* Institute for Anatomy, University of Cologne, 50931 Cologne, Germany; Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; and § Boehringer Mannheim, 82372 Penzberg, Germany
Teratomas are benign tumors that form after
ectopic injection of embryonic stem (ES) cells into mice
and contain derivatives of all primitive germ layers. To
study the role of 1 integrin during teratoma formation,
we compared teratomas induced by normal and
1-null
ES cells. Injection of normal ES cells gave rise to large
teratomas. In contrast,
1-null ES cells either did not grow or formed small teratomas with an average weight
of <5% of that of normal teratomas. Histological analysis of
1-null teratomas revealed the presence of various differentiated cells, however, a much lower number
of host-derived stromal cells than in normal teratomas.
Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited
in
1-null teratomas. Basement membranes were present
but with irregular shape and detached from the cell
surface.
Normal teratomas had large blood vessels with a
smooth inner surface, containing both host- and ES
cell-derived endothelial cells. In contrast, 1-null teratomas had small vessels that were loosely embedded into
the connective tissue. Furthermore, endothelial cells
were always of host-derived origin and formed blood
vessels with an irregular inner surface. Although
1-
deficient endothelial cells were absent in teratomas,
1-null ES cells could differentiate in vitro into endothelial
cells. The formation of a complex vasculature, however,
was significantly delayed and of poor quality in
1-null embryoid bodies. Moreover, while vascular endothelial
growth factor induced proliferation of endothelial cells
as well as an extensive branching of blood vessels in
normal embryoid bodies, it had no effect in
1-null embryoid bodies.
A hallmark of tumor cells is their ability to grow anchorage independent. Proliferation and survival of
tumor cells, determining progression of solid tumors, are independent of signals elicited by interactions
with the surrounding extracellular matrix (ECM1; Folkman and Moscona, 1978 Integrins are the most important family of cell surface
receptors that mediate cell-matrix interactions (Hynes,
1992 Integrin engagement and clustering regulate shape, motility, survival, and proliferation of cells. These events are
executed by integrin-mediated cascades of intracellular
signals that include tyrosine phosphorylation of FAK
(Guan and Shalloway, 1992 In the present study we have used Cells and Cell Culture
The following ES cells were used to induce teratomas: wild-type ES cell
line D3 (+/+; Doetschman et al., 1985 ES cells were cultured in the absence of a fibroblast feeder layer in
DME, supplemented with 20% heat-inactivated FCS (GIBCO BRL,
Gaithersburg, MD), 0.1 mM For differentiation, ES cells were cultured in hanging drops as described previously (Fässler et al., 1996 Alternatively, after outgrowth of cell aggregates for 20 d, cells were
trypsinized and cultured on gelatin-coated glass cover slips for 24 h, fixed,
and stained for von Willebrand Factor (vWF) or PECAM. Positive cells
were counted using an Axiophot fluorescence microscope (Carl Zeiss,
Oberkochen, Germany).
For VEGF treatment, ES cells were cultured in hanging drops for 2 d,
in bacteriological dishes for 3 d, and then plated on gelatinized glass cover
slips for another 12 d. The culture medium was DME, containing 20%
FCS with either 10 or 20 ng/ml VEGF (R&D Sytems, Wiesbaden, Germany).
Antibodies
The following primary antibodies were used: rabbit anti-rat The following secondary antibodies were used: goat anti-rabbit FITC;
goat anti-rabbit Cy3; rabbit anti-hamster FITC; goat anti-chicken FITC;
donkey anti-goat Cy3; goat anti-rat Cy3 (Jackson Immunoresearch Lab.
Inc., West Grove, PA), rabbit anti-digoxygenin FITC (Boehringer Mannheim, Mannheim, Germany), biotinylated goat anti-rabbit Ig (Vector
Laboratories, Inc., Burlingame, CA), biotinylated goat anti-rat Ig (Amersham Intl., Little Chalfont, UK), and streptavidin-horseradish peroxidase
conjugate (Amersham Intl.).
Teratoma Induction
107 ES cells were trypsinized, washed twice, suspended in 100 µl PBS, and
then injected subcutaneously on the back of syngeneic 129/SV male mice.
After 21 or 28 d, tumors were surgically removed and frozen in ice-cold
isopentan. To analyze cell proliferation, 25 mg per 100 g body weight of
the thymidine analogue bromodeoxyuridine (BrdU) was injected intraperitoneally 2.5 h before the excision of the tumors.
Microscopical Analysis of Embryoid Bodies
and Tumor Tissue
Light microscopy.
