It has been shown recently that high metastatic tumor lines can be converted to low metastatic ones by mutagenization and selection for resistance to toxic concentrations of the lectin wheat germ agglutinin (1-3). Lectin-resistant variants were found to differ from the parental lines and respective revertants in terminal cell-surface carbohydrates. Here we describe for the first time a drug-independent selection procedure for the isolation of a low metastatic variant type cell from a high metastatic tumor line. The method is based on the selection of plastic-adherent cells from a suspension culture of highly malignant cells. Similar to the above situation, plasticadherent low-metastatic variant cells were found to differ from the parental cells and from metastatic revertants in terminal cell surface carbohydrates. The importance of terminal carbohydrates, in particular of sialic acid, for cell-surface adhesiveness and metastatic potential will be discussed. Materials and MethodsTumor Lines. Eb is the Heidelberg subline of the methylcholanthrene A-induced T cell lymphoma L5178Y of DBA/2 mice and ESb is a spontaneous high metastatic variant thereof (4). Details of these cells have been described elsewhere (for a review see ref. 5).Selection of a Plastic-adherent Variant. Both tumor lines Eb and ESb grow in suspension culture (6). A strongly plastic-adherent variant (ESb-M) was isolated from the ESb cells by continuously selecting the adherent subpopulation from the suspension cultures over a period of 2 mo and >20 selection steps. ESb-M cells which were released into suspension from monolayer cultures grown to confluency were taken for analysis.Flow Cytofluorography. Immunofluorescent staining was carried out by incubating 2 × 106 cells with appropriately diluted fluorescein isothiocyanate-(FITC) conjugated lectins for 45 rain on ice. The reaction was carried out in saline containing 1 mM Mg and Ca salts. Flow cytofluorographic analysis was performed on an Ortho H-50 cell sorter (Ortho Instruments, Westwood, MA). Fluorescence gains were kept at constant setting for each lectin analyzed. Cell surface differentiation antigens were determined by indirect immunofluorescenceand cytofluorography as described (7).Isolation of Tumor Cells from Organs. Spleen and brain of ESb-M tumor bearing mice were removed 40 d after tumor cell inoculation and pressed through a fine nylon mesh. The cells were suspended and cultured in the appropriate tissue culture medium (4). ResultsWhen inoculated into syngeneic DBA/2 mice, the plastic-adherent ESb-M variant type cells were found to be tumorigenic but not metastatic. The survival curves of * Supported as a guest by the Deutsches Krebsforschungszentrum.Supported by a grant from the Stiftung Meyenburg.J. Exe. MED.

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In many tumor systems, the location and extent of metastases are nonrandom, suggesting that unique cell properties are important in tumor arrest and formation of secondary tumor colonies. The lung and the liver are among the most frequently involved organs. These organs are also the predominant site of metastases by tumor cells of a model system of chemically induced lymphomas in DBA/2 mice that we have been investigating (1-5). The system consists of a low-metastatic parental tumor (Eb), a spontaneous high-metastatic variant thereof (ESb), and a high-metastatic tumor line of independent origin (MDAY-D2).We previously described that ESb and MDAY-D2, but not Eb, tumor cells had the unique properties of adhesion to and invasion of normal lung tissue in vitro (1, 6). Now we describe another unique characteristic of the two metastatic tumor lines, namely, a selective binding to isolated hepatocytes in vitro.Recently Kolb et al. (7) demonstrated a lectin-like receptor on rat liver cells that has specificity for galactosyl residues and may play a role in the trapping of recirculating desialylated lymphocytes in the liver (8). In our study, we investigated whether a similar interaction might be responsible for the trapping of circulating metastatic tumor cells in the liver. Materials and MethodsTumor Cells. Eb is a Heidelberg subline of the methylcholanthrene-induced DBA/2 lymphoma L5178Y, and ESb is a spontaneous variant thereof with highly metastatic potential (1, 5). The etiology of the metastatic DBA/2 tumor MDAY-D2 has been described (9). The tumor cells were passaged in tissue culture using RPMI-1640 medium (Grand Island Biological Co., Grand Island, N. Y.) with 10% fetal calf serum (FCS) and 2 × 10 -~ M 2-mercaptoethanol, Murine Hepatocytes. Liver cell suspensions were prepared as previously described (10). An 8-12-wk-old mouse was injected with 0.3 ml of 3.6% chloral hydrate in phosphated-buffered saline and 0.1 ml of heparin (500 U/ml). The hepatic portal vein and inferior vena cava were cannulated with a 21-gauge needle and a 16-gauge plastic catheter, respectively. The liver was perfused in situ, first with perfusion buffer (10) that was saturated with 02 at 36°C for 2-3 min,

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