Embryoid bodies (EB) derived from teratocarcinoma (TC)OTT6050 were cultured with ascitic liquids (AL) from animals carrying 16-, 22- and 35-day evolved EB. At the same time the presence of fibronectin (FN) in AL were analyzed by immunoblotting. Results indicate the probable existence of growth-stimulatory factors for EB, as well as the presence of FN in the 22-day AL

Anta i Vinyals, Josep Maria de

Martínez Rubio, G.

Monzó Planella, Mariano

Ruano Gil, Domingo

Saltó, C.

Diposit Digital de la Universitat de Barcelona

Histol Histopath (1 992) 7: 23-27 Histology and Histopathology Effect of ascitic liquid on growth in vitro of embryoid bodies derived from teratocarcinoma M. Monzó, G. Martínez-Rubio, J.M. de Anta, C. Saltó and D. Ruano-Gil Department of Morphological Sciences, University of Barcelona, Barcelona, Spain Summary. Embryoid bodies (EB) derived from teratocarcinoma (TC) OTT6050 were cultured with ascitic liquids (AL) from animals carrying 16-, 22- and 35-day evolved EB. At the same time the presence of fibronectin (FN) in AL were analyzed by immunoblotting. Results indicate the . probable existence of growth-stimulatory factors for EB, as well as the presence of FN in the 22-day AL. Key words: Teratocarcinoma, Embryoid bodies, Fibronectin, Ascitic liquid lntroduction Teratocarcinomas (TC) are tumors obtained experimentally by the ectopic grafting of young mouse embryos (Stevens, 1970; Damjanov et al., 1987). The tumors thus obtained can be reimplanted intraperitoneally in isogeneic mice, giving rise to the ascitic form of the tumor known as embryoid bodies (EB) (Stevens, 1960). EB are divided morphologically in cystic and simple (Martin, 1978). Cystic EB are formed by an endodermal (END) cell layer that surrounds a cystic cavity with cells of embryonal carcinoma (EC) and are similar to 3.5-day-old mouse embryos. Simple E B  form an endodermal layer that surrounds EC cells without cystic cavities and are similar to 5 day mouse embryos (Martin, 1978; Monzó et al., 1985). When EB are injected subcutaneously into isogeneic mice, they develop the original tumor (Pierce and Dixon, 1959; Stevens, 1983). EB are maintained in vivo by intraperitoneal passage in mice of the strain 1291Sv; a volume of ascitic liquid (AL) always appears in relation to the days of evolution of EB in the peritoneal cavity. It Offprint requests to: Dr. M. Monzó, Departamento de Ciencias Morfológicas, Facultad de Medicina, Avda. Diagonal, 08028 Barcelona, Spain is now known that the presence of growth and tumor inhibition factors is detected in the AL in certain experimental tumors (Levine et al., 1984). Little is known, however, about the existence of extracellular matrix (ECM) proteins in AL of experimental TC. The aim of this study is to analyze the influence of AL from TC-carrying animals on the growth of EB in vivo and to investigate the presence of fibronectin (FN) in these AL. Materials and methods E6 obtention EB were derived from the TC OTT6050 obtained by Stevens in 1970, and were a gift from Prof. F. Jacob of the Pasteur Institute in Paris. EB had been maintained in vivo in our laboratory since 1984 by intraperitoneal passage in 129ISv isogeneic mice every fifteen days. Animals carrying EB of 12 days of evolution were killed by cervical dislocation and the peritoneal cavity was immediately washed with DMEM (Dulbecco's Modification of Eagle's Medium). EB were spun at 1,000 rpm for 5 min at 4O C and pellets were washed with DMEM. Ascitic liquid obtention and E9  culture AL was obtained by intraperitoneal aspiration of 129ISv mice carrying EB of 16, 25 and 35 days of evolution which had previously been injected with lo6 EB. AL were centrifuged in a microfuge, decomplemented at 56°C for 30 min and filtered through a 0.8 pm membrane (Millipore). EB obtained 12 days postinjection were cultured on Petri Perm dishes (Nunc) and in microdrops of 200 p1, each one of which contained the following culture media: A) 73% DMEM, 10% FCS, 2% L-Gln, 