For light microscopical examination, small pieces of teratomas and 20-µm-thick immunostained cryosections were dehydrated conventionally in a graded ethanol series, and finally infiltrated with and
embedded in araldite (Serva, Heidelberg, Germany). Semithin sections of
1-2 µm were analyzed using a Zeiss Axiophot microscope (Carl Zeiss),
with or without methylene blue staining.
Histochemistry.
Teratomas were surgically removed, frozen in ice-cold
isopentan, and stored at Cell proliferation.
Incorporation of intraperitoneally injected BrdU
into the DNA of replicating teratoma cells was analyzed using anti-BrdU
monoclonal antibodies, following the protocol supplied by the manufacturer (Boehringer Mannheim).
). In contrast, normal diploid cells require anchorage to the ECM for proliferation as well as
survival (Dike and Farmer, 1988
). Several lines of direct
evidence show that integrins transduce these signals (Varner and Cheresh, 1996
).
). They are heterodimers of noncovalently linked
and
subunits. So far 15 different
subunits and 8 different
subunits are known. The
1 subunit can associate
with at least 10 different
subunits forming the largest
subfamily of integrins. Members of the
1 integrin subfamily primarily bind to components of the ECM such as
fibronectin, collagens, and laminins, but some of them also participate in direct cell-cell adhesion (Hynes, 1992
; Haas
and Plow, 1994
). The cytoplasmic domain of
1 integrin
can directly interact with cytoskeletal proteins such as
talin and
-actinin and with signal transducing proteins
such as focal adhesion kinase (FAK; Schaller et al., 1995
)
and integrin-linked kinase (Hannigan et al., 1996
).
), increases in intracellular
Ca2+ levels (Schwartz, 1993
), intracellular pH (Schwartz et
al., 1989
, 1990
), inositol lipid synthesis (McNamee et al.,
1993
), and expression of cyclins (Guadagno et al., 1993
).
Furthermore, it has been demonstrated that integrins can
also mediate the activation of protein kinase C (Vuori and
Ruoslahti, 1993
), mitogen-activated protein kinase (Morino et al., 1995
) and NF-
B (Yebra et al., 1995
). In addition to these adhesion-mediated signaling pathways, many
cells depend on growth factor-mediated signals for appropriate cell cycle progression and proliferation.
1 integrin-deficient
embryonic stem (ES) cells (Fässler al., 1995) to induce teratomas in syngeneic mice. ES cells as well as pre- or early
postimplantation embryos of most mouse strains develop
into tumors when transplanted into an ectopic location of
syngeneic animals (Damjanov and Solter, 1974
; Damjanov, 1978
). These tumors are composed of various differentiated somatic tissues and are called teratomas. We
show that
1-null ES cells give rise to either very small or
no teratomas. The most prominent changes that are associated with the impaired growth in
1-null teratomas are
abnormal depositon of ECM proteins and various defects
in basement membranes. Furthermore,
1-null teratomas showed an inefficient angiogenesis. A number of studies
have demonstrated convincingly that tumor growth is dependent on angiogenesis (Folkman, 1996
). Tumor angiogenesis is regulated by factors produced by tumor cells as
well as by cell adhesion molecules expressed on endothelial cells. Systemic or local administration of antibodies or
cyclic RGD peptides blocking
v
3 integrin function inhibits tumor angiogenesis and as a consequence promotes
tumor regression (Brooks et al., 1994a
). This anti-angiogenic effect of the
v
3 antagonists results from the activation of apoptosis of newly sprouting blood vessels
(Brooks et al., 1994b
). We now show that
1 integrin plays
an essential role during angiogenesis in teratomas. In normal teratomas both host- and ES cell-derived endothelial cells contribute to angiogenesis. In contrast, in
1-null teratomas, all vascular cells are exclusively derived from the
host. Furthermore, we report that vascular endothelial
growth factor (VEGF) treatment-induced proliferation of
endothelial cells and extensive branching of blood vessels
in normal but not in
1-null embryoid bodies.
MATERIALS AND METHODS
); G119 (+/
), which is heterozygous for the
1 integrin gene mutation; G101 (+/+), which is wild-type for
1 integrin gene but mock transfected; and G201 (
/
) which is
1 integrin deficient (Fässler et al., 1995
). In the cell clones G119 and G201, a fusion DNA of
-galactosidase and neomycin is inserted in frame with the
ATG of the
1 integrin gene (Fässler et al., 1995
). The cell clone G101
contains a randomly integrated
-galactosidase gene that is ubiquitously
expressed.
-mercaptoethanol (Sigma Chemical Co., St.
Louis, MO), 1× non-essential amino acids (GIBCO BRL), and 1,000 U/ml
recombinant leukemia inhibiting factor (GIBCO BRL).
