microvessel endothelial cell monolayers, quantitative differences were found in the extractable tumor cell surface components that bound to the different organ-derived microvessel endothelial cells. Cell surface components (1-butanol extractable), of Mr approximately 85,000-90,000 and approximately 37,000-40,000 bound to hepatic sinusoidal endothelial cell monolayers to a greater extent than to murine lung microvessel endothelial or bovine aortic endothelial cell monolayers, whereas tumor cell surface components of Mr approximately 45,000, approximately 33,000, and approximately 25,000 bound similarly to endothelial cells regardless of origin. The results suggest but do not prove that tumor cell/endothelial cell adhesion involves multiple tumor cell surface components, some of which commonly bind to various endothelial cells and others for which binding may be dictated by the tissue origin and type of endothelial cell. Particular RAW117 butanol-extractable cell membrane components were associated with tumor cell-endothelial cell adhesion, and these components could be responsible, in part, for the preferential adhesion of RAW117 cells to liver sinusoidal endothelial cells and metastasis to liver.

Murine RAW117 large-cell lymphoma cells that show organ preferences of metastatic colonization were selected. We examined the role of adhesive systems in determining the organ preference of metastasis using cell lines of low (RAW117-P) and high (RAW117-H10) liver-metastatic potential. Highly metastatic H10 cells adhered at higher rates than low metastatic P cells to target organ microvessel endothelial cells, and these interactions were partially inhibited by RGD-containing polymers but not by small peptides such as GRGDS or GRGES. The most effective polymers, such as (GRGDS)4 and GRGDS(GRGES)2GRGDS, significantly inhibited H10 cell adhesion but had less effect on P cell adhesion to target liver sinusoidal endothelial cell monolayers or on P cell or H10 cell adhesion to bovine aortic endothelial cell monolayers. The (GRGDS)4 polymer reduced the rate of H10 liver sinusoidal endothelial cell adhesion to that of P cells in the absence of inhibitors, suggesting that the quantitative difference in adhesion of H10 cells versus P cells to liver sinusoidal endothelial cells may have been due to integrin-like molecules. Other RGD-containing polymers, such as (GRGES)2(GRGDS)2, GRGES(GRGDS)2GRGES, or (GRGES)4, were less effective, suggesting that the secondary structure of the polymers may be an important consideration. A peptide from the B1 chain of laminin (YIGSR) or its homopolymer, (YIGSR)4, had no effect on endothelial cell adhesion, consistent with the lack of differential laminin adhesion seen with various RAW117 cell lines. The results suggest that integrin-related molecules may play a role in the organ specificity of endothelial cell adhesion seen with RAW117 tumor cells.