| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Molecular/Cancer Biology Laboratory and Department of
Pathology,*
Haartman Institute, University of Helsinki, the
Department of Oncology,
Helsinki University
Central Hospital, Helsinki, the Collagen Research Unit, Biocenter,
Department of Medical Biochemistry,
University of Oulu, Oulu, Finland, the Department of
Pathology,§
University Hospital, Nijmegen,
The Netherlands, and the Department of Gene
Expression,¶
GBF, Braunschweig, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-actin gave a weak staining of the necklace
vessels, suggesting that they were incompletely covered by
pericytes/smooth muscle cells. A highly elevated number of VEGFR-3
positive vessels was found in invasive breast cancer in comparison with
histologically normal breast tissue (P <
0.0001, the Mann-Whitney test). VEGF-C was located in the
cytoplasm of intraductal and invasive cancer cells. The results
demonstrate that the expression of VEGFR-3 becomes up-regulated in the
endothelium of angiogenic blood vessels in breast cancer. The results
also suggest that VEGF-C secreted by the intraductal carcinoma cells
acts predominantly as an angiogenic growth factor for blood
vessels, although this paracrine signaling network between the
cancer cells and the endothelium may also be involved in modifying the
permeabilities of both blood and lymphatic vessels and metastasis
formation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
VEGFR-3 is a receptor tyrosine kinase that is similar to the two VEGF receptors in structure but does not bind VEGF,10 placenta growth factor (PlGF),11 or VEGF-B.12 VEGFR-3 is initially expressed in all embryonic endothelia, but its expression in the blood vessel endothelium decreases during development, and it becomes largely restricted to the lymphatic endothelium in adult tissues.13 In the early embryos, VEGFR-3 plays an important role in blood vessel development.14 We have shown that monoclonal antibodies against VEGFR-3 provide the first specific antigenic marker for lymphatic endothelial cells in several normal tissues.15 In adults, very little or no staining was observed in most blood vessel endothelia, whereas increased expression was found in Kaposi's sarcoma spindle cells and in vascular skin tumors.15,16 In addition, increased VEGFR-3 mRNA has been found in metastatic lymph nodes and in lymphangiomas.13
The two known ligands of VEGFR-3 have a high degree of homology to VEGF. Thus, they have been named as VEGF-C17 and VEGF-D18 and consist of proteolytically processed polypeptides, which form disulfide-linked dimers.18,19 Experiments in transgenic mice have shown that VEGF-C is a growth factor for the developing lymphatic vessels,20,21 although it can also bind to VEGFR-2 expressed in blood vessel endothelia and induce capillary endothelial cell migration in culture17,19 and angiogenesis ischemic rabbit hindlimb22 and in mouse cornea.23 Expression of VEGF-C mRNA has been detected also in malignant human tumors, including nearly half of the breast cancers analyzed.24
While active angiogenesis is known to be a prerequisite for tumor growth beyond a few mm3 in size,25 lymphangiogenesis in normal or pathological adult tissues, including malignant tumors has not been reported.25-27 It has been suggested that solid tumors may compress the nearby lymphatic vessels, which cannot penetrate the tumor mass because of an elevated interstitial fluid pressure within the tumor.28 If lymphangiogenesis takes place during cancer progression, cancers with active lymphangiogenesis could be predisposed to metastatic spread via the lymphatic system and thus to poor survival. We wanted to use VEGFR-3 as a marker to study the lymphatic vasculature of breast carcinomas. To our surprise, we found that in normal breast tissue, VEGFR-3 was very weakly expressed in blood capillary endothelium in addition to lymphatic endothelia. In breast carcinomas, VEGFR-3 was also expressed in the lymphatic endothelium, but more detailed analysis showed that the expression of this receptor becomes up-regulated in angiogenic capillaries and that some of the nearby tumor cells express the VEGF-C protein.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Freshly frozen breast tissue samples were retrieved from the files of the Department of Pathology, University of Helsinki. The samples consisted of ductal carcinoma (n = 6), lobular carcinoma (n = 6), intraductal carcinoma (n = 8), fibroadenoma (n = 4), and histologically normal breast tissue (n = 12). Two of the ductal carcinomas were grade I, three were grade II, and one was grade III. All samples had been frozen immediately after surgical excision in liquid nitrogen and stored at -70°C.
