Infiltration of lymphosarcoma cells into hepatocyte cultures: inhibition by univalent antibodies against liver plasma membranes and lymphosarcoma cells
OTTO P. MIDDELKOOP
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ED ROOS
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ILJA V. VAN DE PAVERT
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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.
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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)