Interaction of malignant MO4 cells with chick hypoblast in culture

Malignant MO4 mouse fibrosarcoma cells were confronted with fragments of hypoblast from stage 4 (Vakaet 1970) blastoderms in different dispositions either permitting or preventing contact of the hypoblast with the tissue culture plastic. Explantation of an MO4 cell aggregate on top of 24 h-old-hypoblast caused retraction of the hypoblast. Contact inhibition of ruffling in hypoblast cells at the inner margin, by MO4 cells migrating radially from the aggregate, prevented closure of the hole brought about by the initial retraction. Disintegration of hypoblast was not observed. Migration of MO4 cells during the first 24 h was faster from an aggregate explanted on top of hypoblast than from an aggregate explanted on tissue culture plastic. Hypoblast fragments explanted on top of confluent layers of MO4 cells attached and spread during the first 12 h. Later, the hypoblast progressively disintegrated. Here, MO4 cells accumulated underneath the hypoblast. We concluded 1) that the hypoblast attracted the MO4 cells by influencing their pattern of migration and 2) that contact with the artificial substrate allowed survival of hypoblast confronting malignant MO4 cells. Ultrastructural analysis suggested that formation of extracellular material played a major role in the interaction between the normal tissue and the malignant cells.     

Confrontation of an invasive (MO4) and a noninvasive (MDCK) cell line with embryonic chick heart fragments in serum-free culture media

Summary Confronting cultures of precultured embryonic chick heart fragments (PHF) with aggregates of malignant cells in vitro have been shown to be relevant for a number of aspects of tumor invasion in vivo. Preculture of the heart fragments, formation of cell aggregates and subsequent culture of confronting pairs have so far been done only in serum-containing culture media. We describe here confronting cultures of PHF with invasive MO₄ mouse cell aggregates or noninvasive MDCK dog kidney cell aggregates in serum-free media. Heart fragments precultured in the absence of serum seemed to be necrotic after confronting culture in serum-free media. However, preculturing in media supplemented with 10% fetal bovine serum allowed us to do subsequent confronting cultures in absence of serum. Cell aggregates were also prepared in serum-containing medium. MO₄ cells occupied and replaced the heart tissue within 4 d, whereas MDCK cells remained at the periphery, of the PHF. This indicates that serum-free confronting cultures can discriminate between invasive and noninvasive cells. The viability of individual PHF and cell aggregates cultured in the same way as in confrontations was ascertained by histology and by explantation and postculturing on a solid tissue culture substrate. Growth of the cultures was smaller in serum-free media than in media supplemented with 10% fetal bovine serum. The main advantage of serum-free culture conditions in vitro is the elimination of the influence of serum components on invasion, and the ability to examine the effect on invasion of drugs that are, susceptible to inactivation by serum. This work was supported by the Fonds van de Sport Vereniging tegen de Kanker, Brussels, Belgium, and the Fonds voor Geneeskundig Wetenschappelijk Onderzoek Brussels, Belgium     

The anti-invasive flavonoid (+)-catechin binds to laminin and abrogates the effect of laminin on cell morphology and adhesion

To study the effect of the flavonoid (+)-catechin on cell-matrix interactions two cell types with a different morphology on and adhesion to laminin were used. MO4 virally transformed fetal mouse cells adhere and spread when cultured on top of laminin-coated coverslips or on human amnion basement membrane. M5076 mouse reticulum cell sarcoma cells poorly adhere to these substrates and remain round. Both cell types are invasive in confronting cultures with embryonic chick heart fragments. (+)-Catechin binds to laminin in a pH-dependent way. Pretreatment of laminin-coated coverslips or amnion basement membrane with 0.5 mM (+)-catechin abrogates the effect of laminin on cell morphology and adhesion. MO4 cells do not adhere to the pretreated substrates and remain round, while M5076 cells now adhere and spread. (+)-Catechin inhibits the invasion of MO4 cells but not of M5076 cells into embryonic chick heart in vitro. We speculate that the anti-invasive activity of the flavonoid to MO4 cells is the result of its interference with MO4 cell adhesion to laminin. Invasion of M5076 cells does not imply adhesion to and spreading on laminin.     