Immunohistochemistry
Mouse monoclonal antibodies (mAbs) against human VEGFR-3 were produced as described earlier.15 The VEGFR-3 extracellular protein domain (VEGFR-3EC) was expressed via a recombinant baculovirus in insect cells and purified from the culture medium. Mouse monoclonal antibodies against VEGFR-3EC were then produced using standard methods, and the immunoglobulin fraction was purified by protein A affinity chromatography from hybridoma ascites fluid or Tecnomouse® culture supernatants.
Five-µm cryosections of the tissues were air-dried and fixed in cold
acetone for 10 minutes. The sections were rehydrated in
phosphate-buffered saline (PBS) and incubated for 30 minutes in 5%
normal horse serum at room temperature. The sections were then
incubated for 2 hours in a humid atmosphere at room temperature with
the 9D9 mAbs at the concentration of 1.0 µg/ml. Other anti-VEGFR-3
mAb against distinct epitopes of the VEGFR-3EC were also studied;
clones 2E11 and 7B8 were used at the concentrations of 9.5 and 8.5
µg/ml, respectively. A subsequent incubation for 30 minutes in
biotinylated anti-mouse serum was followed by a 60-minute incubation
using reagents of the Vectastain Elite Mouse IgG ABC kit (Vector
Laboratories, Burlingame, CA). Peroxidase activity was developed
with 3-amino-9-ethyl carbazole (AEC, Sigma, St. Louis, MO) for 10
minutes. Finally, the sections were stained with hematoxylin for 20
seconds. Negative controls were done by omitting the primary antibody
or by using irrelevant primary antibodies of the same isotype. The
purified baculoviral immunogen15
was used to block the
binding of the 9D9 antibodies as another negative control. In these
experiments the antibodies were incubated overnight with a 10-fold
molar exess of the VEGFR-3EC protein in PBS. After centrifugation for 4
minutes at 4000 rpm, 4°C, the supernatant was carefully collected and
then used as primary antibody. The 5-µm cryosections adjacent to the
ones stained with the anti-VEGFR-3 antibodies were immunostained for
PAL-E (0.15 µg/ml, Monosan, Uden, The Netherlands), laminin (1:4000
dilution of the supernatant of clone LAM-89, Sigma), collagen XVIII
(1.9 µg/ml), -smooth muscle actin (SMA, 0.5 µg/ml, clone 1A4,
Sigma), VEGFR-1 (1:200 dilution of the supernatant of clone 19), or
VEGFR-2 (dilution 1:100).29
Following the staining procedures, all samples were examined by a trained pathologist (P.H.). The blood vascular densities were obtained from the slides stained for PAL-E,27 following the guidelines recommended by Gasparini and Harris.30 The VEGFR-3 positive vessel densities were studied in the same way. A slide was first scanned at low magnification, and intratumoral vessel density was then assessed by counting the number of stained vessels per a x400-magnification high power field (hpf) in the areas with the highest vascular density ("vascular hotspots") or in the areas with highest VEGFR-3 positive vessel density. A minimum of five fields was counted per slide, after which the three highest counts were averaged.
Double-Staining Procedures
VEGFR-3 and PAL-E Double Staining
Double staining with PAL-E and VEGFR-3 mAbs was used to differentiate immunohistochemical staining of lymphatic and blood vessels in two intraductal carcinomas. Briefly, acetone-fixed 5-µm cryosections were incubated for 1 hour with anti-PAL-E antibodies with biotinylated horse anti-mouse antibody (Vectastain Elite Mouse IgG ABC kit, Vector Laboratories) for 30 minutes with ABC-peroxidase (Vectastain, 1:100) for 45 minutes and developed finally with 3-amino-9-ethyl carbazole (AEC) for 10 minutes. For the second step, the sections were incubated with anti-VEGFR-3 antibodies for 1 hour (0.14 µg/ml), followed by biotinylated anti-mouse antibody for 30 minutes (1:200 dilution of the supernatant of clone), ABC-peroxidase for 30 minutes (1:100), biotinylated tyramin solution (1:2.000) containing 0.01% peroxide for 5 minutes, ABC-alkaline phosphatase (1:100) for 20 minutes, and developed with Fast Blue (Sigma) for 20 minutes, according to a procedure previously described for in situ hybridization signal enhancement.31 Five-µm cryosections adjacent to the double stained ones were also immunostained with VEGFR-3 antibodies only, as described above.
