Download Gastrulation: Is It Analogous to Malignant Invasion?1

AMER. ZOOL., 24:563-570 (1984)
Gastrulation: Is It Analogous to Malignant Invasion?1
RUTH BELLAIRS
Department of Anatomy and Embryology, University College London,
Gower Street, London WC1 6BT, U.K.
AND
MARIE-CHRISTINE VAN PETEGHEM
Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine,
University Hospital, De Pintelaan 185, B-900 Gent, Belgium
SYNOPSIS. The process of gastrulation has often been compared with that of malignant
invasion. In this paper, the terms "malignant" and "invasion" are denned and the characteristics of malignant cells are discussed. One of the best examples of invasion during
gastrulation takes place during the formation of the endoderm in the chick, when the
definitive endoblast invades the hypoblast. Experiments are described in which the hypoblast is invaded by a) definitive endoblast, b) other normal embryonic cells, and c) three
types of human malignant cells. It was found that not only does the hypoblast react
differently to normal and malignant cells, but that the cell interactions differ also according
to the type of malignant cells. In particular, there are differences in the behaviour of the
cells and in the amount of extracellular material laid down between the hypoblast and
malignant cells. It is concluded that even within the limits of this experiment, chick
gastrulation is not wholly analogous to malignant invasion.
Many investigators have compared certain processes that take place during
embryonic development with those that
occur during malignant invasion (e.g.,
Armstrong, 1977; Sherbet, 1982), and a
number of now well-known examples tend
to be quoted. These include the invasion
of the uterine wall by the trophoblast; the
migration of neural crest cells, primordial
germ cells and growth cones of axons; and
the migration of cells during gastrulation.
In this paper, we propose to assess the
value of such an analogy, but we shall
restrict ourselves to the events in chick gastrulation and compare them with features
that characterise malignant invasion.
Let us start by establishing what we mean
by the term "invasion" when it is used in
the context of cell biology and pathology.
It is the process whereby cells or groups of cells
of one type pass into another region of the body
and displace cells of another type. For exam-
ple, cells from a carcinoma of the skin may
break through the basal lamina and displace some of the underlying connective
1
From the Symposium on Gastrulation presented
at the Annual Meeting of the American Society of
Zoologists, 27-30 December 1982, at Louisville, Kentucky.
tissue and muscle. A similar sequence of
events occurs at several stages in chick gastrulation. One oft-quoted example occurs
as cells migrate through the primitive streak
and then pass laterally as the newly formed
mesoderm. But this event is not an ideal
example of invasion however, since the new
cells pass between the ectoderm and
endoderm, and do not appear to displace
other cells. Recently, however, it has
become apparent that one of the best
examples of embryonic invasion takes place
in the chick embryo, during the formation
of the definitive endoblast.
The initial lower layer of the chick
embryo is called the hypoblast. It is completed soon after incubation begins and
does not contribute to the embryonic endoderm. Instead, it spreads out to the
periphery of the area pellucida and subsequently forms extra-embryonic endoderm (Vakaet, 1962, 1970; Rosenquist,
1971, 1972; Fontaine and Le Douarin,
1977). The future embryonic endoderm
(or definitive endoblast) is initially located
around the anterior end of the primitive
streak. At the time of its formation it inserts
itself into the hypoblast and gradually
spreads, so that it forms a patch in the central part of the lower layer while the hypo-
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R. BELLAIRS AND M.-C. VAN PETEGHEM
b
c
Definitive
Endoblast
Hypoblast
Hypoblast
FIG. 1. Diagram of the lower layer of the area pellucida of a young chick blastoderm, a. At about 12 hr of
incubation, b. At about 18 hr of incubation, c. At about 20 hr.
blast cells move away (Fig. 1). This is a
process therefore which exhibits the essential features of invasion, as defined above.