Migration inhibition of an epithelial cell line by s-Con A and the effect on its invasiveness

In an attempt to delineate the role of tumor-cell motility in the process of invasion, we compared the migration of NBT II in a two-dimensional migration assay with its migration in a three-dimensional invasion assay. Both systems were maintained with and without succinylated concanavalin A (s-Con A) dissolved in the culture medium. This lectin has a reversible inhibitory effect on the migration of cells in vitro. The migration of NBT-II aggregates, seeded in flasks containing 200 micrograms/ml s-Con A or without s-Con A, was studied by time-lapse photomicrography. In the presence of s-Con A, migration was immediately stopped. When the treated medium was replaced by culture medium to which 10 mM alpha-methyl-mannose was added, the inhibition of migration was abolished. The invasive capacity of NBT II in the presence or absence of s-Con A was studied by confronting precultured fragments of 9- to 11-day-old embryonic chick heart (0.4 mm in diameter) with NBT-II aggregates (0.2 mm) made in the presence or absence of s-Con A. Light microscopy showed no difference in the extent of invasion. To demonstrate the presence of s-Con A in the invading tumor cells, immunoperoxidase staining for Con A was done. The treated cultures stained positively while the controls were negative. The data presented here question the correlation between tumor-cell motility in two-dimensional system and the invasive behavior of these cells in three dimensions, and implies that the ability or inability of cells to migrate on plastic does not necessarily reflect their invasiveness in vitro.     

Interaction of three human malignant cell lines with chick hypoblast in culture

The hypoblast (lower layer) was dissected from young chick blastoderms and explanted in vitro, where it formed an epitheloid sheet. Cells from the following malignant lines were explanted on top of the sheet both as aggregates and as cell suspensions: Hu456 human bladder carcinoma, SAOS-2 human osteosarcoma, LICR(LOND)-HN-4 laryngeal carcinoma. The interaction of the malignant cells with the hypoblast was studied by time lapse cinephotography, light microscopy, and transmission electron microscopy. All malignant cells penetrated through the hypoblast, so that a gradually enlarging hole formed in it. Apart from this common pattern of behaviour, the three types of malignant cells differed in their interactions with the hypoblast in the following ways. 1) Both the Hu456 and to a lesser extent the SAOS-2 cells brought about an initial retraction of the hypoblast so that a temporary cell-free space was formed. No such retraction occurred in response to the LICR-(LOND)-HN-4 cells. 2) Each of the three types of malignant cells migrated for some distance beneath the hypoblast, and in this area of underlap, there were differences in the amount and disposition of extracellular material. Thus, there was more extracellular material between the hypoblast and underlying SAOS-2 cells than between the hypoblast and underlying Hu456 cells, whilst there was no extracellular material between the hypoblast and underlying LICR(LOND)-HN-4 cells. Indeed, the hypoblast and LICR(LOND)-HN-4 cells often shared desmosomes. 3) When explanted as aggregates on hypoblast Hu456 and SAOS-2 cells left the corona and migrated as solitary cells underneath the hypoblast in contrast with control aggregates explanted on plastic. These cells which had migrated beneath the hypoblast were flatter than their corresponding control cells which had spread on the plastic substrate. The flatter cells appeared to have been using the extracellular materials as a substrate, rather than the plastic. Such differences in the migratory behaviour between experimental and control cultures were not observed with LICR(LOND)-HN-4 cells.     

Hypoblast cells of chick pre-streak stage embryos invaded basement membrane analogues in vitro: Implications for hypoblast layer formation

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro . Primary and secondary hypoblast cells from embryos at various pre-streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24–48 h. As a result, we found that after 24 h of serum-supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum-free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.     

The dynamics of antigenic changes in the epiblast and hypoblast of the chick during the processes of hypoblast, primitive streak and head process formation, as revealed by immunofluorescence.