VEGFR-3 and Ki-67 Double Staining
To study if the endothelial cells in VEGFR-3 positive necklace vessels in intraductal carcinoma were undergoing angiogenesis, two intraductal samples were chosen for double staining for the nuclear proliferation marker Ki-67 (0.5 mg/ml, Dako Immunoglobulins, Glostrup, Denmark) and VEGFR-3. Five-µm frozen sections were rehydrated and incubated for 20 minutes in 5% normal goat serum at room temperature. The sections were incubated for 1 hour at room temperature with the rabbit polyclonal Ki-67 antibody at a concentration of 5 µg/ml. A subsequent incubation for 30 minutes in biotinylated anti-rabbit serum was followed by a 30-minute incubation using reagents of the Vectastain Elite ABC anti-rabbit kit (Vector Laboratories). Peroxidase activity was developed with 3-amino-9-ethyl carbazole (Sigma) for 10 minutes. Subsequently, the sections were rehydrated and incubated for 20 minutes in 5% in normal horse serum at room temperature. The sections were incubated for 1 hour with the anti-VEGFR-3 monoclonal antibodies at the concentration of 2.6 µg/ml. A subsequent incubation for 30 minutes in biotinylated anti-mouse serum was followed by a 30-minute incubation using reagents of the Vectastain Elite ABC anti-mouse IgG kit (Vector Laboratories). Peroxidase activity was developed with 3,3'-diaminobenzidine (Sigma) for 10 minutes. Finally, the sections were stained with hematoxylin. Negative controls were performed by omitting the primary antibodies or by using irrelevant primary antibodies.
Production, Analysis, and Use of Polyclonal Anti-VEGF-C Antibodies
Polyclonal antibodies were produced in rabbits against a synthetic peptide corresponding to the amino acid residues 2–18 of the N-terminus of mature secreted human VEGF-C (residues 104–120 of the VEGF-C prepropeptide) as described earlier.17,19 The antisera were affinity-purifed using the immunogenic polypeptide coupled to an epoxy-activated sepharose-6B column and tested for specific staining of VEGF-C using cells infected with an adenoviral vector expressing VEGF-C or control ß-galactosidase (unpublished data of Michael Jeltsch, Seppo Ylä-Herttuala and the authors). The possibility of a cross-reaction between VEGF-C and VEGF-D was studied by immunoprecipitation analysis. 293T cells grown in Dulbecco's modified Eagle's medium - 10% fetal calf serum were transfected using the calcium phosphate precipitation method with equivalent amounts of plasmids pcDNA3.1-VEGF-C, pcDNA3.1-VEGF-D, CMV-ßgal, pIg-ßgal, pIg-VEGFR-3(1–3 loop), and pIg-VEGFR-1(1–5 loop). When producing the receptor bodies, serum-free culture medium containing 0.2% bovine serum albumin was changed to the cells 48 hours after transfection, and after an additional 30 hours the medium was collected, clarified by centrifugation, and used in the experiments. Metabolic labeling of VEGF-C and VEGF-D produced by the transfected cells was carried out by addition of 100 µCi/ml of Pro-Mix L-[35S] in vitro cell labeling mix (Amersham) to culture medium devoid of cysteine and methionine. After 6 hours, the medium was collected and immunoprecipitated using affinity purifed rabbit antibodies against VEGF-C or rabbit anti-VEGF-D antisera (a generous gift from Drs. Marc Achen and Steven Stacker).
The eight intraductal carcinomas and all of the invasive carcinomas analyzed for VEGFR-3 were chosen for further analyses of the expression of VEGF-C. Five-µm cryosections adjacent to the sections stained with the anti-VEGFR-3 antibodies were air-dried and fixed in cold acetone for 10 minutes. The sections were rehydrated in PBS and incubated for 30 minutes in 5% normal goat serum and then for 2 hours in a humid atmosphere at room temperature with the rabbit polyclonal antibodies against human VEGF-C diluted 1:200 in PBS. A subsequent incubation for 30 minutes in biotinylated anti-rabbit serum was followed by a 60-minute incubation using reagents of the Vectastain Elite Rabbit IgG ABC kit (Vector Laboratories). The sections were further processed as described above. As a negative control, the purified immunogen was used to block the binding of the VEGF-C antibodies. In these experiments, VEGF-C antibodies were incubated overnight with a 10-fold molar excess of the VEGF-C protein in PBS. After centrifugation for 4 minutes at 4000 rpm, 4°C, the supernatant was carefully collected and used in the immunostainings.