Malignant invasion may or may not be
accompanied by other phenomena, such as
the destruction of host tissue with the consequent occurrence of patches of necrosis
and often of inflammatory responses at the
invaded site by the host organism. The
invasion of the hypoblast however does not
include such features, though small patches
of necrotic cells are indeed present in the
chick gastrula (Bellairs, 1961). Indeed,
localised patches of dead cells are a wellknown component of many tissues in developing embryos (Gliicksman, 1951) but are
not necessarily associated with invasiveness.
The term malignancy is less easy to define
than invasion. It was originally applied by
pathologists to tumours which are able to
invade and usually also to metastasize, i.e.,
after the cells leave the primary tumour
and pass to another region of the body,
they form there a secondary tumour. Ultimately, malignant cells lead to the death
of the host. Ideally then, the term malignant should be used only for a cell line or
tumour that has been shown to possess these
properties when it has been transplanted
back into a whole animal. When we speak
of malignant cells in this discussion therefore, we shall be concerned only with cells
which come from lines which fulfill these
criteria. We shall not be concerned with
the behaviour of so-called "transformed
cells" nor with that of non-malignant
(benign) tumours (i.e., tumours which do
not invade and metastasize).
Invasiveness is an essential property of
malignant cells and it is a property which
is retained by them when they are transplanted into other tissues. It is thus a characteristic of the cells themselves rather than
something imposed on them by their surroundings (Mareel, 1980).
What then are the characteristics of true
malignant cells which permit them to be
invasive? Unfortunately, this is not an easy
question to answer since different tumours
exhibit different characteristics and it has
not so far been possible to find a precise
description which fits all malignant cells.
The characteristics which have appeared
promising have been lack of contact inhibition of locomotion (Abercrombie, 1975),
loss of cellular adhesion (Coman, 1944),
absence of directionality (Trinkaus, 1976),
dependence on growth (i.e., increase in
mass) (Eaves, 1973), active cell movements
(Strauli and Weiss, 1977; Mareel and De
Brabander, 1978) and secretion of proteolytic enzymes (Reich et al., 1975). There
can be little doubt however that while some
or even all of these characteristics may be
important in certain situations others may
be irrelevant, and are even shared by normal tissues. Mareel (1982) has criticised the
idea that invasion depends on an active
growth of the tumour and has suggested
GASTRULATION AND MALIGNANT INVASION
instead that growth may contribute indirectly to invasion merely because the number of invasive cells increases.
The problem is further complicated by
the fact that although certain aspects of
malignancy, such as metastasis, can be studied only in vivo, most direct observations
have had to be made in vitro. Indeed a wide
variety of culture techniques have been
used, and although there are indeed reasons for supposing that the behaviour of
the cells in vitro parallels their behaviour
in vivo, doubts must always remain (Mareel,
1982).
Embryologists are of course more fortunate because it is easier to follow the
behaviour of gastrulating cells directly in
vivo, at least in the chick blastoderm (for a
recent review, see Bellairs, 1982). Nevertheless, if we are to compare the behaviour
of malignant and gastrulating cells, we perforce must carry out most of our experiments under the same conditions, i.e., in
culture. Before discussing these experiments, there is one further point to be
emphasized. So far we have spoken mainly
of the characteristics of the invasive cells
and have scarcely mentioned the host cells.
But these too play a role. For example some
tissues may be protected from invasion by
the basement membrane which acts as a
barrier (Ingber and Jamieson, 1982; Liotta
et al., 1982) while other tissues, cartilage
in particular (Kuettner et al., 1978), are
highly resistant in their own right to invasion. In considering any invasive situation
therefore we must take account not only
of the invading tissue but also of the invaded
one and the way it reacts.
The questions we will ask therefore are:
How does the hypoblast react to being
invaded by:
(a) Definitive endoblast (its normal invader)?
(b) Other normal embryonic cells? and
(c) Malignant cells? and
(d) Do all malignant cells react in the same
way to the hypoblast?
