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Stefano J.Mandriota
1
, Lotta Jussila
2
,
Michael Jeltsch
2
, Amelia Compagni
3
,
Danielle Baetens, Remko Prevo
4
,
Suneale Banerji
4
, Joachim Huarte,
Roberto Montesano, David G.Jackson
4
,
Lelio Orci, Kari Alitalo
2
, Gerhard Christofori
3
and Michael S.Pepper
5
Department of Morphology, University Medical Centre, 1 rue Michel
Servet, 1211 Geneva 4, Switzerland,
2
Molecular/Cancer Biology
Laboratory and Ludwig Institute for Cancer Research, Haartman
Institute, University of Helsinki, Finland,
3
Institute of Molecular
Pathology, Vienna, Austria and
4
MRC Human Immunology Unit,
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK
1
Present address: The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive, Oxford, UK
5
Corresponding author
e-mail: michael.pepper@medecine.unige.ch
Metastasis is a frequent and lethal complication
of cancer. Vascular endothelial growth factor-C
(VEGF-C) is a recently described lymphangiogenic
factor. Increased expression of VEGF-C in primary
tumours correlates with dissemination of tumour cells
to regional lymph nodes. However, a direct role for
VEGF-C in tumour lymphangiogenesis and subse-
quent metastasis has yet to be demonstrated. Here we
report the establishment of transgenic mice in which
VEGF-C expression, driven by the rat insulin pro-
moter (Rip), is targeted to b-cells of the endocrine
pancreas. In contrast to wild-type mice, which lack
peri-insular lymphatics, RipVEGF-C transgenics
develop an extensive network of lymphatics around
the islets of Langerhans. These mice were crossed
with Rip1Tag2 mice, which develop pancreatic b-cell
tumours that are neither lymphangiogenic nor meta-
static. Double-transgenic mice formed tumours
surrounded by well developed lymphatics, which
frequently contained tumour cell masses of b-cell
origin. These mice frequently developed pancreatic
lymph node metastases. Our ®ndings demonstrate
that VEGF-C-induced lymphangiogenesis mediates
tumour cell dissemination and the formation of lymph
node metastases.
Keywords: islet of Langerhans/lymphangiogenesis/
tumour metastasis/VEGF-C
Introduction
The metastatic spread of tumour cells is responsible for the
majority of cancer deaths. Tumour cell dissemination is
mediated by a number of mechanisms, including local
tissue invasion, lymphatic spread, haematogenous spread,
or direct seeding of body cavities or surfaces. Clinical and
pathological observations have long suggested that, for
many tumours, the most common pathway of initial
dissemination is via lymphatics, with patterns of spread
via afferent vessels following routes of natural drainage
(reviewed by Fidler, 1997; Cotran et al., 1999; Sleeman,
2000). However, the lymphatic system has traditionally
been overshadowed by the greater emphasis placed on the
blood vascular system. This has been due in part to the
absence of suitable markers that distinguish lymphatic
from blood vascular endothelium, and to the lack of
identi®cation of lymphatic-speci®c growth factors.
In recent years, these limitations have been relieved by
the discovery of a small number of potential lymphatic-
speci®c markers (reviewed by Jackson, 2001). These
include: LYVE-1, a lymphatic endothelial receptor for
the extracellular matrix/lymphatic ¯uid mucopolysac-
charide hyaluronan (Banerji et al., 1999); Prox-1, a
homeobox gene product involved in regulating early
lymphatic development (Wigle and Oliver, 1999); podo-
planin, a glomerular podocyte membrane mucoprotein
(Breiteneder-Geleff et al., 1999); and the vascular
endothelial growth factor receptor-3 (VEGFR-3), a
transmembrane tyrosine kinase receptor for the lymphatic
endothelial growth factors vascular endothelial growth
factor-C (VEGF-C) and VEGF-D (reviewed in Veikkola
et al., 2000). Targeted inactivation of the VEGFR-3 gene
has revealed that it also plays an important role in the
development of the early blood vascular system, prior to
the emergence of lymphatic vessels (Dumont et al., 1998).
VEGF-C, the ®rst ligand to be discovered for VEGFR-3
(Joukov et al., 1996; Lee et al., 1996), is a member of the
VEGF family of polypeptide growth factors, which
comprises VEGF-A, -B, -C, -D and orf virus VEGFs (or
VEGF-E) (reviewed in Eriksson and Alitalo, 1999;
Ferrara, 1999). VEGF-C is produced in a pre-pro-peptide
form that is proteolytically processed to a mature
homodimer of ~40 kDa (Joukov et al., 1997). Proteolytic
processing increases the af®nity of VEGF-C for VEGFR-3
some 400-fold, and also enables it to bind to and activate
VEGFR-2 (Joukov et al., 1997). Based on its expression
pro®le and its binding to VEGFR-3, VEGF-C has been
implicated in the development of the lymphatic system
(Kukk et al., 1996; Lymboussaki et al., 1999). In addition,
transgenic overexpression of VEGF-C using the keratin 14
promoter induces lymphatic vessel enlargement/dilatation
in the skin (Jeltsch et al., 1997), and recombinant VEGF-C
induces lymphangiogenesis in the chick chorioallantoic
membrane (Oh et al., 1997). The capacity of VEGF-C to
bind to and activate VEGFR-2 may partially explain why
it also stimulates angiogenesis under certain experimental
conditions (Cao et al., 1998; Witzenbichler et al., 1998).