For comparison, two intraductal carcinomas were immunostained with commercially available anti-VEGF-C polyclonal antibodies (c = 200 µg/ml, Santa Cruz Biotechnology, Santa Cruz, California) raised against the amino terminus of the human VEGF-C precursor. After acetone fixation sections were incubated with 5% normal rabbit serum and with 4.0 µg/ml anti-VEGF-C antibodies for 2 hours at room temperature, a subsequent incubation for 30 minutes in biotinylated anti-goat serum was done and was followed by a 60-minute incubation using reagents of the Vectastain Elite Goat IgG ABC kit (Vector Laboratories).
Production of Collagen Type XVIII Monoclonal Antibodies
Monoclonal antibodies to human type XVIII collagen were generated by DiaBor Ltd. (Oulu, Finland) by immunization of mice with the recombinant polypeptide QH48.18,32 corresponding to the common region of the N-terminal NC1 domain of human type XVIII collagen. The clones were screened by enzyme-linked immunosorbent assay and Western analysis using the polypeptide QH48.18 and also by immunofluorescence staining of frozen human tissue sections (data not shown). The screening of the hybridoma clones resulted in three monoclonal antibodies, which were positive in all three assays mentioned (enzyme-linked immunosorbent assay, Western, immunofluorescence staining). One of the antibodies that gave the strongest signals, DB144-N2, was used in subsequent experiments. It gave an identical staining pattern to that of the polyclonal anti-all hu(XVIII), eg, in adult human skin and kidney samples (data not shown).33
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Immunohistochemical staining of VEGFR-3 in normal breast tissue
showed a very weak staining in capillaries of the interductal stroma.
These vessels did not form any specific pattern but were scattered
throughout the stroma (Figure 1A)
. The
density of the VEGFR-3 positive vessels in the normal breast tissue
samples ranged from 6 to 17 per hpf, median 9 (n
= 12). Most of such vessels were strongly stained for the blood
vascular endothelial marker PAL-E and for the basal lamina component,
collagen XVIII (black arrows in Figure 1
), suggesting that VEGFR-3 was
expressed weakly in the blood vessels of normal breast tissue. However,
some thin vessels in the stroma, which were clearly stained for VEGFR-3
were negative for PAL-E and only weakly positive for the collagen type
XVIII, suggesting that they were lymphatic vessels (green arrows).
VEGFR-3 positive vessels were also uniformly found in benign
fibroadenomas (Figure 1D)
in which their density (median 8 vessels per
hpf; range 3 to 19; n = 4) did not differ from that of
the histologically normal breast tissue (median 8 versus 9;
P > 0.1, the Mann-Whitney test).
|
In intraductal carcinomas, a special pattern of strongly stained
VEGFR-3 positive vessels was observed. The vessels formed arch-like
structures around the affected ducts (Figure 2A)
. This necklace pattern was also
observed in staining of adjacent sections for the blood vessel
endothelial marker, PAL-E (Figure 2B)
, suggesting that VEGFR-3
expression was enhanced in capillary endothelium. To more definitively
differentiate between blood and lymphatic vessels and to search for the
presence of smooth muscle cells and pericytes in the vessel walls,
additional stainings were done using antibodies against smooth muscle
-actin and basal lamina components laminin and type XVIII collagen.