). Briefly, 600 cells were cultured in
20 µl of DME, supplemented with 20% FCS hanging from the lid of the
culture dish for 5 d, which allows the formation of cell aggregates (embryoid bodies). Subsequently, the aggregates were plated on Tissue Tek
chambers (Nunc, Wiesbaden, Germany) and incubated for 7, 15, or 20 d,
respectively, fixed in 4% paraformaldehyde and immunostained for von
Willebrand Factor (Behringwerke Ag, Marburg Lahn, Germany) and
platelet endothelial cell adhesion molecule (PECAM) (Vecchi et al.,
1994
).
1 integrin;
hamster anti-mouse
3 integrin; hamster anti-mouse
v integrin (both
from PharMingen, San Diego, CA); hen anti-human FN (Johansson and
Höök, 1984
); rabbit anti-mouse FN (GIBCO BRL); goat anti-collagen
type I; goat anti-collagen type III (both from Southern Biotechnology Associates Inc., Birmingham, AL); rat anti-mouse nidogen (Dziadek et al.,
1988
); rat anti-mouse PECAM-1 (Vecchi et al., 1994
); and rabbit anti-
mouse vWF (Behringwerke).
80°C. Tissue specimens were cut into 10-µm-thick sections and collected on glass slides (Shandon, Frankfurt, Germany). Hematoxylin/Eosin and lacZ staining followed published protocols (Fässler
et al., 1995
). Stained sections were analyzed using a Zeiss Axiophot microscope.
Apoptosis.
Cell death was analyzed following the protocol supplied by
the manufacturer (Oncor, Heidelberg, Germany). Briefly, tissue sections
were fixed in 4% paraformaldehyde in PBS, postfixed in ethanol/acetic
acid (2:1), and incubated with terminal deoxynucleotidyl transferase,
which catalyzes the addition of digoxygenin-conjugated nucleotides to
free 3-hydroxyl groups in apoptotic cells. Incorporation of labeled nucleotides was detected by FITC-conjugated, anti-digoxygenin antibodies.
The number of apoptotic cells was determined from random sections of
three normal and four
1-null teratomas.
Immunofluorescence.
Tumor sections, which were 6-µm thick, were incubated with primary antibody in PBS, supplemented with 1% BSA at dilutions recommended by the suppliers for 1 h, at room temperature. Subsequently, the sections were washed three times in PBS and incubated
with fluorescence-labeled secondary antibodies at dilutions recommended by the manufacturer for 1 h, at room temperature. After washing three
times in PBS, the fluorescent specimens were mounted using elvanol
(Fässler et al., 1995).
Immunohistochemistry. For immunohistochemistry, 20-µm-thick cryosections of sucrose-embedded teratomas prefixed with 4% paraformaldehyde in 0.1× PBS, and paraformaldehyde-fixed embryoid bodies were stained. Endogenous peroxidase was blocked by 3% H2O2 in 60% methanol. Subsequently, cells were permeabilized with 0.2% Triton X-100 (Sigma Chemical Co.) in 0.1× PBS. The sections were incubated with von Willebrand antibody (1:1,000) or PECAM antibody (1:800) in PBS. Embryoid bodies were incubated in PBS, supplemented with 0.8% BSA overnight at 4°C. Subsequent sections and embryoid bodies were washed four times in PBS and incubated with biotinylated secondary antibodies for 1 h at room temperature. A streptavidin-horseradish peroxidase complex was then used as a detection system (1:200; incubation for 1 h at room temperature). The staining was developed for 15 min with DAB in 5 mM Tris-HCl buffer, pH 7.4, supplemented with 0.1% H2O2. Analysis was performed using a Zeiss Axiophot microscope. Morphometrical analyses of vessels identified by PECAM antibody were performed with Optimas 5.2 (Optimas Corporation, Bothwell, WA).
Electron Microscopy
Tumor tissue was obtained from 6-wk-old chimeric animals, which were killed by cervical dislocation and subsequently transcardially perfused with a 0.1 mol/liter cacodylate buffer containing 2% paraformaldehyde and 2% glutaraldehyde at a perfusion pressure of 60 cm H2O. Tumors were removed, fixed for an additional 4 h in the same fixative, and then cut into small pieces and fixed in 0.1 mol/liter cacodylate buffer, pH 7.3, containing 2% osmium tetroxide for 2 h at 4°C. Tissue pieces were rinsed three times in cacodylate buffer, block stained for 8 h in 70% ethanol containing 1% uranyl acetate, dehydrated in a series of graded ethanol, and embedded in araldite. Ultrathin sections (30-60 nm) obtained with a diamond knife on a Reichert ultramicrotome (Reichert, Bensheim, Germany) were placed on copper grids and examined with a Zeiss EM 902A electron microscope.
