DEVELOPMENTAL BIOLOGY 81, 127-132 (1981) Effects of Anti-Embryonal Carcinoma Serum on Aggregation and Metabolic Cooperation between Teratocarcinoma Cells JEAN-FRANCOIS NICOLAS, ROLF KEMLER, AND FRANCOIS JACOB Unite’ de GhnStique cellulaire du Coll&e de France et de l’lnstitut Pasteur, 25 we du Dr. Rozrx. ?‘572.$ Paris Ceder 15, France Received March 24. 1980; accepted in revised form May 27, 1980 Effects of rabbit anti-embryonal carcinoma IgG on embryonal carcinoma cells and their differentiated derivatives were studied at different levels of cell-cell interaction. Fab fragments of anti-EC IgG were found to inhibit aggregation of the majority of EC cell lines. Two, however, were insensitive. Anti-EC Fab fragments act also on the transfer of metabolites between EC cells: the rescue of HPRT- EC cells by HPRT+ EC cells in selective medium is abolished. These findings are correlated with the disappearance of tight and gap junctions from the surface of EC cells (Dunia et a/., 19’79). The presence of the surface structure involved in the action of anti-EC Fab fragments was tested by absorption experiments followed by decompaction test on PCC4 Aza Rl cells. All EC cell lines and two embryonic cell lines-a trophectodermal and an endodermal line-were found to bear material absorbing the decompacting activity. The results are discussed in terms of state of differentiation of the cell lines and of complexity of aggregation of embyronic cells. INTRODUCTION Embryonal carcinoma (EC) cells, the malignant ter- atocarcinoma stem cells, are in many respects similar to early embryonic cells. Both share common surface structures not present on adult cells, as shown by im- munological (Artzt et al., 1973; see Refs. in Jacob, 197’7) and biochemical analysis (Muramatsu et a.b., 1978,1979). Both have the potentiality to differentiate into many types of tissues either in vitro or ,in. vivo (Pierce and Verney, 1961; Nicolas et al., 1976; see Refs. in Martin, 1978) or after microinjection into normal blastocysts (Illmensee and Mintz, 1976). Recently, it was shown that monovalent fragments of a rabbit anti-EC IgG inhibit compaction of morulae cells and block in vitro the formation of mouse blas- tocysts (Kemler et al., 1977; Johnson et a.b., 1979). To further characterize the mode of action of the antise- rum, its effects on EC cells were investigated. In par- ticular, it was of importance to precisely determine at which level of cell-cell interaction Fab fragments act. Thus, their effect on EC aggregates has been investi- gated. Furthermore, it has been reported that EC cells, when in contact, exchange small molecules (metabolic cooperation and metabolic killing) (Hooper and Slack, 1977; Nicolas et al., 1978). We have developed a quan- titative method to study such a transfer, by measuring the rescue of hypoxanthine-phosphoryltransferase (HPRT)-defective cells by HPRT+ cells in a medium inhibiting HPRT- cell growth (Nicolas et al., 1978). With this technique, it is possible to study the effects of Fab fragments on such a transfer. Finally, the tissue distribution of the structures absorbing the activity of the Fab preparation was also investigated with differ- entiated cell lines derived from teratocarcinoma. MATERIALS AND METHODS Cells and cu.bture conchtions. The origin and proper- ties of the cell lines used are specified in Table 1. PSA- 1 and LT l-2 A are a gift of Dr. G. R. Martin. C 17 S- 1 is a gift of Dr. McBurney (McBurney, 1975). All cells were cultivated in Eagle’s medium (Dulbecco’s modi- fication) supplemented with 15% fetal calf serum (Gibco). The cultures were grown in a humidified in- cubator in an air/l2% CO2 mixture at 37°C on Falcon tissue culture dishes. Cells were passaged every 2 days by disaggregation with trypsin in Tris-buffered saline EDTA for F9, PSA-1, and 3/A/l D-3. LT l-2 A and PSA-1 were cultivated on y-irradiated (6000 rad) feeder layers of ST0 cells as described by Martin and Evans (1975). For further details about the isolation, proper- ties, and culture conditions see Nicolas et al., 1976. Antisera. Rabbits were hyperimmunized with F9-41 cells or mouse embryonic liver cells. The characteristics of the antisera, as well as the purification of an im- munoglobulin fraction and the preparation of mono- valent IgG Fab fragments, have already been described (Kemler et al., 1977). The studies reported were done with Fab preparations of a unique rabbit. Antisera of three other rabbits, however, give similar results. Measure of metabolic cooperation. The technique used 127 0012-1606/81/010127-06$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.