According to this staining, the small vessels close to the intraductal
carcinomas expressed simultaneously VEGFR-3 and the basal lamina
proteins but stained more weakly for SMA, suggesting that they are
incompletely covered by pericytes/smooth muscle cells in the vessel
wall (black arrows in Figure 2, C-F
). In contrast, larger blood vessels
at some distance from the intraductal lesions were in general negative
for VEGFR-3 but were positive for laminin, collagen XVIII, and SMA (red
arrows). In addition, vessels were found that were positive for VEGFR-3
but only very weakly stained for the basal lamina proteins laminin and
type XVIII collagen and not at all for SMA (green arrows). These were
considered to represent lymphatic vessels.
|
Two intraductal carcinomas were chosen for the immunohistochemical
double-staining procedure to more clearly differentiate lymphatic
vessels from blood vessels.27
Using this method, the
VEGFR-3 positive vessels are stained blue, whereas the PAL-E positive
vessels and basal laminae are stained brown. Both tested samples showed
a similar pattern of staining: the vessels lining the tumor filled
ducts were predominantly PAL-E positive (Figure 2
, arrowhead in G and
H), whereas the presumably lymphatic, VEGFR-3 positive vessels a short
distance away in the interductal stroma were PAL-E negative (Figure 2
,
black arrows in G and H). To exclude misinterpretation due to possible
double-staining artifacts, adjacent 5-µm sections were stained using
anti-VEGFR-3 alone. This staining confirmed that several of the PAL-E
positive blood vessles are also positive for VEGFR-3.
Double Staining Using Antibodies Against Ki-67 and VEGFR-3
To show if the endothelial cells of the necklace vessels in
intraductal carcinomas were undergoing angiogenic growth, double
staining of the nuclear proliferation marker Ki-67 and VEGFR-3 was
carried out for two intraductal carcinomas. Using this method, the
nucleae in S phase are stained red, and VEGFR-3 positive endothelial
cells are stained dark brown. Both of the samples showed a similar
pattern of staining; several nucleae of the tumor cells inside the
ducts were strongly positive for Ki-67 (Figure 2A
, inset, green arrows)
as were several endothelial cell nucleae of the necklace vessels (red
arrow). Some distance away, the VEGFR-3 positive vessels had only Ki-67
negative nucleae (black arrow), suggesting that they were in a
quiescent state.
Specificity of Anti-VEGF-C Antibodies
The rabbit antisera against human VEGF-C have been previously
characterized and found to lack cross-reactivity against VEGF or
VEGF-B.17,19
To test for a possible cross-reaction with
the related VEGF-D polypeptides, immunoprecipitations were carried out.
Anti-VEGF-C antibodies precipitated the proteolytically processed 29-
and 31-kd VEGF-C polypeptides from metabolically labeled conditioned
media of transfected 293T cells (Figure 3
, lane 1) but did not react with the
fully processed 21.5-kd form, which was however bound by the VEGFR-3-Ig
receptor bodies (Figure 3
, lane 4). On the other hand, anti-VEGF-C did
not recognize VEGF-D (Figure 3
, lane 2) unlike the anti-VEGF-D
antibodies (Figure 3
, lane 3), indicating a lack of cross-reaction with
VEGF-D.
|
Polyclonal affinity-purified antibodies against human VEGF-C were
used to stain the eight intraductal carcinoma samples. According to the
results obtained, all samples contained at least some VEGF-C, but
considerable heterogeneity was observed in the intensity of staining
and in the expression patterns. In some cases, most of the carcinoma
cells were strongly positive for VEGF-C (Figure 4A)
, whereas in others, only some carcinoma
cells gave a staining signal (Figure 4B)
. In contrast, very little or
no staining was observed in the normal tissues surrounding the affected
ducts, although weak signal was also obtained in unaffected normal
ductal epithelium (data not shown). Antigen-blocking experiments
indicated that the staining for VEGF-C was specific (inset in Figure 4B
). Antibodies against the amino terminus of the VEGF-C precursor form
of human origin showed a similar pattern of staining (data not shown).
The other VEGF-C receptor, VEGFR-2, as well as the other VEGF receptor
VEGFR-1 were expressed in the same necklace vessels adjacent to the
intraductal carcinoma cells (Figure 4, C-F
).