Embryoid bodies were fixed in a 100 mM Hepes/Pipes buffer, pH 7.35, containing 1.75% paraformaldehyde, 2% glutaraldehyde, and 15% picric acid for 1 h at room temperature. Afterwards embryoid bodies were treated with 100 mM Hepes/Pipes buffer containing 1% tannic acid for 30 min at room temperature and finally osmificated with 0.5% OsO4. Before embedding in Epon resin (Agar Scientific, Stansted, UK) embryoid bodies were dehydrated in a graded series of ethanol. For light microscopy, semithin sections were stained with methylene blue. For electron microscopy, ultrathin sections (30-60 nm) were mounted on formvar-coated copper grids, stained with 0.2% uranyl acetate and lead citrate, and then examined with a Zeiss EM 902A electron microscope.
ES Cells Lacking 1 Integrin Form No or
Only Small Teratomas
To test whether the lack of 1 integrin has an influence on
the development of teratomas normal and
1-null ES cells
were injected subcutaneously into syngeneic male mice.
After 21 d of incubation, wild-type tumors were clearly
visible on the back of the mice. In contrast,
1-null tumors
either did not form or were significantly reduced in size
(Fig. 1).
To investigate the growth of 1-null tumors in more detail, mice were killed by cervical dislocation and examined
under the stereomicroscope. In 9 out of 18 animals injected
with
1-null ES cells, no tumor could be detected either at
the site of injection, or in distant organs. In the remaining
nine animals, a small tumor had formed, which was significantly reduced in weight (mean value 47 mg) when compared with tumors derived from normal (0.93 g) or
1 integrin-heterozygous (1.18 g) ES cells (Fig. 2). No difference in tumor growth was observed in mice carrying either a
1-null tumor alone or together with a normal or heterozygous tumor.
Cell Proliferation and Apoptosis Is Not Significantly
Altered in 1-null Teratomas
To test whether cell proliferation is reduced in 1-null tumors BrdU was injected into tumor-bearing mice. Detection of BrdU in tissue sections with specific antibodies
showed that the distribution of replicating cells is similar
in normal and
1-null teratomas. Some areas in both tumor types contained nests of many proliferating cells,
whereas other areas show a homogenous distribution of a few proliferating cells (Fig. 3). This heterogeneity of the
tumor architecture results from the wide range of different
tissues that form in teratomas. As a consequence, the
number of proliferating as well as apoptotic cells varied
tremendously from one area to another; normal tumors
grown for 10 d had between 84 and 176 BrdU-postive cells
per mm2 tissue section and tumors grown for 21 d had between 220 and 279 BrdU-positive cells per mm2 tissue section. In corresponding sections of
1-null teratomas, the
number of proliferating cells ranged between 42 and 116 cells in 10-d-old teratomas, and between 156 and 265 cells
in 21-d-old teratomas. This large range in proliferating
cells in both tumor types did not provide a statistically significant difference but a tendency towards a reduced proliferation rate in
1-null teratomas.
Similarly, the number of apoptotic cells was very inhomogenously distributed in the tissue of normal and 1-null
teratomas:
1+/+,
1+/
, and
1
/
teratomas showed
higher apoptosis rates in areas that were lacZ-positive, and
thus ES cell-derived and lower rates in areas that were
lacZ-negative, and thus host cell-derived (data not shown).
Both tumor types showed areas with small cell clusters
with a high rate of apoptosis, whereas in other areas the
apoptotic cells were evenly distributed (Fig. 3). The number of apoptotic cells ranged between 250 and 712 cells per
mm2 tissue in normal teratomas, and between 249 and 805 cells per mm2 tissue section in
1-null teratomas. Also
here, the large variation did not provide a statistically significant difference but again a tendency towards a higher
apoptosis rate in
1-null teratomas was apparent.
1-Null Teratomas Contain Differentiated Cells
but Have an Altered Tissue Architecture with Less
Host-derived Cells
Hematoxylin/Eosin staining revealed that 1 +/+ teratomas (G101) (Fig. 4 A) and
1 +/
teratomas (not shown)
are composed of a variety of differentiated cells and tissues, e.g., glandular epithelium, cartilage, connective tissue,
and neuronal cells.
1-Null tumors appeared cell dense
and contained fewer cords of fibrotic material (Fig. 4 B).