In contrast to VEGF-A, whose crucial role in tumour
angiogenesis is well established (Ferrara, 1999), very little
is known about the function of VEGF-C in tumour
Vascular endothelial growth factor-C-mediated
lymphangiogenesis promotes tumour metastasis
The EMBO Journal Vol. 20 No. 4 pp. 672±682, 2001
672 ã European Molecular Biology Organization
formation and/or progression. Nonetheless, a correlation
between VEGF-C expression, tumour lymphangiogenesis
and the formation of metastases in regional lymph nodes
has recently been described. Thus, levels of VEGF-C in
primary tumours are signi®cantly correlated with lymph
node metastases in a variety of cancers, including thyroid,
prostate, gastric, colorectal and lung (Bunone et al., 1999;
Tsurusaki et al., 1999; Yonemura et al., 1999; Akagi et al.,
2000; Niki et al., 2000; Ohta et al., 2000). One study has
described a strong correlation between lymphatic vessel
density and VEGF-C expression (Ohta et al., 1999).
However, in this study, no correlation was observed
between lymphatic vessel density and lymph node
metastases. Despite the continuing accumulation of
correlative clinical data, a functional role for VEGF-C in
tumour lymphangiogenesis and/or lymphatic enlargement,
and its role in tumour cell dissemination, have yet to be
demonstrated directly.
In order to test the hypothesis that VEGF-C-induced
lymphangiogenesis can promote tumour metastasis, we
generated transgenic mouse lines in which VEGF-C
expression, driven by the rat insulin promoter, is targeted
to b-cells of the islets of Langerhans, and tested their
capacity to form metastases by crossing them with the
Rip1Tag2 transgenic line, a transgenic mouse model of
non-metastatic b-cell carcinogenesis (Hanahan, 1985).
Results
A full-length human VEGF-C cDNA was cloned between
the rat insulin II gene promoter (Rip) and the SV40 small
T antigen intron and polyadenylation signal (Figure 1A).
Two founder (F0) mice (designated nos 23 and 24) were
identi®ed, which were capable of germline transmission.
RipVEGF-C transgenic mice were viable, similar in size to
wild-type littermates, normoglycaemic and fertile. Mice
derived from F0 no. 23 had a single copy of the
transgene, whereas those derived from F0 no. 24 had
four to six copies integrated in a head-to-tail array
(Figure 1B and data not shown). Pancreas-speci®c
expression of the VEGF-C transgene was con®rmed by
reverse transcription±polymerase chain reaction (RT±
PCR) screening of several organs, using oligonucleotide
primers designed to amplify VEGF-C cDNA of human but
not of mouse origin (Figure 1C and data not shown). In
contrast to wild-type littermates (Figure 2A), immuno-
histochemical analysis of the pancreata of RipVEGF-C
transgenic mice revealed strong VEGF-C staining in the
majority of islet cells (Figure 2B), as would be expected
from the relative abundance of insulin-expressing b-cells
in islets (~80%).
Striking morphological differences were observed
between transgenic and wild-type pancreata. Clearly
demarcated spaces, lined by a single layer of ¯attened
cells, which frequently contained lymphocytes rather than
red blood cells, were observed around the majority of
transgenic islets (Figure 3C and E). These morphological
features were observed in all RipVEGF-C mice killed
between E15.5 and 18 months. These structures, which
were never observed around islets of wild-type mice
(Figure 3A), were identi®ed as lymphatic vessels on the
basis of three separate criteria. First, they were lined by
endothelial cells that were immunoreactive for VEGFR-3
(Figure 2C). Weak VEGFR-3 immunostaining was
also observed within some islets of both wild-type and
transgenic mice (Figure 2C and data not shown).
Secondly, as revealed by transmission electron micro-
scopy (TEM), the basement membrane of these vessels
was either absent or discontinuous, and the endothelial
cells were devoid of the characteristic fenestrations found
in contiguous capillary blood vesselsÐboth hallmarks of
lymphatic endothelium (Figure 4A and B). Thirdly, as
revealed by immunohistochemistry and immuno-EM, they
stained with an antibody to the novel lymphatic endothe-
lial marker LYVE-1 (Banerji et al., 1999; Prevo et al.,
2001) on both their luminal and abluminal surfaces
(Figure 3D, F and 4C), similar to lymphatic vessels in
normal wild-type mouse tissues (Prevo et al., 2001).
Fig. 1. Molecular characterization of RipVEGF-C transgenic mice.
(A) The transgene was constructed by cloning the complete human
VEGF-C cDNA (nucleotides 1±1997; DDBJ/EMBL/GenBank
accession No. X94216) between the ~695 bp BamHI±XbaI fragment
of Rip (Hanahan, 1985) and the SV40 small T antigen intron and
polyadenylation signal. The L-shaped arrow indicates insulin gene
transcription initiation. E, EcoRI restriction sites. (B) Ten micrograms
of genomic DNA from two RipVEGF-C transgenic mice from families
23 and 24 or from a wild-type littermate (wt) (the latter spiked with the
RipVEGF-C transcriptional unit as indicated in picograms) were
digested with EcoRI and analysed by Southern blotting using the SV40
moiety of the transgene as a probe. Single or double asterisks indicate
the 3¢ end of the insertion site in family 23 and 24, respectively.
Markers on the left indicate kilobases. (C) Reverse transcription
products from oligo dT-primed total RNAs from pancreata of
RipVEGF-C transgenic mice from families 23 and 24 or from wild-
type mice (wt) were analysed by PCR using hVEGF-C speci®c primers
or acidic ribosomal phosphoprotein P0 (P0) primers. Where indicated,
RT was omitted, or PCR mix alone (mix) was analysed. Markers on the
left indicate base pairs.