Antisera were prepared to epiblast, primary hypoblast, yolk, yolk entoderm, and extraembryonic yolk sac ectoderm and were submitted to various absorption procedures. The absorbed antisera were used in the indirect immunofluorescent method to stain microscopic sections of developing chick blastoderms at different developmental stages. The antigens revealed by the staining at the periods studied were divided into groups of persistent, nonspecific, and specific antigens. The epiblast does not appear to form or include specific antigens until stage XIII (full hypoblast). The primary hypoblast is the layer which during its formation acquires specificity by the inclusion of antigenic components through a cytoplasmic segregation and probably by one or two waves of appearance of primary hypoblast specific antigens. The inductive role of the hypoblast is discussed in relation to the above antigenic manifestations. The anti-hypoblast and anti-epiblast sera after absorption with yolk were found to be suitable reagents for the detection of morphogenetic movements.     

The sorting-out of thymidine-labelled chick hypoblast cells in mixed epiblast--hypoblast aggregates.

Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa. The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.     

The Sorting-out of Thymidine-labelled Chick Hypoblast Cells in Mixed Epiblast–Hypoblast Aggregates

Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa . The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.     

Gastrulation: Is It Analogous to Malignant Invasion

The process of gastrulation has often been compared with thatof malignant invasion. In this paper, the terms "malignant"and "invasion" are denned and the characteristics of malignantcells are discussed. One of the best examples of invasion duringgastrulation takes place during the formation of the endodermin the chick, when the definitive endoblast invades the hypoblast.Experiments are described in which the hypoblast is invadedby a) definitive endoblast, b) other normal embryonic cells,and c) three types of human malignant cells. It was found thatnot only does the hypoblast react differently to normal andmalignant cells, but that the cell interactions differ alsoaccording to the type of malignant cells. In particular, thereare differences in the behaviour of the cells and in the amountof extracellular material laid down between the hypoblast andmalignant cells. It is concluded that even within the limitsof this experiment, chick gastrulation is not wholly analogousto malignant invasion.     

An electron-microscopical analysis of embryonic chick tissues explanted in culture

Summary Three types of tissue (hypoblast, germ wall and epiblast) were dissected from early chick embryos and explanted on Falcon plastic dishes. After they had settled and spread, the explants were fixed, usually within 18–24 h after explantation, and sections were cut through the tissue and the Falcon dish. The closeness of the cells to the substrate varied even within the same explant, but the epiblast tended to be closer to the substrate than did the hypoblast or germ wall. Plaques were present in all three tissues in regions where the cell processes contacted the substrate. Extensive desmosomes were visible in the epiblast explants, small desmosomes were present in the germ wall explants, but desmosomes were never seen in hypoblast explants. These differences in cell/substrate and cell/cell morphology are discussed in relation to the different behavioural characteristics of the three tissues. Some mixed cultures were also examined by electron microscopy. When the epiblast was confronted with either hypoblast or germ wall, it underlapped them at the region of contact.     

Invasiveness of malignant ST/A mouse lung cells in vitro

ST/A mouse lung cells underwent apparently spontaneous malignant alteration in tissue culture. We have compared the capacity of these cells to form malignant tumours in syngeneic animals with their behaviour in vitro. ST-L1, ST-L22, and ST-L104 cells were malignant, whilst ST-L108 and ST-L109 cells were not. ST-L1 and ST-L22 cells showed anchorage-dependence of growth, whilst ST-L104, ST-L108 and ST-L109 cells did not. ST-L22, ST-L104, ST-L108 and ST-L109 performed directional migration from a spheroid explanted on glass. This capacity was lost in ST-L1 cells, which produced so-called round-cell transformants. All but ST-L108 cells produced type C viral particles. The tumorigenic cell lines ST-L1, ST-L22 and ST-L104 invaded fragments of embryonic chick heart in three-dimensional culture, whereas the non-tumorigenic ST-L108 and ST-L109 cells did not. Furthermore, the histology of ST-L cells invading in three-dimensional culture resembled that of the invasive sarcomas which they produced in vivo. The present observations with ST-L cells confirm that invasiveness in three-dimensional culture is a reliable criterion for malignant of tissue culture lines.     