15% AL (16 days). B) 73% DMEM, 10% FCS, 2% L-Gln, 15% AL (22 days). Effects of ascitic liquid on embtyoid bodies A R E A  ( r n n 2 )  Fig. 1. Aspect of EB cultured with DMEM + FCS for 24 Fig. 2. Growth areas of E0 cultured with DMEM + FCS for 24 h h by scanning electron rnicroscopy (SEM). x 200 and 48 h. Fig. 1B. Aspect of E0 cultured with DMEM + FCS for 48 h by inverted rnicroscope. A great arnount of ernbryonal carcinoma (EC) cell centres are observed. C) 73% DMEM, 10% FCS, 2% L-Gln, 15% AL (35 days). D) DMEM plus (15% AL [16 days] or 15% AL (22 days) or 15% AL [35 days]). DMEM plus 10% FCS was used as control medium. Cultures were photographed after 24 and 48 hours and the growth area was calculated by means of a Kontron Mop 20. After 24 h cultures were processed for scanning electron microscopy (SEM) as follows: they were fixed in 2% glutaraldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide, washed in PBS, dehydrated in acetone series, conserved in isoamyl acetate, and critical-point dried. Detection of FN in AL AL of 16, 22 and 35 days of evolution were processed by 7.5% SDS-polyacrylamide gel Fig. 3. Growth areas of EB cultured with DMEM + FCS + ascitic liquid (AL) of different days for 24 and 48 h. Thick line, AL of 16 days; double line, AL of 22 days; narrow line, AL of 32 days. electrophoresis. Proteins obtained were transferred to nitrocellulose filters by electroblotting at 12 volts for 16h at 4O C. Filters were then blocked overnight in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), incubated with a polyclonal antibody of rabbit anti-FN (Biogenex) diluted 1:200 in 1% BSA in PBS and washed in 1% BSA in PBS. They were then postincubated with peroxidase-conjugated goat anti- rabbit IgG antibodies (Miles) diluted 1:400 in 1% BSA in PBS, washed in PBS and developed with diaminobenzidine (DAB) . Results AL, at 16 days of evolution, presented a clear, transparent aspect, which contrasted with that of 25 and 35 days which usually appeared more turbid. Some 35-day AL were occasionally haemorrhagic and were thus discarded for culture. Animals carrying AL Effects of ascitic liquid on embryoid bodies Fig. 4A. General appearance of EB cultured with DMEM + FCS + AL of 22 days for 48 h. x 40 Fig. 5A. General aspect of EB cultured with DMEM + FCS . AL of 16 days for 48 h. x 40 Fig. 48 .  Detall of Fig. 4A. EC cell centres and endodermic (END) cells are observed. x 400 after day 22 usually presented tumoral implantatic in the peritoneal cavity. M O S ~  of the E B  cultured with DNIEM plus FCS for 24 h had anchored and begun to grow, and END-like cells appeared with microvilli on the surface (Fig. 1A). After 48 hours the culture had increased its growth considerably and nodules of E C  cells were observed together with END-like cells (Fig. 1B). Areas of growth corresponding to 24 and 48 h are shown in Fig. 2. Cultures with DMEM, FCS and AL EB cultured with 73% DMEM, 10% FCS, 2% L-Gln and 15% A L  of 22 and 35 days were completely anchored after 24 h ,  although the cultures with 16-day AL were still not adhered. The anchored E B  presented a similar morphology to that observed in Fig. l A ,  as did the areas of growth, as shown in Fig. 3. However, we observed a slight increase in the areas of growth of the cultures with 22-day AL cornpared to Fig. 5B. Detall of Fig. 5A. EC and END cells are observed. x 450 those with 16- and 33-day AL. The same cultures presented important variations in their growth at 48 h (Fig. 3). Maximum area of growth corresponded to cultures with 22-day AL (Figs. 3, 4A, 4B). Cultures with 35-day A L  were slightly smaller than those with 22-day AL. (Fig. 3) while those with 16-day AL showed very little growth compared with the others (Figs. 5A, 5B). Cultures with DMEM plus AL in absence of FCS E B  cultured with 73% DMEM, 2% L-Gln and 15% A L  of 16-, 22- and 35-days, but without FCS, showed a higher area of growth (Fig. 6) than controls after 24 h (Fig. 2), but was lower that of