VEGF-C-mediated lymphangiogenesis and metastasis
673
A quantitative analysis of lymphatic vessel density in
RipVEGF-C mice, based on LYVE-1 immunoreactivity,
revealed that 98% of islets were in intimate association
with LYVE-1-positive lymphatics (Figures 3D, F and 5;
Table I), which contrasts with the ®nding that only 4% of
islets had closely apposed lymphatics in wild-type
littermates (Figures 3B and 5; Table I). In some
RipVEGF-C mice, peri-insular lymphatics were particu-
larly well developed, and extended between the lobules of
exocrine acini (Figure 3F). Previous studies in the rodent
pancreas have revealed the absence of an association
between lymphatics and islets of Langerhans (Bertelli
et al., 1993; Navas et al., 1995; Ji and Kato, 1997;
reviewed by O'Morchoe, 1997), which is consistent with
our LYVE-1 and VEGFR-3 immunohistochemical data.
From these data we conclude that RipVEGF-C mice form
lymphatics de novo (lymphangiogenesis). In contrast,
mice expressing VEGF-C in the skin under the control of
the keratin 14 promoter display an increase both in the size
and number of lymphatic vessels (Jeltsch et al., 1997;
T.Veikkola, unpublished data).
Vascular density and the percentage islet volume
occupied by blood vessels were determined by immuno-
histochemistry with the vascular endothelial-speci®c anti-
body MECA-32 (Hallmann et al., 1995). In contrast to
LYVE-1, which stains lymphatic vessels (Banerji et al.,
1999), MECA-32 only stains blood vessels (Figure 6A and
B). No differences were observed between wild-type and
RipVEGF-C transgenic islets (Table I). Similar results
were obtained using the pan-endothelial marker CD31
(data not shown), indicating that forced expression of
VEGF-C did not promote vascular angiogenesis.
No alterations were observed in the normal proportion
or spatial distribution of a, b, d or PP cells in the islets of
RipVEGF-C mice when compared with wild-type litter-
mates, as assessed by immunohistochemical analysis for
glucagon, insulin, somatostatin and pancreatic polypep-
tide, respectively (data not shown).
We thus conclude that increased expression of VEGF-C
in the b-cells of RipVEGF-C transgenic mice speci®cally
promotes lymphangiogenesis, which is observed around
but not within the islets of Langerhans, and which does not
affect islet architecture or cellular composition.
To assess the role of VEGF-C-induced lymphangio-
genesis in tumour metastasis, we crossed RipVEGF-C
mice with Rip1Tag2 mice, a well characterized transgenic
model of non-metastatic b-cell carcinogenesis (Hanahan,
1985; Perl et al., 1998, 1999) that displays morphological
features typical of human pancreatic b-cell tumours (Holm
et al., 1988). Of note, b-cell tumours that develop in
Rip1Tag2 mice are capable of local invasion, but do not
induce extensive lymphangiogenesis (Figure 7A and E).
When RipVEGF-C mice were crossed with Rip1Tag2
mice, the resulting double-transgenic animals displayed a
modest but signi®cant increase in tumour incidence
(de®ned as the number of tumours per pancreas having a
size >0.5 mm) compared with single Rip1Tag2 transgenic
animals, yet neither tumour volume (expressed in cubic
millimetres) nor the ratio of benign adenoma to invasive
carcinoma was altered (Table II). RipVEGF-C 3
Rip1Tag2 double-transgenic tumours consisted almost
exclusively of insulin-producing b cells (data not shown),
similar to what has been reported for Rip1Tag2 single-
transgenic tumours (Hanahan, 1985). Detailed immuno-
histochemical analyses revealed heterogeneous VEGF-C
immunoreactivity in the majority of double-transgenic
tumour cells, whereas VEGF-C immunoreactivity was
absent from Rip1Tag2 tumours (Figure 7C and D).
Furthermore, there was an increase in the number of
VEGFR-3 immunoreactive peri-tumoural lymphatic
vessels in double-transgenic mice compared with single-
transgenic mice (data not shown). The extent of this new
lymphatic vessel formation was demonstrated dramatic-
ally by LYVE-1 immunostaining, which revealed that
insulinomas were either partly surrounded by or fully
enclosed within such vessels (Figure 7F). Indeed, quan-
titative analyses con®rmed that 62% of insulinomas/islets
in double-transgenic animals had closely apposed lym-
phatics encompassing most or all of the insulinoma/islet
circumference (Figures 5, 7B and F). In contrast, only 29%
of insulinomas/islets in the Rip1Tag2 mice had associated
lymphatics (Figures 5, 7A and E). The lower levels of
lymphangiogenesis in the latter tumours correlate with the
®nding that endogenous VEGF-C was not upregulated
during tumourigenesis in Rip1Tag2 single-transgenic mice
(Figure 7C and data not shown). In small adenomas of
double-transgenic mice, LYVE-1-positive lymphatics
surrounded the tumour tissue without penetrating it
(Figure 7F). In contrast, in large adenomas and carcin-
omas, in addition to surrounding the tumours, lymphatics
(positive for LYVE-1 and VEGFR-3) were infrequently
observed in the peripheral rim of the tumour, but never
extended into the body of the tumour itself (data not
shown).
Fig. 2. Expression of VEGF-C and VEGFR-3 in RipVEGF-C
transgenic mice. Immunohistochemistry for (A and B) VEGF-C and
(C) VEGFR-3. (A) Ten-month-old female wild-type-littermate; (B and
C) 11-month-old female RipVEGF-C mouse, family 24. (B) and (C)
are serial sections. Bar: (A and B) 50 mm; (C) 30 mm.