Histochemical analysis of several
1-null tumors revealed
that many differentiated cells are present in
1-null teratomas. ES cell-derived cells could be differentiated by lacZ
activity, since the lacZ gene was introduced in the
1 integrin gene and in the genome of the mock-transfected ES
cells (Fässler et al., 1995
; and data not shown). Mock-transfected, as well as
1 integrin-heterozygous tumors
contained large areas devoid of lacZ activity proving their
host-derived origin (Fig. 4 C). In contrast, the number of
lacZ-negative cells was low in all
1-null tumors analyzed
(Fig. 4 D) indicating that the migration of normal host
cells into mutant tumor tissue is reduced.
Deposition of Extracellular Proteins Is Altered in
1-null Teratomas
To test whether the deposition of matrix proteins is affected
by the lack of 1 integrin, the distribution of fibronectin, collagen I, and nidogen were compared between normal and
1-null tumors. In normal tumors most of the fibronectin
(Fig. 5 C) was associated with cells expressing large amounts
of
1 integrin (Fig. 5 A). Only in a few areas fibronectin
staining did not colocalize with
1 integrin staining (data
not shown). In
1-null tumors, on the other hand, fibronectin
was deposited throughout the tumor tissue (Fig. 5 D) and
did not correlate with host cell-derived
1 integrin expression
(Fig. 5 B). Similarly, in sections derived from
1-null tumors,
collagen I (Fig. 5 F) and nidogen (not shown) were diffusely
distributed. This was not the case in normal tumors in
which collagen I (Fig. 5 E) and nidogen (not shown) were
mainly restricted to areas with strong
1 integrin staining.
To evaluate whether the ultrastructural morphology of
basement membranes is altered by the lack of 1 integrin
expression, electronmicroscopy was performed with tumor
tissues and embryoid bodies derived from normal and
1-null ES cells. In normal tumors and normal embryoid bodies the basement membrane was continuously present
along the basal surface of epithelial cells (Fig. 6, A and D)
or around muscle cells (not shown) with a constant width that never exceeded 30 nm. In contrast, most cells in
1-null teratomas and embryoid bodies were covered by partially detached basement membranes, which resulted in
the formation of irregular clefts between plasma membrane and lamina densa (Fig. 6, B and F). In other areas,
cells were covered by several layers of basement membranes (Fig. 6 C) or by only partially developed basement
membranes (Fig. 6, E and F).
Blood Vessels of 1-null Teratomas Are Small
and Irregular in Shape and Lack ES Cell-derived
Endothelial Cells
Hematoxylin/Eosin staining, as well as immunostaining for
the endothelial markers vWF and PECAM revealed a
large number of circular vessels of various sizes in 1 +/+
teratomas (Fig. 7 A). In contrast, in
1-null tumors, vessels
were smaller in size and of irregular shape (Fig. 7 B).
To test whether 1-null ES cells can contribute to the
formation of blood vessels, endothelial cells were characterized in
1 +/+ and
/
teratomas. In
1 +/+ and +/
teratomas, all cells that expressed vWF or PECAM showed
strong staining for
1 integrin (Fig. 7 C). Furthermore,
many cells that expressed vWF were also lacZ-positive
(Fig. 8, A and C) clearly indicating that ES cells can contribute to angiogenesis in developing teratomas. In
1-null tumors, however, cells that expressed vWF (Fig. 7 B) always expressed high levels of
1 integrin, indicating their
host-derived origin (Fig. 7 D). These results were confirmed in serial sections of six
1-null teratomas, which were
analyzed by lacZ and vWF stainings. In all
1-null teratomas analyzed so far, cells that were positive for vWF staining (Fig. 8 B) were negative for lacZ expression (Fig. 8 D).
During the analysis of tissue sections derived from 1-null
teratomas it became evident that also the host cell-derived
vasculature has abnormalities. Whereas blood vessels in
normal teratomas have a smooth inner surface and are
tightly anchored to the surrounding connective tissue (Fig.
9 A), blood vessels in
1-null tumors have an irregular inner surface and display large gaps between endothelial
cells and the surrounding tissue (Fig. 9 B).
1-Null ES Cells Can Differentiate into Endothelial
Cells In Vitro but Formation of Vessels Is Delayed
ES cells were cultured for 5 d in suspension to form embryoid bodies, and then were plated on tissue culture
dishes. Initially, PECAM-positive cells were aggregated in
dense clusters (Fig. 10 A). These clusters then started to
form thin tubes and branched. After 7 d on the culture
dish about half of the PECAM-positive cells were part of
blood vessels in embryoid bodies derived from normal ES
cells. After 15 d of incubation, PECAM-positive cell clumps were no longer visible, the blood vessels were significantly grown in diameter, extensively branched (Fig. 10
B), and often contained many blood cells (Fig. 10 C).
