Infiltration of lymphosarcoma cells into hepatocyte cultures: inhibition by univalent antibodies against liver plasma membranes and lymphosarcoma cells

Journal of Cell Science, Aug 1982

O.P. Middelkoop, E. Roos, I.V. Van de Pavert

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Infiltration of lymphosarcoma cells into hepatocyte cultures: inhibition by univalent antibodies against liver plasma membranes and lymphosarcoma cells

OTTO P. MIDDELKOOP 0 ED ROOS 0 ILJA V. VAN DE PAVERT 0 0 Division of Cell Biology, The Netherlands Cancer Institute , Plesmanlaan 121, 1066 CX Amsterdam , The Netherlands The number of murine MB6A lymphosarcoma cells that infiltrated rat hepatocyte cultures was found to be diminished after treatment of the lymphosarcoma cells with univalent antibodies raised against these tumour cells (anti-MB6A Fab), and also after treatment of the hepatocyte cultures with univalent antibodies directed against rat liver plasma membranes (anti-LPM Fab). The inhibition of infiltration by anti-MB6A Fab and an anti-LPM Fab raised against sinusoidal face-enriched membranes could be entirely attributed to their interference with adhesion of MB6A cells to the exposed surface of the hepatocytes, because infiltration of the adherent cells was not inhibited. Anti-LPM Fab raised against contiguous face-containing LPM, on the other hand, inhibited the adhesion to the exposed surface and the subsequent infiltration of adherent cells. These observations suggest that specific membrane constituents of both MB6A cells and hepatocytes take part in liver infiltration, and that there may be two different hepatocyte components involved, one mediating adhesion to the exposed surface and the other taking part in the infiltration process proper. - Previously (Roos, Van de Pavert & Middelkoop, 1981), we described the infiltration of MB6A lymphosarcoma cells into cultures of isolated adult hepatocytes. We use these cultures as a model for the infiltration of MB6A cells into the intact liver during the formation of hepatic metastases (Dingemans, 1973; Roos, Dingemans, Van de Pavert & Van den Bergh Weerman, 1977). After adhesion to the hepatocytes the MB6A cells rapidly infiltrated between and under the liver cells and accumulated there at interhepatocyte boundaries. After 24 h, virtually all tumour cells were located within the cultures. We assumed that specific adhesion molecules were involved in this process. Such molecules that mediate mutual adhesion of cells and their adhesion to extracellular substrates, have been identified on several different cell types (Takeichi, 1977; Obrink, 1980; Nielsen, Pitts, Grady & McGuire, 1981). To explain the accumulation of lymphosarcoma cells within the cultures, we assumed further that the contiguous surface of hepatocytes is more adhesive for lymphosarcoma cells than the exposed surface, causing the tumour cells to be arrested at the contiguous surface. This increased adhesiveness could be due to a higher density of the same adhesion molecule or to the presence of a different molecule for which these tumour cells have a higher affinity. To establish whether these assumptions are correct, we used an immunological method, which has been successfully applied to the identification of several different adhesion molecules on various cell types (Rosen, Haywood & Barondes, 1976; Brackenbury, Thiery, Rutishauser & Edelman, 1977; Thiery et al. 1977; Mviller & Gerisch, 1978; Obrink & Ocklind, 1978; Urushihara, Ozaki & Takeichi, 1979; Wylie et al. 1979; Bertolotti et al. 1980; Ocklind et al. 1980), including embryonal (Bertolotti et al. 1980) and adult (Obrink & Ocklind, 1978; Ocklind et al. 1980; Nielsen et al. 1981) hepatocytes. This method is based on inhibition of adhesion by Fab fragments of immunoglobulins raised against whole cells or plasma membranes. Pretreatment of the Fab fragments with the isolated adhesion molecule neutralizes the inhibiting activity. Thus, using a multispecific antiserum, adhesion molecules can be identified by pretreatment of the Fab fragments with purified membrane components. In this report we present data on the inhibition of both adhesion and infiltration of lymphosarcoma cells into hepatocyte cultures, by Fab fragments prepared from antibodies raised against lymphosarcoma cells or against liver plasma membrane preparations, containing either contiguous face membranes or sinusoidal face-enriched membranes. The effects of the different antisera point to the existence of at least one, but possibly two, MB6A adhesion molecule(s) binding to two different components on liver cells: one on the exposed surface mediating the initial adhesion of MB6A cells to the cultures, and another on the contiguous surface involved in infiltration. MATERIALS AND METHODS Hepatocyte isolation and culture Rat hepatocytes were isolated as described (Roos et al. 1981), and cultured using the modified method described by Roos & Van de Pavert (1982). Murine MB6A ascitea lymphosarcoma cells were maintained and harvested as described before (Roos et al. 1981). Preparation of liver plasma membranes (LPM) Contiguous face-containing LPM (CLPM) were prepared according to Emmelot, Bos, Van Hoeven & Van Blitterswijk (1974), omitting overnight washing in Cal+-free phosphate-buffered saline (PBS). Antisera kindly provided by Dr B. Obrink were raised against CLPM, prepared according to Ray (1970), and against l (...truncated)


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O.P. Middelkoop, E. Roos, I.V. Van de Pavert. Infiltration of lymphosarcoma cells into hepatocyte cultures: inhibition by univalent antibodies against liver plasma membranes and lymphosarcoma cells, Journal of Cell Science, 1982, pp. 461-470, 56/1,