S.J.Mandriota et al.
674
It has recently been reported that VEGFR-3, although
absent from mature blood vessels, is induced in angiogenic
tumour blood vessel endothelium (Partanen et al., 1999;
Valtola et al., 1999). We have also observed VEGFR-3-
positive intra-tumoural vessels in our system, although
a large degree of heterogeneity was observed within
individual islet cell tumours: some regions were positive,
while other regions in the same tumour were devoid of
immunoreactivity (data not shown). Since we have never
observed LYVE-1-positive vessels within normal or
tumourigenic islets (Figures 3B, D, F, 6B, 7E and F), we
are con®dent that the VEGFR-3-positive intra-islet or
intra-tumoural staining we have seen is con®ned to vessels
of the blood vascular system. VEGFR-3-positive blood
vessels were observed within tumours in both Rip1Tag2
and RipVEGF-C 3 Rip1Tag2 double-transgenic mice
(data not shown).
Since VEGF-C can be both lymphangiogenic and
angiogenic, our next objective was to compare blood
vessel density in Rip1Tag2 mice versus RipVEGF-C 3
Rip1Tag2 double transgenics. Blood vessel density is
frequently used as a measure of tumour-induced angio-
genesis (reviewed by Weidner, 1998). Using the pan-
endothelial marker CD31 (Figure 6C and D), we found no
differences between the two genotypes (Table III).
Similarly, no differences were observed when vessel
density was determined in the peripheral or central parts of
the tumour (Table III). These CD31-positive structures are
blood vessels, since we have never observed LYVE-1-
positive vessels within tumours (see above). We thus
conclude that VEGF-C does not induce angiogenesis in
double-transgenic RipVEGF-C 3 Rip1Tag2 mice.
Further examination of pancreata from RipVEGF-C 3
Rip1Tag2 double transgenics aged between 12 and 15
weeks revealed aggregates of tumour cells within the
lumen of lymphatic vessels in every case (Figure 8 and
data not shown). These intra-lymphatic tumour cells,
which were very rarely seen in Rip1Tag2 single-transgenic
mice, were clearly derived from primary b-cell tumours, as
established by their ultrastructural characteristics and
insulin immunoreactivity (Figure 8C and data not
shown). A proportion of the cells within these aggregates
contained mitotic ®gures (Figure 8A and C), indicating
that they were actively dividing. As before, the lymphatic
nature of these vessels was con®rmed by both LYVE-1
and VEGFR-3 immunoreactivity (Figure 8B and data not
shown).
We reasoned that intravascular tumour cells, by follow-
ing routes of natural drainage, would reach the marginal
sinus of pancreatic mesenteric lymph nodes, thus forming
Fig. 3. Histological analysis of wild-type and RipVEGF-C mice. (A and C) Haematoxylin and eosin staining; (B, D and F) immunohistochemistry for
LYVE-1 (DAB brown) and insulin (B and D) (fast red); (E) semithin section stained with methylene blue. (A) Twelve-month-old male wild-type
littermate; (B) 2-month-old male wild-type littermate; (C) 12-month-old male RipVEGF-C mouse, family 24; (D) 2-month-old male RipVEGF-C
mouse, family 24; (E) 11-month-old male RipVEGF-C mouse, family 24; (F) 10-month-old male RipVEGF-C mouse, family 24. Arrows in (B)
indicate ducts; En, endocrine; Ex, exocrine. Bar: (A±D) 50 mm; (E) 25 mm; (F) 100 mm.
VEGF-C-mediated lymphangiogenesis and metastasis
675
metastases at these sites. We therefore analysed regional
lymph nodes in double-transgenic mice for the presence of
tumour cells. Tumour cells were indeed observed in
regional mesenteric lymph nodes in 37% of RipVEGF-C
3 Rip1Tag2 mice (Table II; Figure 9A). In relatively
early metastases, which contained a thin rim of tumour
cells con®ned to the periphery of affected lymph nodes,
tumour cells displayed ultrastructural features of b-cells or
were insulin immunoreactive (Figure 9B and C), thus
con®rming their metastatic nature and their derivation
from primary b-cell tumours. No PP-, somatostatin- or
glucagon-immunoreactive cells could be detected in
metastases (data not shown). At later stages of metastatic
growth, lymph node tissue was almost completely
replaced by tumour cells (data not shown). These
advanced metastases displayed morphological hetero-
geneity, including an altered nucleus/cytoplasm ratio and
nuclear atypia, both hallmarks of the cellular anaplasia that
accompanies the later phases of tumour progression (data
not shown). Consistent with these observations, insulin
immunoreactivity was only retained by a minority of
tumour cells in these metastases (data not shown). At the
time points analysed, no overt metastases were observed in
distant lymph nodes or in other organs of RipVEGF-C 3
Rip1Tag2 double-transgenic mice.
Peri-tumoural lymphatics, intra-lymphatic tumour cell
aggregates and lymph node metastases have been observed
Fig. 4. Ultrastructural analysis of RipVEGF-C transgenic mouse
pancreas. (A and B) TEM of the pancreas from a 2-month-old male
RipVEGF-C transgenic mouse from family 24. (B) is a higher
magni®cation of the area delimited in (A). Arrows in (B) indicate the
basement membrane surrounding a capillary blood vessel (Bv). Note
the absence of a basement membrane along the basal surface of the
lymphatic endothelium. Arrowheads indicate endothelial fenestrations
in the capillary blood vessel. Note the absence of fenestrations in the
lymphatic endothelium. (C) Immunoelectron microscopy analysis of
LYVE-1 distribution on lymphatic endothelium in the same animal as
shown in (A) and (B). Cross-striated collagen ®brils are on the
abluminal side of the vessel. Lv, lymphatic vessel; Ex, exocrine tissue;
En, endocrine tissue. Bar: (A) 5 mm; (B) 500 nm; (C) 250 nm.