When 1-null embryoid bodies were analyzed 7 or 15 d
after plating, only PECAM-positive cell clusters were
found without any signs of vessel formation (Fig. 10 D).
After 20 d in culture, a fine network of small vessels began
to form in some areas of the embryoid bodies (Fig. 10 E).
These vessels were small in diameter and never contained
blood cells. In other areas, PECAM-positive cells were
still organized in small cell patches, often with loose cell-
cell contacts (Fig. 10 F). The mean value of the diameter of normal and
1-null vessels in seven embryoid bodies after a 3-wk culture period was 18.1 ± 3.0 µm and 6.8 ± 2.1 µm, respectively (Fig. 11).
To determine whether the absence of 1 integrin expression interferes with the differentiation of endothelial
cells, we counted the number of PECAM-positive cells in
normal as well as
1-null embryoid bodies. After a plating
period of 28 d, 100 embryoid bodies (derived from normal
and
1-null ES cells, respectively) were trypsinized; single
cells were plated and cultured overnight, and then stained for
PECAM expression. Out of 9,600 cells counted, 96 PECAM-
positive cells were found in normal embryoid bodies and 9 cells in
1-null embryoid bodies. This 10-fold decrease of
PECAM-positive cells in the
1-null embryoid bodies indicates a delayed or less efficient differentiation of
1-null
endothelial cells.
Expression of 3 and
v Integrin Is not Significantly
Altered on
1-null Endothelial Cells
Proliferating endothelial cells express large amounts of
v
3 and
v
5 integrins. To determine whether the expression of
3 or
v integrin is altered on
1-null endothelial cells, normal and
1-null embryoid bodies were double-immunostained for either PECAM and
3 integrin
(Fig. 12, A-D), or PECAM and
v integrin (Fig. 12, E-H),
respectively. Vessels in embryoid bodies derived from normal and
1-null ES cells express similar amounts of
3 (Fig. 12, B and D) and
v integrin (Fig. 12, F and H).
These data indicate that the absence of
1 integrin is not
associated with an altered expression of other endothelial
cell integrins.
VEGF Does Not Promote Angiogenesis in 1-null
Embryoid Bodies
VEGF induces proliferation of endothelial cells and sprouting of vessels and therefore is a potent promoter of angiogenesis. To test whether VEGF can overcome abnormal
angiogenesis in 1-null embryoid bodies, normal and
1
integrin-deficient ES cells were differentiated in the presence of 10 or 20 ng/ml VEGF, respectively. After 5 d in
suspension culture and 12 d on gelatinized cover slips, embryoid bodies were treated with BrdU for 2 h and subsequently stained for PECAM expression and BrdU incorporation. Treatment of normal embryoid bodies with either
10 or 20 ng/ml VEGF significantly increased the number
and the extent of blood vessel branching (Fig. 13, A and C;
and Table I). In contrast, VEGF at both concentrations had no significant effect on blood vessel branching in
1-null embryoid bodies (Fig. 13, B and D; and Table I) indicating that VEGF cannot overcome the lack of
1 integrin
function. VEGF treatment also increased the number of
BrdU-positive cells in normal (Fig. 13, A and C, arrows)
but not in
1-null embryoid bodies (Fig. 13, B and D, arrows).
Table I.
Quantification of Branches of Blood Vessels in Normal
(+/+) and |
In the present study we show that the growth of ES cell-
derived teratomas is dependent on the expression of 1 integrin. Whereas subcutaneous injection of normal ES cells
under the skin of the back of syngeneic mice gave rise to
large teratomas,
1-null ES cells either produced no, or
only very small tumors. This reduced growth of
1-null
teratomas suggests a growth-supporting role of
1 integrin.
These results were unexpected since many tumors lack
5
1 integrin function, a phenomenon that has been
linked with anchorage-independent growth and high tumorigenicity (Hirst et al., 1986
; Plantefaber and Hynes,
1990
; Varner et al., 1995
). Conversely, overexpression of
5
1 integrin in Chinese hamster ovary cells is accompanied by a loss of tumorigenicity and reduced proliferation
in vitro (Giancotti and Ruoslahti, 1990
). A similar growth-suppressing role was recently demonstrated for
2
1 integrin, which is frequently absent in breast cancer cells. Reexpression of
2
1 integrin in such cells also leads to a
diminution of the malignant phenotype (Zutter et al., 1995
).
Several explanations could account for this discrepancy.