|
Strongly stained VEGFR-3 positive vessels were also present in all
invasive ductal carcinomas and lobular carcinomas studied (Figure 5, A-C and D
, respectively). The VEGFR-3 positive vessels did not appear
to form any specific distribution pattern; most of these vessels were
also immunoreactive for the PAL-E antigen (Figure 5B)
. The intratumoral
VEGFR-3 positive vessel density (median 21, range 9 to 56 vessels per
hpf; n = 12) was significantly elevated in the invasive
breast carcinomas when compared with normal breast tissue (median 21
versus 9; P < 0.0001, the Mann-Whitney
test). Occasionally, invasion of the carcinoma cells into the VEGFR-3
positive vessels could be observed (asterisk in Figure 5D
).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A second group of vessels lying a distance away from the affected ducts were positive for VEGFR-3 but very weakly positive for the basal lamina components and negative for PAL-E, suggesting that they are lymphatic vessels. These vessels also lacked SMA-positive pericytic components. Also in invasive breast carcinomas, VEGFR-3 was up-regulated in PAL-E positive blood vessels, although the vessel patterns seen were more randomly organized in the connective tissue stroma around the tumor cells. The results suggest that VEGFR-3 expression is up-regulated in breast carcinomas during angiogenesis associated with tumor growth. The highly elevated number of VEGFR-3 positive vessels found in carcinoma in situ is compatible with the hypothesis that the carcinoma cells produce factors, which activate the growth of blood vessels in the immediate vicinity of the carcinoma cells.
VEGF-C is obviously a candidate growth factor for the VEGFR-3 and VEGFR-2 positive vessels in the carcinomas. Indeed, we found that both intraductal and invasive carcinoma cells contain the VEGF-C protein. This is in agreement with our recent study in which nearly half of unselected malignant invasive tumors contained VEGF-C mRNA in Northern blotting analyses.24 So far, VEGF-C has been considered to be a relatively specific lymphatic endothelial growth factor in normal tissues. Expression of a VEGF-C transgene in the basal layer of mouse skin keratinocytes caused a selective development of a hyperplastic subpapillary lymphatic network,20 and recombinant VEGF-C protein also promoted predominantly lymphangiogenesis in the chick chorioallantoic membrane.21
VEGF-C is synthesized as a precursor protein, which requires proteolytic cleavages for full activity. In a recent study we showed that proteolytic processing of the VEGF-C precursor is required for activation of VEGFR-2, whereas a partially processed precursor can also activate VEGFR-3.19 The fully processed VEGF-C was less potent in stimulating capillary endothelial cell growth but almost as active as VEGF in promoting capillary endothelial cell migration. In malignant tumors, the fully processed VEGF-C may be available and may participate in the stimulation of angiogenesis via both of its two receptors, VEGFR-3 and VEGFR-2, which according to the present data are up-regulated in angiogenic blood capillary endothelium. Such a paracrine relationship appears to exist also for VEGF and its two receptors VEGFR-1 and VEGFR-2, which have been shown to be expressed at mRNA level by breast carcinoma cells and the endothelia of adjacent vessels.8
In some cases we observed invasion of tumor cells into the VEGFR-3 positive vessels, which had a morphology characteristic of the lymphatic vessels and lacked red blood cells. It is well established that breast cancer may spread via the lymphatic vessels into the axillary lymph nodes. Lymph node-positive cancer is associated with an inferior survival rate as compared with node-negative disease, and the number of involved axillary lymph nodes is still probably the most important and most widely used independent prognostic factor in breast cancer.34 Even small breast cancers may be associated with axillary nodal metastases. About 23% of cancers that are 2 cm or smaller in size are associated with positive axillary nodes in conventional histopathological examination,35 and even a higher frequency of metastatic nodes may be found if the nodes are investigated by serial sectioning and immunohistochemistry. Interestingly, enhanced expression of the VEGFR-3 mRNA has been found in the lymphatic endothelium of metastatic lymph nodes.13 This could be due to activation of the lymphatic endothelium by cytokines secreted by the cancer cells, but it could also be a consequence of a relative obstruction of the lymphatic flow.
Relatively little is known about the mechanisms of metastatic spread via the lymphatics. It appears that the tumor cells need to penetrate the lymphatic endothelium twice to translocate into the interstitium of a lymph node. Because lymphatic vessels do not contain tight junctions or continuous basal laminae,36 their penetration may only require tumor cell adhesion to the endothelium and transmigration through intracellular gaps that have been described to occur between lymphatic endothelial cells (reviewed in Ref. 37 ). It is not yet known how such interendothelial gaps are regulated, but VEGF has been shown to increase microvascular permeability by enhancing the transmigration activity of the vesicular-vacuolar organelles.38 Both VEGF and VEGF-C are strong vascular permeability factors,19 and VEGF-C might also have a similar function in the lymphatic system. In general, VEGF-C and VEGFR-3 could be components of a paracrine signaling network between cancer cells and the endothelium, and they may be involved in modifying the permeabilities of both blood and lymphatic vessels and metastasis formation.