The main series of experiments involve
first, dissection of the hypoblast from the
young chick embryo (usually before the
primitive streak has begun to form) and
565
the explanting of it in culture so that it
spreads as a monolayer. Second, another tissue is brought into contact with this monolayer.
In the original series of experiments
(Sanders et al., 1978), this second tissue was
explanted at the same time as the hypoblast
so that as the two tissues spread and formed
monolayers, they came into confrontation
with one another. When the hypoblast
explant was confronted with a definitive
endoblast explant the hypoblast cells
became displaced by the definitive endoblast. The hypoblast explant tended to
fragment into smaller groups of cells, many
of which migrated around the definitive
endoblast, so that the eventual layout of
the tissues mimicked the situation in vivo.
In a series of control experiments hypoblast was confronted with hypoblast, or with
somites, while definitive endoblast was confronted with definitive endoblast, or with
somites. It was found that the hypoblast
behaved consistently, whatever the other
tissue, by fragmenting and allowing the
other tissue to invade it. It was suggested
that the ability of the hypoblast cells to
separate easily from one another might play
an important role in the penetration of the
hypoblast sheet by the definitive endoblast,
both in vivo and in vitro.
Unfortunately, we do not as yet possess
any direct evidence as to the relative cellto-cell adhesiveness of the hypoblasts and
of the definitive endoblast, but our interpretation receives some indirect support.
Thus, the hypoblast of the early embryo
both in vivo and in vitro rarely possesses
desmosomes though simple tight junctions
are not uncommon (Sanders et al., 1978;
Stolinski et al., 1981; Al-Nasser and Bellairs, 1982). Small spaces can sometimes be
seen between the cells. By contrast, in the
definitive endoblast, the desmosomes are
more common, tight junctions are more
elaborate and intercellular spaces are not
normally seen. Further, although the
hypoblast in vivo is of an epithelial-like
structure, it conspicuously lacks a true
basement membrane (Sanders, 1979) and
for that reason may be expected to offer
little resistance to invasion. It was concluded therefore that certain properties of
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R. BELLAIRS AND M.-C. VAN PETEGHEM
the hypoblast may be important in permitting invasion of this tissue, in vivo as well
as in vitro.
When we approached the problem in a
different way by explanting pieces of other
embryonic tissues on top of the hypoblast
monolayer, once again we found that these
cells readily invaded it (Bellairs et al., 1981).
Indeed, they passed right through the
hypoblast monolayer, apparently by taking
advantage of the small gaps in the sheet,
and spread on the glass or plastic substrate
forming a patch in the substance of the
monolayer. However, the situation turned
out to be a complex one, because when an
extensive series of experiments using different tissues was carried out, it was found
that irrespective of the identity of the tissue grown as an initial monolayer and that
of the other tissue explanted on top of it,
the upper tissue generally penetrated the
monolayer and spread on the substrate.
Thus, even hypoblast itself, when placed
on the top of other tissues, tended to invade
them and spread.
Meanwhile, Mareel et al. (1970, 1975)
had approached the problem from the
other end. They had been interested in the
use of the young chick blastoderm as a suitable host in which to implant malignant
tissues. And indeed they had reported
striking differences in the behaviour of the
hypoblast in situ when normal or when
malignant cells were inserted into it. Thus,
when normal cells were inserted, the
hypoblast healed, but when malignant cells
were inserted, healing was inhibited.
More recently, Van Peteghem et al.
(1980) drew attention to the potential of
the hypoblast as a test tissue for studying
malignant invasion in culture, pointing out
that the intracellular yolk spheres provided a good natural marker, and they carried out a series of confrontations between
hypoblast and malignant cells in 3-D culture.
Very recently however, Van Peteghem
et al. (1983) have investigated the interaction of hypoblast monolayers with three
types of malignant cells in monolayer cultures. These experiments were carried out
in a comparable manner to those performed by Bellairs et al. (1981) in mono-
layer culture. The results may therefore
be compared directly. The malignant cell
lines used were:
HU456, which is derived from a transitional cell carcinoma of the human bladder (Schroyens et al., 1984).