Invasion of malignant C3H mouse fibroblasts from aggregates transplanted into the auricles of syngenic mice

Spheroid aggregates of malignant fibroblasts (MO4), shown to be invasive in vitro, were implanted subcutaneously into the auricle of the external ear of syngenic C3H mice. The course of early invasion into the surrounding tissues and the formation of tumours was studied in serial sections of auricles fixed 6 h to 30 days after implantation. MO4 cells are first observed to make contact with the surrounding tissues after 6 h. They exhibit, cytoplasmic extensions and spread from the original aggregate. During the first day invading MO4 cells preferentially follow tissue crevices created by the inoculation procedure. Later they also invade the surrounding tissues. Polymorphonuclear leucocytes, and later monocytes infiltrate the aggregate, which is completely destroyed after 4 days. Palpable tumours arise from MO4 cells that have left the original implant and invaded the tissues of the auricles. These observations indicate that invasion of malignant cells is important for the take of a transplant.     

Synthesis,characterization and in vitro anti-invasive activity screening of polyphenolic and heterocyclic compounds

Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 microM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 microM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.     

Selective Cellular Affinities in the Unincubated Chick Blastoderm

Cell suspensions from whole unincubated chick blastoderms (stage 1,⁴) were obtained by EDTA or trypsin dissociation. They were cultured in rotating flasks and aggregates were examined after 1 to 7 days in culture. Early aggregates were solid and consisted of a continuous phase of loosely packed cells with abundant yolk granules, among which groups of tightly packed and less yolky cells were present. Loosely packed cells became peripheral while the tightly packed cells formed continuous cords traversing the aggregate. Aggregates also developed a cavity surrounded mainly by the loosely packed cells. The latter gave rise to cells similar to those of the columnar endoderm of the yolk sac. Our data suggest that sorting-out occurs in aggregates prepared from unincubatedblastoderms, before the onset of gastrulation.     

Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo.

It is well established that hemopoietic cells arising from the yolk sac invade the avian embryo. To study the fate and role of these cells during the first 2.5-4.5 days of incubation, we constructed yolk sac chimeras (a chick embryo grafted on a quail yolk sac and vice versa) and immunostained them with antibodies specific to cells of quail hemangioblastic lineage (MB1 and QH1). This approach revealed that endothelial cells of the embryonic vessels are of intraembryonic origin. In contrast, numerous hemopoietic cells of yolk sac origin were seen in embryos ranging from 2.5 to 4.5 days of incubation. These cells were already present within the vessels and in the mesenchyme at the earliest developmental stages analyzed. Two hemopoietic cell types of yolk sac origin were distinguishable, undifferentiated cells and macrophage-like cells. The number of the latter cells increased progressively as development proceeded, and they showed marked acid phosphatase activity and phagocytic capacity, as revealed by the presence of numerous phagocytic inclusions in their cytoplasm. The macrophage-like cells were mostly distributed in the mesenchyme and also appeared within some organ primordia such as the neural tube, the liver anlage and the nephric rudiment. Comparison of the results in the two types of chimeras and the findings obtained with acid phosphatase/MB1 double labelling showed that some hemopoietic macrophage-like cells of intraembryonic origin were also present at the stages considered. These results support the existence in the early avian embryo of a phagocytic cell system of blood cell lineage, derived chiefly from the yolk sac. Cells belonging to this system perform phagocytosis in cell death and may also be involved in other morphogenetic processes.     