We conclude that the number of VEGFR-3 positive vessels is elevated in breast cancer. Part of the VEGFR-3 positive vessels are angiogenic blood vessels with up-regulated expression of VEGFR-3 in the endothelial cells. However, a part of the VEGFR-3 positive structures appear to be lymphatic vessels, although we found no evidence that lymphangiogenesis occurs in breast cancers. The presence of VEGF-C in intraductal carcinoma cells as well as the VEGFR-3 positive capillaries surrounding the affected ducts suggest that VEGF-C secreted by the cancer cells acts predominantly as an angiogenic growth factor, but it could also affect the lymphatic vessels during tumor metastasis into the axillary lymph nodes.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the Finnish Academy of Sciences, the Finnish Cancer Research Foundation, the Helsinki University Central Hospital Research Funds (TYH8105), and the European Union (Biomedicine Grant PL 963380). M. Rehn and T. Pihlajaniemi were supported by the Sigrid Juselius Foundation and FibroGen Inc. (South San Francisco, CA).
Accepted for publication February 3, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  C. A. Heckman, T. Holopainen, M. Wirzenius, S. Keskitalo, M. Jeltsch, S. Yla-Herttuala, S. R. Wedge, J. M. Jurgensmeier, and K. Alitalo The Tyrosine Kinase Inhibitor Cediranib Blocks Ligand-Induced Vascular Endothelial Growth Factor Receptor-3 Activity and Lymphangiogenesis Cancer Res., June 15, 2008; 68(12): 4754 - 4762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-H. Jeon, C. Jang, J. Han, R. P. Kataru, L. Piao, K. Jung, H. J. Cha, R. A. Schwendener, K. Y. Jang, K.-S. Kim, et al. Profound but Dysfunctional Lymphangiogenesis via Vascular Endothelial Growth Factor Ligands from CD11b+ Macrophages in Advanced Ovarian Cancer Cancer Res., February 15, 2008; 68(4): 1100 - 1109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Koyama, N. Kobayashi, M. Harada, M. Takeoka, Y. Kawai, K. Sano, M. Fujimori, J. Amano, T. Ohhashi, R. Kannagi, et al. Significance of Tumor-Associated Stroma in Promotion of Intratumoral Lymphangiogenesis: Pivotal Role of a Hyaluronan-Rich Tumor Microenvironment Am. J. Pathol., January 1, 2008; 172(1): 179 - 193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lahdenranta, R. L. Sidman, R. Pasqualini, and W. Arap Treatment of hypoxia-induced retinopathy with targeted proapoptotic peptidomimetic in a mouse model of disease FASEB J, October 1, 2007; 21(12): 3272 - 3278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Qin, Y. Wan, M. Li, X. Xue, S. Wu, C. Zhang, Y. You, W. Wang, C. Jiang, Y. Liu, et al. Identification of a Novel Peptide Ligand of Human Vascular Endothelia Growth Factor Receptor 3 for Targeted Tumour Diagnosis and Therapy J. Biochem., July 1, 2007; 142(1): 79 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. Donoghue, F. L. Lederman, B. J. Susil, and P. A.W. Rogers Lymphangiogensis of normal endometrium and endometrial adenocarcinoma Hum. Reprod., June 1, 2007; 22(6): 1705 - 1713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. van Weel, L. Seghers, M. R. de Vries, E. J. Kuiper, R. O. Schlingemann, I. M. Bajema, J. H.N. Lindeman, P. M. Delis-van Diemen, V. W.M. van Hinsbergh, J. H. van Bockel, et al. Expression of Vascular Endothelial Growth Factor, Stromal Cell-Derived Factor-1, and CXCR4 in Human Limb Muscle With Acute and Chronic Ischemia Arterioscler. Thromb. Vasc. Biol., June 1, 2007; 27(6): 1426 - 1432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Strumberg, J. W. Clark, A. Awada, M. J. Moore, H. Richly, A. Hendlisz, H. W. Hirte, J. P. Eder, H.