cultures with FCS plus A L  (Fig. 3). After 48 h we observed that the Effects of ascitic liquid on embryoid bodies 2 0 0 ~ ~  A R E  A (rnrnz)  Fig. 6. Growth areas of EB cultured with DMEM + AL of different days and in absence of FCS. area of growth of cultures with 16-day AL was similar to that of controls and to those of AL plus FCS. Areas of growth of cultures with 22- or 35-day AL were smaller than those obtained with AL plus FCS, and maximum growth area continued to be that associated with 22- day AL (Fig. 6). Immunoblot performed on 16-, 22-and 35-day AL to FN indicated that its presence could be detected from day 22 onwards (Fig. 7). Discussion Most cells in culture need a set of exogenous substances to enable them to adhere to and grow on an artificial substrate. Depending on this process of adhesion, cells show varying responses to exogenous stimuli such as growth factors. Thus, cells of the corneal epithelium in vitro respond to the addition of fibroblastic growth factor (FGF) but not to epidermal growth factor (EGF). However, in these same cells are grown in the presence of ECM proteins, they respond to EGF, as is observed in vivo (Gospodarowicz, 1978). In cultures of EB, our results indicate that the maximum adhesion is obtained in cultures of EB with DMEM, FCS plus 22-day AL. The presence of FN is also detected from day 22 onwards. This suggests the possible influence of this protein in facilitating the anchorage of the EB. The function of FN as an anchor protein has already been demonstrated in cultures of pre-implantation embryos (Armant et al.. 1986; Tsuiki et al., 1989), in EC cells (Tienari et al., 1989) and observed during EB growth (Monzó et al., 1991). It is also demonstrated in the rat that histiocytoma cells do not adhere to the host animal if they are injected together with an anti-FN antibody (Pande and kllar, 1988). The maximum area of growth is also obscrved in cultures with DMEM, FCS plus 22-day AL, and it is interesting to note that cultures with 22-day A L  but without FCS grow more than control cultures, with only contain FCS. This finding suggests the possible Fig. 7. lrnrnunoblot of AL of 16 days (2), 22 days (3) and 35 days (4) to fibronectin (FN). FN is detected in AL of 22 and 35 days. (1) Standard proteins. existence of growth factors in AL. In fact Levine et al. (1984) isolated a tumor-stimulatory factor in AL of Novikoff's hepatoma. On the other hand we cannot discard the possibility that FN itself may act as a growth-stimulatory factor as this phenomenon has been demonstrated both in the growth of experimental TC (Wartiovaara et al., 1978; Rizzino and Crowley, 1980; Leivo and Wartiovaara, 1989) and in humans (Maclhinney et al., 1983; Chakrabarty et al., 1988). Cultures with 35-day AL show less growth than those with 22-day AL. The mechanisms by which this phenomenon may be explained are unknown, although it has been observed that growth-stimulatory and -inhibitory factors can coexist in AL, and that the latter operates by preventing the anchorage of tumor cells, and also that the growth of the tumor depends on the relative concentration of these factors (Levine et al., 1984). In conclusion. we would argue that the AL of animals carrying TC contains growth-stimulatory factors apart from FN. and that this is thus a suitable model for the study of the relation between tumor cells and anchorage and growth factors. Acknowledgements. We thank Robin Rycroft for his help in the preparation of the English rnanuscript, and Ms. Dolors Fuster for her technical assistance. This study was supported by the DGICYT PB87-0163. References Arrnant R.D., Kaplan H.A. and Lennarz W. (1986). Fibronectin and larninin prornote in vitro attachrnent and outgrowth of mouse blastocysts. Dev. Biol. 11 6, 51 9-523. 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Effect of ascitic liquid on growth in vitro of embryoid bodies derived from teratocarcinoma

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