The role of non-muscle myosin IIA in aggregation and invasion of human MCF-7 breast cancer cells

Human MCF-7/6 breast cancer cells differ from their MCF-7/AZ counterparts by their invasiveness in a number of assays in vitro, such as invasion of MCF-7 spheroids into embryonic chick heart fragments or type I collagen gels. Comparative proteomic analysis of these two variants revealed an identical pattern, except for a 230 kDa protein present in the invasive MCF-7/6 variant, but hardly detectable in the non-invasive MCF-7/AZ one. This protein appeared to be the non-muscle myosin IIA heavy chain (NMIIA), also coined MYH9. Experimental inhibition of NMIIA by reducing either its expression (via stable shRNA transduction) or its function (via the specific ATPase inhibitor blebbistatin) underpinned the decisive role of NMIIA in MCF-7 cell invasion. Inhibition of NMIIA indeed blocked the invasion of MCF-7/6 cells in three-dimensional invasion substrata such as embryonic chick heart fragments and type I collagen gels. Invasiveness of MCF-7/6 cells has been related to poor formation and compaction of aggregates, due to a functionally defective E-cadherin/catenin complex. Both genetic and pharmacological inhibition of NMIIA stimulated MCF-7/6 cell aggregation. Together, these data indicate that NMIIA is a decisive protein for MCF-7 cells to invade, indicating that this molecule is a candidate for targeted anti-invasive treatment.     

Early development of the chick embryo

The ultrastructure of the early chick embryo was investigated, using scanning (SEM) and transmission electron microscopy (TEM). Eggs were obtained from the shell gland by injecting hens intravenously with a synthetic prostaglandin or arginine vasopressin. Embryos were examined during late cleavage (stages IV–VI, Eyal-Giladi and Kochav, '76), formation of the area pellucida (stages VII–XI), and formation of the hypoblast (stages X–XIV). SEM highlighted the reduction in cell number at the underside of the embryo during formation of the area pellucida although it became apparent that the thickness of the embryo is not reduced to a single layer of cells at stage X. In addition, blastomeres at the perimeter of embryos (stages V–VI) project filopodial extensions onto a smooth membrane that separates the sub-embryonic cavity from the yolk. During hypoblast formation, epiblast cells generate stellate projections at their basal aspect, thus providing a meshwork for the advancing secondary hypoblast cells. By stage XII the epiblast was one cell thick and reminiscent of a columnar epithelium when viewed transversely. Cells of the deep portion of the posterior marginal zone were distinguished morphologically in the stage XII embryo by their many cell surface projections and ruffled appearance. Blastomeres at the perimeter of stage V–VI embryos projected filopodial extensions onto a smooth membrane which separates the sub-embryonic cavity from the yolk. This membrane is presumed to be confluent with the cytolemma. Evidence is presented demonstrating the presence of intracellular membrane-bound droplets which are hypothesised to contain sub-embryonic fluid. © 1993 Wiley-Liss, Inc.     

Thalidomide may inhibit proliferation of mesenchyme in human limb buds

Limb buds from 4- and 4.5-week-old human embryos were cultured on agar medium consisting of Medium 199, chick embryo extract and horse serum for 4 days with or without thalidomide (1-1.5 microgram/ml), and the direct effect of thalidomide was examined morphologically in histological preparations. In the explants treated with thalidomide, mitotic figures of mesenchymal cells were significantly decreased both in overall explant and in mesenchymal cell aggregates, but the extracellular matrix in the mesenchymal cell aggregates was seen in the experimental and control explants. These findings suggest that thalidomide affects undifferentiated and differentiated mesenchymal cell proliferation but not the chondrogenic capacity of the mesenchyme.     

Interaction between embryonic rat superior cervical ganglion and syngeneic heart fragments in a confronting culture

Freshly dissected 17-19 days embryonic superior cervical ganglia are confronted with 0.5 mm diameter heart fragments of the same Wistar rat. Incubation in vitro at 37 degrees C of confronting heart-ganglion pairs is carried out in Dulbecco's medium on a gyratory shaker. Fixation and staining followed after 2 hr, 1, 3 and 6 days of incubation. Histological analysis with light and electron microscopy revealed the interaction between the sympathetic ganglion and its target organ. Ingrowth of axons rich in neurofilaments and neurotubules and containing light and dense core vesicles, is observed.