Fig. 5. Quantitation of lymphatic vessel density and disposition
in wild-type, single- (RipVEGF-C and Rip1Tag2) and double-
(RipVEGF-C/Rip1Tag2) transgenic mice. Lymphatic vessels were
identi®ed by LYVE-1 immunoreactivity, and the per cent islet/
insulinoma perimeter surrounded by LYVE-1-positive structures was
determined. For wild type, 185 islets were analysed from ®ve mice; for
RipVEGF-C, 99 islets were analysed from ®ve mice; for Rip1Tag2,
232 islets were analysed from seven mice; for RipVEGF-C/Rip1Tag2,
322 islets were analysed from eight mice.
Table I. Blood vascular and lymphatic phenotypes in wild-type versus
RipVEGF-C mice
Wild type RipVEGF-C
MECA-32-positive 13.2 6 3.6 13.6 6 4.5
vascular pro®les/10 000 mm
2
(n = 30) (n = 30)
% islet volume occupied by 13.0 6 3.4 12.9 6 4.4
MECA-32-positive blood vessels (n = 30) (n = 30)
% islets with tightly apposed 3.6 98
LYVE-1-positive lymphatics (n = 185) (n = 99)
For MECA-32 staining, six RipVEGF-C mice and six wild-type
littermates were assessed (three mice from family 23 and three from
family 24 in each case); ®ve islets were analysed per animal. Values
are mean 6 SD. For LYVE-1 staining, a total of 185 islets were
assessed in ®ve wild-type mice, and 99 islets were assessed in ®ve
RipVEGF-C mice.
n = number of islets.
S.J.Mandriota et al.
676
in double-transgenic mice derived from the two founder
families (Nos 23 and 24). Thus, we can ®rmly exclude a
transgene integration effect as the reason for the metastatic
phenotype.
Discussion
In the present study we have generated transgenic mice to
investigate the consequences of VEGF-C overexpression
on tumour cell dissemination in a well de®ned model of
b-cell carcinogenesis. Two lines were generated, which
express VEGF-C under the control of Rip. Although in
post-natal life this promoter speci®cally directs transgene
expression to pancreatic b-cells, during embryogenesis it
is transiently active in cells of the neural tube and neural
crest (Alpert et al., 1988). In almost all (98%) islets of
Langerhans analysed in RipVEGF-C transgenic mice,
extensive lymphatic channel formation was observed
around (but not within) the islets, the anatomical units in
which the transgene is expressed. In contrast, in wild-type
littermates, islets are very rarely in direct contact with
LYVE-1-positive vessels, indicating that in RipVEGF-C
mice lymphatics have formed de novo (lymphangio-
genesis). The latter result is consistent with the lymph-
angiogenic effects of VEGF-C observed in transgenic
mice which express VEGF-C in the skin under the control
of the keratin 14 promoter (T.Veikkola, unpublished data)
and in the differentiated avian chorioallantoic membrane
(Oh et al., 1997). K14-VEGF-C mice also display an
increase in the size of lymphatic vessels (Jeltsch et al.,
1997). The parameters that dictate whether VEGF-C will
induce lymphangiogenesis or lymphatic enlargement/
dilatation are not known. However, it has been well
documented that the activity of many cytokines is context
dependent, and it will therefore be important in the future
to determine what (additional) factors are involved in
determining this selectivity.
To assess the role of VEGF-C-induced lymphangio-
genesis in tumour metastasis, we crossed RipVEGF-C
mice with Rip1Tag2 mice, a well characterized transgenic
model of b-cell carcinogenesis (Hanahan, 1985; Perl et al.,
1998, 1999). Of note, b-cell tumours that develop in
Rip1Tag2 mice are capable of local invasion but are not
metastatic. In our model, increased VEGF-C expression in
double transgenics resulted in the de novo formation of
lymphatics in intimate association with b-cell tumours.
Concomitant with lymphangiogenesis, all of these mice
exhibited tumour cell aggregates within lymphatic vessels.
This was associated with the formation of metastases in
the draining regional mesenteric lymph nodes of the
pancreata in 37% of mice. While intra-lymphatic tumour
cell masses were occasionally seen in Rip1Tag2 mice,
lymph node metastases have never been observed in these
animals.
Although the metastatic dissemination of tumour cells
to regional lymph nodes is a common feature of many
human cancers, it is not clear whether tumours utilize
existing lymphatic channels or whether tumour dissemin-
ation requires the de novo formation of lymphatics
(lymphangiogenesis) (Saaristo et al., 2000; reviewed by
Fig. 6. MECA-32 and CD31 immunohistochemistry in wild-type and transgenic islets. (A) MECA-32 and (B) LYVE-1 immunolabelling in
consecutive frozen sections of a RipVEGF-C islet. CD31 immunolabelling of adenomas from 14-week-old (C) Rip1Tag2 and (D) double-transgenic
RipVEGF-C 3 Rip1Tag2 mice. L, lymphatic vessel; A, adenoma. Bar: 100 mm.
VEGF-C-mediated lymphangiogenesis and metastasis
677
Pepper, 2001). Our ®ndings suggest that Rip1Tag2 tumour
cells are intrinsically metastatic, but are unable to realize
their metastatic potential because they lack access to
appropriate portals and routes to distant sites. When such
routes are provided, in this instance via the lymphangio-
genesis induced by ectopically expressed VEGF-C, then
metastases are observed. The ®nding that increased peri-
tumoural lymphatic density results in metastasis in only
37% of the mice that develop islet tumours suggests that
additional, rate-limiting steps must also exist.