First, ES cells are normal embryonal cells that differentiate into many somatic tissues and thus give rise to a tumor
called teratoma (Damjanov and Solter, 1974; Damjanov,
1978
). Since these cells are not transformed, they may still
be dependent on anchorage to the extracellular matrix for
growth (Dike and Farmer, 1988
) and survival (Meredith et
al., 1993
; Boudreau et al., 1995
). Integrins execute cell-
matrix interactions and lack of anchorage in
1-null ES
cells could therefore be an explanation for the reduced tumor growth. To test this hypothesis we analyzed cell proliferation by injecting BrdU into teratoma-bearing mice
and stained for apoptosis. Extensive analysis of normal
and
1-null teratomas revealed that cell proliferation and
apoptosis are not significantly affected in
1-null tumors.
It has to be noted, however, that the extents of proliferation and apoptosis were very different between different sections of the same tumor, as well as between individual
tumors. This heterogeneity is due to the ability of ES cells
to differentiate into a wide range of somatic tissues with
different apoptosis and proliferation rates. For this reason
we obtained, in normal as well as
1-null teratomas, enormous variations in the numbers of proliferating and apoptotic cells. Despite this variation, there were more areas
with higher numbers of proliferating and lower numbers
of apoptotic cells in normal teratomas when compared with
1-null teratomas. Such alterations would be in agreement with reports that have linked cell survival with integrin function (for review see Ruoslahti and Reed, 1994).
The binding of
5
1 integrin to fibronectin is inducing bcl-2
expression, which is one of the best studied cell survival
factors (Zhang et al., 1995
). Other studies showed that disrupting the binding to basement membranes can induce
apoptosis (Boudreau et al., 1995
; Coucouvanis and Martin, 1995
). Biochemical studies of
1-null teratomas (Sasaki,
T., E. Forsberg, W. Bloch, K. Addicks, R. Fässler, and R. Timpl, submitted for publication), together with our electronmicroscopical analysis indicate that the integrity of
basement membranes is severely affected by the absence
of
1 integrin. Whereas epithelial cells of normal teratomas are tightly attached to a basement membrane,
1-null
cells have either partly or completely lost this tight attachment leading to the dislocation of basement membranes
into the interstitium. This absence of normal interaction
between cells and basement membranes could not only explain the increased cell death in teratomas, but also the altered differentiation of keratinocytes (Bagutti et al., 1996
)
and cardiac muscle cells (Fässler et al., 1996
). Similar defects in basement membrane morphology have been reported in
3-null mice, which have disorganized and fragmented glomerular basement membranes, and podocytes
that completely lack foot processes (Kreidberg et al.,
1996
). In addition, these mice develop skin blisters that are
due to an inefficient maintenance of the epidermal basement membrane (DiPersio et al., 1997
).
A second explanation for the reduced tumor growth
could be the altered composition or distribution of ECM
proteins in 1-null teratomas. The ECM has an important
role as reservoir for growth factors, proteases, and protease inhibitors (Alexander and Werb, 1991
; Nathan and
Sporn, 1991
). Basement membranes, for example, can
store and release many growth factors. The various abnormalities of basement membranes as seen in
1-null teratomas and
1-null embryoid bodies may contribute to the diminished growth and to defects in differentiation (Fässler
et al., 1996
). In addition, the disturbed matrix deposition
can alter mechanochemical properties, which may reduce
the growth rate of
1-null teratomas (Ingber, 1991
). Indeed,
immunostainings for fibronectin and collagens revealed an
altered distribution in
1-null teratomas. In normal teratomas both matrix proteins concentrate in streaklike
structures and often colocalize with high
1 integrin staining. In contrast, both proteins are diffusely deposited in
1-null teratomas and never concentrate along
1 integrin-positive, host-derived cells. This striking finding was
also supported by routine histological analysis. Tissue
specimens of
1-null teratomas contained little ECM, with
many mutant and only a few normal, host-derived (lacZ negative) cells.
A third explanation for the formation of small teratomas could be abnormal angiogenesis in the absence of 1
integrin. In contrast to tumors derived from somatic tissue,
the vasculature of teratomas is derived from the host and
from the ES cells (Risau et al., 1988
; Vittet et al., 1996
).