SAOS-2, which is derived from a human
osteosarcoma (Fogh and Trempe, 1975)
and
LICR (LOND.)-HN4, which is derived
from a squamous cell carcinoma of the
human larynx (Easty et al., 1981).
A similar series of experiments was carried out by Van Peteghem and Mareel
(1982) using malignant MO4 cells, which
are virally transformed C3H mouse cells
(Billiau et al., 1973) which have been shown
to produce invasive and metastasizing
fibrosarcomas in syngeneic mice (Meyvisch
and Mareel, 1982).
A major aim of the experiments of Van
Peteghem et al. (1983) was to determine
whether the invasion of the hypoblast by
the malignant cells was similar to its invasion by normal embryonic cells. In some
respects it was so. Each type of malignant
cell passed through the monolayer sheet of
hypoblast soon after settling on it. It is likely
that, as with non-malignant explants, they
took advantage of the small gaps between
the cells which are a transient feature of
cultured sheets of hypoblast. In control
explants of hypoblast these gaps tended to
heal within a few hours, but if an explant
had settled and given rise to an outgrowth,
the gap enlarged to form a hole and usually
failed to heal. With both normal and malignant tissues the hypoblast cells at the edge
of the hole tended to become aligned parallel to it. It seems likely however that the
failure to heal may have been due at least
in part to the conditions of culture, since
when similar experiments were carried out
in 3-D culture (Van Peteghem etal, 1980),
or in situ in the embryo (Mareel et al., 1975),
non-malignant tissues did not prevent healing of the hypoblast, which grew back over
them.
In other respects however, the results
obtained with malignant cells differed from
those with normal ones. Indeed, the interactions that took place in the experiments
GASTRULATION AND MALIGNANT INVASION
567
FIG. 2. Phase contrast micrographs of aggregates of malignant cells (M) explanted on top of hypoblast sheets
(H) and fixed after 3 hr. a. HU456. b. SAOS-2. c. LICR (LOND.)-HN4. Note that with both HU456 and
SAOS-2 cultures a cell-free space (arrowhead) lies between the malignant cells and the edge of the hole in
the hypoblast (arrow). Scale bars: 100 nm.
with malignant cells differed according to
the type of malignant cells.
One of the main differences between the
malignant cells was that aggregates of
HU456 cells, and to a lesser extent of
SAOS-2 cells provoked a retraction of the
hypoblast cells away from them so that a
cell-free space was visible partly or totally
around the malignant tissue for several
hours (Fig. 2). A similar retraction occurred
when aggregates of MO4 cells were
explanted on top of hypoblast sheets (Van
Peteghem and Mareel, 1982). (These cellfree spaces were later abolished as cells
migrated out from the nodule of malignant
cells.) Such retractions were never seen
when definitive endoblast, or indeed any
other normal embryonic tissue, was
explanted onto the hypoblast.
By contrast to the other malignant cell
lines tested, the LICR (LOND.)-HN4
aggregates did not appear to cause such a
retraction, and in this respect were more
comparable with the non-malignant cells
used by Bellairs el al. (1981).
Whatever the type of malignant tissue
which had been placed on a hypoblast sheet
however, it was found that within about 24
hr the cells had spread from it for a short
distance beneath the hypoblast so that there
was clear nuclear underlapping. This
underlapping was usually more extensive
than the very limited underlapping of
lamellae reported for normal embryonic
cells (Bellairs et al., 1981; Al-Nasser and
Bellairs, 1982). It was in this region of
underlap that the interactions of hypoblast
and malignant cells were most apparent.