RipVEGF-C 3 Rip1Tag2 double-transgenic tumours
were similar to Rip1Tag2 single-transgenic tumours in
terms of tumour volume, transition from adenoma to
carcinoma and the relative distribution of pancreatic
hormone immunoreactive cells. Interestingly, however,
tumour incidence was increased in RipVEGF-C 3
Rip1Tag2 mice. Although we have no explanation for
this increase, we speculate that this could be due to the
formation of local intra-pancreatic metastases, which
occur as a consequence of tumour cell dissemination
through the extensive peri-tumoural lymphatic network
induced by VEGF-C. Alternatively, trophic and/or mito-
genic signals released by lymphatic endothelial cells in
the nearby parenchyma of islets/tumours might have a
positive effect on islet/tumour cell survival and/or pro-
liferation, thus increasing the fraction of islets that form
primary tumours.
In addition to its capacity to induce lymphangiogenesis
and an increase in the size of lymphatic vessels, VEGF-C
has also been demonstrated to be angiogenic under certain
circumstances (Cao et al., 1998; Witzenbichler et al.,
1998). However, we found no evidence for increased
angiogenesis in RipVEGF-C or RipVEGF-C 3 Rip1Tag2
double-transgenic mice, as determined by blood vessel
density. Thus, there was no increase in the number of
MECA-32- or CD31-positive blood vascular pro®les
Fig. 7. Histological analysis of RipVEGF-C/Rip1Tag2 double-
transgenic mice. (A and B) Haematoxylin and eosin staining; (C and
D) anti-VEGF-C immunohistochemistry; (E and F) anti-LYVE-1
immunohistochemistry. (A, C and E) Rip1Tag2 single-transgenic mice.
(B, D and F) RipVEGF-C/Rip1Tag2 double-transgenic mice. Ad,
adenoma; Lv, lymphatic vessel; Ex, exocrine tissue; D, duct. The white
space around the adenoma in (E) is artefactual. All mice were killed at
14 weeks; double-transgenic mice are from RipVEGF-C family 23.
Bar: 50 mm.
Table II. Tumour phenotypes and metastasis in Rip1Tag2 versus
RipVEGF-C 3 Rip1Tag2 mice
Rip1Tag2 RipVEGF-C 3
Rip1Tag2
Tumour incidence
a
5.4 6 1.4 12.3 6 2.9
(per mouse) (n = 27) (n = 33)
Tumour volume
b
56.5 6 35.8 38.3 6 29.1
(per mouse) (n = 27) (n = 33)
Adenoma 36% 46%
(n = 17) (n = 27)
Carcinoma 64% 54%
(n = 17) (n = 27)
Lymph node metastases
c
0% 37%
(0/17) (10/27)
a
Tumour incidence per mouse was determined macroscopically by
counting all apparent tumours >0.5 mm in the whole pancreas.
b
Tumour volume per mouse (in mm
3
) was calculated from all
macroscopically apparent tumours in the whole pancreas, with a
diameter >0.5 mm.
c
As detected by haematoxylin and eosin staining.
Values are mean 6 SD; n = number of mice.
Table III. Vascular phenotype in Rip1Tag2 versus
RipVEGF-C 3 Rip1Tag2 mice
CD31-positive vascular pro®les/
10 000 mm
2
Rip1Tag2 RipVEGF-C 3
Rip1Tag2
Tumour periphery 6.6 6 2.8 4.8 6 2.4
(n = 30) (n = 30)
Tumour centre 3.9 6 2.5 3.7 6 2.3
(n = 30) (n = 30)
For CD31 staining, two Rip1Tag2 and three RipVEGF-C 3 Rip1Tag2
mice were assessed. Ten adenomas were analysed per genotype;
vascular pro®les were counted in three (~300 3 300 mm) randomly
selected peripheral and three central areas per adenoma. Values are
mean 6 SD; n = number of ®elds.
S.J.Mandriota et al.
678
within RipVEGF-C islets when compared with wild-type
littermate controls, nor could we detect alterations in the
percentage of islet volume occupied by these vessels
(demonstrating that vessel size was likewise unaltered).
Similarly, there was no difference in vessel density
between Rip1Tag2 versus RipVEGF-C 3 Rip1Tag2
mice, either in the centre of the tumour or in the tumour
periphery. We have recently generated Rip1Tag2 3
RipVEGF-A double-transgenic mice (manuscript in pre-
paration). Extensive phenotypic characterization of these
animals has revealed that VEGF-A has an exclusively
angiogenic effect during tumour progression. This observ-
ation points to the fact that in the setting of the pancreas,
exquisite selectivity with regard to angiogenesis and
lymphangiogenesis can be achieved with VEGF-A and
VEGF-C, respectively, despite the fact that both cytokines
can activate VEGFR-2. Although currently there is no
explanation for this selectivity, this may depend on the
extent of VEGF-C proteolytic processing or on the
repertoire of VEGFRs expressed (including heterodimers
between different VEGFRs).
VEGFR-3 is downregulated in blood vascular endo-
thelium during development, and becomes restricted
almost exclusively to lymphatic endothelium and fenes-
trated blood vascular endothelium in post-natal life
(Kaipainen et al., 1995; Kukk et al., 1996; Jussila et al.,
1998; Partanen et al., 2000). It has been shown that
VEGFR-3 is re-expressed in the blood vascular endo-
thelium of several types of tumours (Partanen et al., 1999;
Valtola et al., 1999). Although we observed a variable
degree of VEGFR-3 staining on blood vascular endo-
thelium in Rip1Tag2 tumours, this was not increased in the
tumours of double-transgenic RipVEGF-C 3 Rip1Tag2
mice.