Immunostaining demonstrated host-derived endothelial
cells expressing
1 integrin in normal as well as
1-null tumors. Whereas lacZ staining indicated ES cell-derived
(lacZ positive) endothelial cells in
1 integrin-heterozygous as well as mock-transfected tumors, no ES cell-derived
endothelial cells were detected in
1-null teratomas. This
complete absence of
1-null endothelial cells is also seen
in the highly vascularized fetal and adult liver of
1-null
chimeric mice (Fässler and Meyer, 1995
; Hirsch et al.,
1996
). These data suggest, that
1-deficient ES cells either
do not differentiate in vivo into endothelial cells or that
they differentiate into endothelial cells but cannot organize
into blood vessels. A detailed inspection of the vasculature revealed furthermore that blood vessels were significantly
smaller in
1-null teratomas as compared to normal teratomas, which may result from the small tumor size. In addition, however, we found that the shape of smaller vessels
is also different in
1-null tumors: the inner surface is irregular and the vessels are loosely embedded into the surrounding tissue. Apparently, even endothelial cells expressing
1 integrin are not able to make normal blood vessels
in
1-null teratoma, indicating lack of an essential interaction between endothelial cells and the surrounding cells or
the extracellular matrix.
To test whether the 1-null ES cells have a cell autonomous impairment of differentiation into endothelial cells,
we differentiated ES cells into embryoid bodies in vitro
and assessed for the presence of endothelial cells (Fässler
et al., 1996
). To our surprise, we found that differentiation
of endothelial cells occurs in normal and
1-null embryoid
bodies. After 12 d (5 d in suspension culture, and 7 d on
plastic surface) normal endothelial cells start to sprout and
to form a large network of vessels of various diameters that are also often filled with blood cells. In contrast,
1-null endothelial cells initially form compact cell nests with
tight cell-cell contacts. Only after a culture period of 3 wk,
1-null endothelial cells start to migrate and to form small
vessels. The ability of
1-null endothelial cells to differentiate may also occur in vivo. This hypothesis is supported
by the presence of
1-null hematopoietic stem cells in chimeric mice (Hirsch et al., 1996
) that, together with endothelial cells, develop from a common precursor cell called
hemangioblast. Currently, we have no experimental explanation for the complete lack of
1-null endothelial cells
both in teratomas and in livers of chimeric mice (Fässler and Meyer, 1995
), despite the fact that they are readily
formed in vitro. It is possible that the in vitro environment
favors the survival of these cells, which is not the case in
vivo.
An important role for angiogenesis has been reported
for v
3 and
v
5 integrins (Varner and Cheresh, 1996
).
Immunostaining of
1-null embryoid bodies with antibodies detecting
v or
3 integrin clearly revealed that both
integrin subunits are not downregulated. Therefore, we
can exclude that the absence of
1 integrin expression influences endothelial cell function by altering the expression of other integrins. Growth factors and angiogenic signals can upregulate
v
3 expression on endothelial cells
and induce blood vessel branching (Brooks et al., 1994a
;
Friedlander et al., 1995
). Moreover, several growth factors
were shown to exert their angiogenic signals in concert
with endothelial integrins. For example, tumor necrosis
factor-
and basic FGF depend on
v
3 integrin, and
VEGF and TGF-
depend on
v
5 integrin (Friedlander et al., 1995
). VEGF is the most potent stimulator of
neovascularization by regulating proliferation of endothelial cells and branching of blood vessels. Testing whether
VEGF can override the defective branching of
1-null endothelial cells, we found that VEGF had no effect on
1-null endothelial cells: neither the proliferation rate nor the
branching could be enhanced in
1-null embryoid bodies. Endothelial cells in normal embryoid bodies, however, responded to VEGF by an increased proliferation rate and
by an extensive formation of new vessel branches. These
data demonstrate for the first time that
1 integrins play
an important role during angiogenesis and moreover, that
VEGF signaling depends on the presence of
1 integrin
function. This finding is unexpected since earlier reports showed that
1 integrin function is not essential for tumor-induced angiogenesis (Brooks et al., 1994b
). Brooks et al.
(1994b)
treated chicken embryos that were exposed to
various tumors with anti-
1 integrin antibodies and were
unable to influence tumor growth or angiogenesis. One explanation for this discrepancy may be an incomplete inhibition of
1 integrin function with a single injection of
antibodies. An alternative explanation could be the ubiquitous expression of
1 integrin. Antibodies against
1 integrin find their antigen on almost all cells and may be
eliminated before endothelial cells proliferate and form
new blood vessels.
Received for publication 20 March 1997 and in revised form 10 July 1997.
W. Bloch and E. Forsberg have contributed equally to this work.We thank M. Grüne and S. Benkert for technical assistance and E. Dejana for antibody gifts.
This work was supported by the Deutsche Forschungsgemeinschaft (Fa 296/1-2) and the Hermann and Lilly Schilling Stiftung.
BrdU, bromodeoxyuridine; ECM, extracellular matrix; ES, embryonic stem; PECAM, platelet/endothelial cell adhesion molecule; VEGF, vascular endothelial growth factor; vWF, von Willebrand Factor.
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