-J. Lenz, and B. Schwartz Safety, Pharmacokinetics, and Preliminary Antitumor Activity of Sorafenib: A Review of Four Phase I Trials in Patients with Advanced Refractory Solid Tumors Oncologist, April 1, 2007; 12(4): 426 - 437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Zhao, G. Smith, J. Cai, A. Ma, and M. Boulton Vascular endothelial growth factor C promotes survival of retinal vascular endothelial cells via vascular endothelial growth factor receptor-2 Br. J. Ophthalmol., April 1, 2007; 91(4): 538 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Laakkonen, M. Waltari, T. Holopainen, T. Takahashi, B. Pytowski, P. Steiner, D. Hicklin, K. Persaud, J. R. Tonra, L. Witte, et al. Vascular Endothelial Growth Factor Receptor 3 Is Involved in Tumor Angiogenesis and Growth Cancer Res., January 15, 2007; 67(2): 593 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hinojar-Gutierrez, M.-E. Fernandez-Contreras, R. Gonzalez-Gonzalez, M.-J. Fernandez-Luque, A. Hinojar-Arzadun, M. Quintanilla, and C. Gamallo Intratumoral Lymphatic Vessels and VEGF-C Expression Are Predictive Factors of Lymph Node Relapse in T1-T4 N0 Laryngopharyngeal Squamous Cell Carcinoma Ann. Surg. Oncol., January 1, 2007; 14(1): 248 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Shayan, M. G. Achen, and S. A. Stacker Lymphatic vessels in cancer metastasis: bridging the gaps Carcinogenesis, September 1, 2006; 27(9): 1729 - 1738. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hoshida, N. Isaka, J. Hagendoorn, E. di Tomaso, Y.-L. Chen, B. Pytowski, D. Fukumura, T. P. Padera, and R. K. Jain Imaging Steps of Lymphatic Metastasis Reveals That Vascular Endothelial Growth Factor-C Increases Metastasis by Increasing Delivery of Cancer Cells to Lymph Nodes: Therapeutic Implications Cancer Res., August 15, 2006; 66(16): 8065 - 8075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Marcellini, N. De Luca, T. Riccioni, A. Ciucci, A. Orecchia, P. M. Lacal, F. Ruffini, M. Pesce, F. Cianfarani, G. Zambruno, et al. Increased Melanoma Growth and Metastasis Spreading in Mice Overexpressing Placenta Growth Factor Am. J. Pathol., August 1, 2006; 169(2): 643 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B Zhao, A Ma, J Cai, and M Boulton VEGF-A regulates the expression of VEGF-C in human retinal pigment epithelial cells Br. J. Ophthalmol., August 1, 2006; 90(8): 1052 - 1059. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Orlandini, A. Spreafico, M. Bardelli, M. Rocchigiani, A. Salameh, S. Nucciotti, C. Capperucci, B. Frediani, and S. Oliviero Vascular Endothelial Growth Factor-D Activates VEGFR-3 Expressed in Osteoblasts Inducing Their Differentiation J. Biol. Chem., June 30, 2006; 281(26): 17961 - 17967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhang, E. Giraudo, J. A. Hoffman, D. Hanahan, and E. Ruoslahti Lymphatic Zip Codes in Premalignant Lesions and Tumors Cancer Res., June 1, 2006; 66(11): 5696 - 5706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kamba, B. Y. Y. Tam, H. Hashizume, A. Haskell, B. Sennino, M. R. Mancuso, S. M. Norberg, S. M. O'Brien, R. B. Davis, L. C. Gowen, et al. VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H560 - H576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Audet, N. J. Beasley, C. MacMillan, D. G. Jackson, P. J. Gullane, and S. Kamel-Reid Lymphatic Vessel Density, Nodal Metastases, and Prognosis in Patients With Head and Neck Cancer Arch Otolaryngol Head Neck Surg, December 1, 2005; 131(12): 1065 - 1070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Gombos, X. Xu, C. S. Chu, P. J. Zhang, and G. Acs Peritumoral Lymphatic Vessel Density and Vascular Endothelial Growth Factor C Expression in Early-Stage Squamous Cell Carcinoma of the Uterine Cervix Clin. Cancer Res., December 1, 2005; 11(23): 8364 - 8371. [Abstract] [Full Text] [PDF]
|
|
|
|