It has long been argued that lymphatic vessels may be
lost and/or collapsed in expanding primary tumours
because of high endogenous tumour interstitial pressure
(Leu et al., 2000). However, their presence can be
Fig. 8. Intra-lymphatic tumour cell masses in RipVEGF-C/Rip1Tag2 double-transgenic mice. (A) TEM showing a tumour cell mass within an
endothelial-lined lymphatic space. (B) Anti-LYVE-1 immunohistochemistry. (C) Enlargement of the area delimited in (A) showing a mitotic ®gure
and endocrine secretory granules. Mice were killed at 14 weeks and were generated from RipVEGF-C family 23. Arrowheads in (A) indicate mitotic
®gures. Bars: (A) 20 mm; (B) 50 mm; (C) 2 mm.
VEGF-C-mediated lymphangiogenesis and metastasis
679
demonstrated in the peri-tumoural stromal component
intimately associated with several types of tumours. In our
system, LYVE-1-positive lymphatics were occasionally
observed in the periphery of large adenomas, but were
never seen within islets of Langerhans or b-cell tumours.
The reasons for this selective localization of lymphatic
vessels around but not within normal and tumorigenic
islets of Langerhans is not known.
A correlation between VEGF-C expression, tumour
lymphangiogenesis and the formation of metastases in
regional lymph nodes has recently been described (Bunone
et al., 1999; Ohta et al., 1999, 2000; Tsurusaki et al., 1999;
Yonemura et al., 1999; Akagi et al., 2000; Niki et al.,
2000). Although these ®ndings provide no information on
the mechanisms of tumour cell dissemination, they raise
the possibility that VEGF-C may increase metastasis by
increasing the number and size of lymphatic vessels, or
alternatively by altering the functional properties of
existing lymphatics. For example, it is possible that
lymphatic endothelial cells locally release paracrine
factors, which in¯uence primary tumour cell invasiveness,
possibly through altered tumour cell±extracellular matrix
adhesion. Alternatively, VEGF-C may facilitate tumour
cell intravasation by altering the functional properties of
pre-existing or newly formed lymphatic endothelium (e.g.
tumour cell±endothelial cell adhesion). Future studies in
this area will need to be conducted on cultured lymphatic
endothelial cells. Although to date all in vitro studies have
been performed on large-vessel lymphatic endothelial
cells (reviewed by Pepper, 2001), it will be important in
the future to work with primary endothelial cells from
lymphatic capillaries. These cells would be useful (i) for
the identi®cation of VEGF-C-induced phenotypic alter-
ations (e.g. molecules involved in adhesive interactions
with tumour cells) and (ii) for application of techniques of
differential gene expression to identify molecular differ-
ences between blood and lymphatic capillary endothelial
cells. The utility of these techniques in identifying gene
expression pro®les in normal versus tumour endothelial
cells has recently been demonstrated (St Croix et al.,
2000).
Tumour cell metastasis to regional lymph nodes is an
early event in metastatic tumour spread, and is frequently
used as a prognostic factor to predict disease outcome or to
determine therapeutic strategies. Our observations provide
the ®rst direct evidence for a causal role for VEGF-C-
mediated lymphangiogenesis in the dissemination of
tumour cells. They also provide a relevant animal model
in which the mechanisms of lymphangiogenesis and
lymphatic tumour metastasis can be dissected, and in
which potential inhibitors of these processes can be tested.
Materials and methods
Transgenic mice
RipVEGF-C transgenic mice were generated according to standard
procedures (Hogan et al., 1994). The transgene was constructed by
cloning the human VEGF-C cDNA between the ~695 bp BamHI±XbaI
fragment of Rip (Hanahan, 1985) and the SV40 small T antigen intron and
polyadenylation signal. Genotypes were con®rmed by Southern blotting
and PCR analysis. Ten micrograms of genomic DNA from mouse tails
Fig. 9. Lymph node metastases in RipVEGF-C/Rip1Tag2 double-transgenic mice. (A) Haematoxylin and eosin staining of a lymph node containing
a peripheral ring of tumour cells (T). (B) Immuno¯uorescent antibody staining for insulin. (C) TEM showing tumour cells (T) surrounded by
lymphocytes. The inset is an enlargement of the delimited area, and shows the presence of endocrine secretory granules in tumour cells. Mice were
killed at 14 weeks and were generated from RipVEGF-C family 23. Bar: (A) 200 mm; (B) 50 mm; (C) 10 mm; inset = 2 mm.
S.J.Mandriota et al.
680
were digested with EcoRI, run in 0.7% agarose gels and analysed by
Southern blotting using the ~450 bp SV40 moiety of the RipVEGF-C
transgene as a probe. Routine PCR screening of RipVEGF-C
heterozygotes was performed using a pair of primers speci®c for the
human VEGF-C cDNA (forward: 5¢-TCCGGACTCGACCTCTCGGAC;
reverse 5¢-CCCCACATCTATACACACCTCC), starting from standard
tail or ®nger genomic DNA preparations. PCR cycles were: 94°C, 3 min
(13); 94°C, 1 min, 60°C, 1 min, 72°C, 1 min (303); 72°C, 3 min (13).
PCR products were analysed on 6% polyacrylamide gels.