Probably the most important differences
between the different malignant cell lines
was in the amount and distribution of
extracellular materials found in the cultures. Thus, cultures of hypoblast grown
with SAOS-2 cells had more extracellular
material than control hypoblasts growing
alone. Cultures of hypoblast with HU456
cells had a comparable amount to control
hypoblasts. And hypoblasts growing with
LICR (LOND.)-HN4 cells had the least
extracellular material (Fig. 3). In control
explants of these malignant tissues growing alone for the same length of time extracellular material was absent apart from a
small amount beneath the LICR (LOND.)HN4 cells.
The location of the extracellular material in the narrow region of underlap is of
particular interest. With the hypoblast/
SAOS-2 cell cultures, the large amounts of
extracellular material lay between the
568
R. BELLAIRS AND M.-C. VAN PETEGHEM
3a
3b
\
: ^
FIG. 3. Transmission electron micrographs of transverse sections from malignant cells (M) explanted on top
of hypoblast sheets (H). The malignant cells have penetrated through the hypoblast sheet and spread beneath
it. a. HU456 aggregates fixed after 3 days. b. SAOS-2 aggregates fixed after 3 days. c. LICR (LOND.)-H\4
GASTRULATION AND MALIGNANT INVASION
hypoblast and the subjacent SAOS-2 cells.
Although we have no direct evidence as to
which of the two tissues secreted this material, nevertheless it appeared that an interaction had occurred between them which
had resulted in this exceptionally high
amount.
This reaction is in striking contrast to
that between the hypoblast and the LICR
(LOND.)-HN4 cells where no extracellular
material separated the two tissues. Indeed
even the amount which would normally
have been present under the hypoblast was
missing in the region of underlap, and the
two tissues lay so close together that not
only were their cell membranes in close
parallel apposition, but they even shared
desmosomes. It appeared as if the interaction of the two tissues was favourable to
both.
Further evidence suggests that not only
the amount and distribution of extracellular materials varied according to the type
of malignant cell line, but the malignant
cells themselves appeared to be affected
differently by the hypoblast. Thus, cells of
HU456 or of SAOS-2 which lay beneath
the hypoblast were more spread than those
which were not covered by hypoblast. It
appeared that this was because those
beneath the hypoblast were spreading at
least partially on the extracellular materials.
In striking contrast, the cells of LICR
(LOND.)-HN4 apparently migrated directly on the undersurface of the hypoblast
cells.
In conclusion, let us return to the question of whether embryonic invasion, as
exemplified by the invasion of hypoblast
by definitive endoblast, is analogous to
malignant invasion. The results of our
experiments show that at least under the
tissue culture conditions we have used,
there are certain similarities, viz., the
invading cells, whatever they are, are able
to penetrate the hypoblast sheet and displace the hypoblast cells.
569
When we compare the reactions that
occur between the hypoblast and normal
embryonic cells, with those between
hypoblast and malignant cells however, an
important difference emerges. The hypoblast appears to react in much the same
way to definitive endoblast and other normal cells, though a more extensive investigation is needed to establish that this is
always so. By contrast, the hypoblast has
no standard reaction to invasion by malignant cells. The interaction varies according to the cell line. In this respect therefore
it is difficult to define the precise nature of
malignant invasion even when we restrict
ourselves to a small number of malignant
cell lines. Even within the arbitrarily
imposed limits of our experimental model,
chick gastrulation is not wholly analogous
to malignant invasion.
ACKNOWLEDGMENTS
We are most grateful to Dr. M. M. Mareel
for reading and criticising the manuscript.
We wish to thank B. Buysse, O. Claeys, M.
Nijs and A. Verspeelt for technical assistance, J. Roels van Kerkvoorde for preparing the photographic illustrations, R.
Cleevely for Fig. 1, and G. Matthys-De Smet
for typing the manuscript.
This work was supported by grants from
the Cancer Research Campaign, U.K., and
the Kankerfonds van de Algemene Spaaren Lyfrentekas, Brussels, Belgium.
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