Expression of the RipVEGF-C transgene was assessed by RT±PCR
using primers speci®c for the human VEGF-C cDNA, and by
immunohistochemical analysis as described below. Total RNA puri®ed
from mouse pancreas using TRIZOL Reagent (Gibco-BRL, Life
Technologies) was reverse transcribed using oligo-dT (Boehringer) and
Superscript II (Life Technologies). RT products were subjected to PCR
analysis using a pair of primers speci®c for human VEGF-C (see above)
or a pair of primers for the acidic ribosomal phosphoprotein P0 (Pepper
and Mandriota, 1998). Where indicated, RT was omitted. PCR cycles
were: 94°C, 3 min (13); 94°C, 1 min, 60°C, 1 min, 72°C, 1 min (303 for
VEGF-C; 25±303 for P0); 72°C, 3 min (13). Equal volumes of PCR
products were analysed on 6% polyacrylamide gels.
The generation and phenotypic characterization of Rip1Tag2 mice
have been described previously (Hanahan, 1985). Glucose (5% w/v) was
added to the drinking water of all tumour-bearing mice, beginning at
10 weeks of age, to counteract hypoglycaemia, which results from
insulinoma development.
Histopathological analyses
For light microscopy, pancreata were either frozen or ®xed in Bouin's
®xative or 4% paraformaldehyde, and embedded in paraf®n. Antibodies
used for immunohistochemistry were as follows: mouse monoclonal anti-
human insulin (Storch et al., 1985); rabbit polyclonal anti-human
VEGF-C (Joukov et al., 1997); rabbit polyclonal anti-human VEGFR-3
(Pajusola et al., 1993); rat monoclonal anti-mouse VEGFR-3 (Kubo et al.,
2000); MECA-32 (Hallmann et al., 1995); rat monoclonal anti-mouse
CD31 (clone MEC13.3; PharMingen); rabbit polyclonal anti-mouse
LYVE-1 ectodomain. The latter was generated by immunizing rabbits
with the mouse LYVE-1 ectodomain fused with human IgFc followed by
pre-absorption of the antiserum on human Ig±Sepharose (Prevo et al.,
2001). MECA-32 was applied to cryostat sections. All other antibodies
were applied to paraf®n-embedded material following antigen retrieval
(microwave), with the exception of MEC13.3, for which no antigen
retrieval was used. Immunoreactivity was visualized using either
peroxidase±DAB or fast red- or ¯uorescein isothiocyanate-conjugated
streptavidin or secondary antibodies. For CD31 immunostaining,
antigen±antibody complexes were revealed by means of a rabbit anti-
rat linker antibody (Dako) followed by anti-rabbit/HRP Envision system
(Dako).
For determination of blood vessel density, the number of MECA-32- or
CD31-positive vascular pro®les per 10 000 mm
2
of islet or adenoma tissue
was determined using an in-house morphometric apparatus. For
adenomas, three randomly selected ®elds (~300 3 300 mm each) at the
periphery and in the centre of each tumour were counted. The per cent
islet volume occupied by MECA-32-positive vessels was determined
using the point counting method (Weibel, 1973). Values are mean 6 SD.
For electron microscopy, mice were either perfused with 1±2%
glutaraldehyde in 100 mM phosphate buffer pH 7.4 and the pancreas
isolated, or freshly isolated pancreatic tissue was minced and ®xed by
immersion in 2% glutaraldehyde for 2 h. After washing in 100 mM
phosphate buffer, tissue fragments were post-®xed in 2% osmium
tetroxide, dehydrated in graded ethanols and embedded in Epon 812. Thin
sections were stained with uranyl acetate and lead citrate, and viewed in a
Philips CM10 electron microscope. For immunoelectron microscopy,
islets were dissected from perfused pancreatic tissue, infused with 2.3 M
sucrose and processed for cryo-ultramicrotomy (Tokuyasu, 1980).
Ultrathin cryosections were incubated using the protein A±gold technique
(Roth et al., 1978) with LYVE-1 antibody (diluted 1/50) followed by
protein A coupled to 10 nm gold particles.
Acknowledgements
The project presented in this manuscript was conceived and started in
Helsinki, and the work is the result of an equal contribution from the
laboratories in Helsinki, Vienna and Geneva, together with a major
contribution from the Oxford group. We thank Drs Hajime Kubo and
Shin-Ishi Nishikawa for the anti-VEGFR-3 antibody, and Dr Jocelyn
Holash for advice with CD31 immunohistochemistry. We also thank
Danielle Ben Nasr, Mireille Quayzin, Marie Ebrahim Malek, Gorana
Perrelet and Petra Wilgenbus for excellent technical assistance, and
Nadine Dupont and Gerard Negro for photographic work. R.M., M.S.P.
and L.O. are supported by grants from the Swiss National Science
Foundation (Nos 31-43364.95 and 31-43366.95). A.C. and G.C.
gratefully acknowledge support by Boehringer Ingelheim and by the
Austrian Industrial Research Promotion Fund. K.A., L.J. and M.J. thank
the Finnish Academy, the Sigrid Juselius Foundation, the University of
Helsinki Hospital (TYH 8105), the State Technology Development
Centre as well as the EU Biomed programs (BMH-CT96-0669 and 98-
3380) for support. D.G.J. is supported by the UK Medical Research
Council (MRC Human Immunology Unit) and a project grant
(No. 00-311) from the Association for International Cancer Research.
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Received October 20, 2000; revised November 22, 2000;
accepted January 3, 2001
Note added in proof
The reader is referred to the following two papers, which, using VEGF-C-
and VEGF-D-transfected tumour cells in xenotransplantation models,
obtained ®ndings comparable to those described in the present paper:
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682
