[CANCER RESEARCH 60, 4932– 4938, September 1, 2000]
Vascular Endothelial Growth Factor, Interleukin 8, Platelet-derived Endothelial
Cell Growth Factor, and Basic Fibroblast Growth Factor Promote
Angiogenesis and Metastasis in Human Melanoma Xenografts1
Einar K. Rofstad2 and Ellen F. Halsør
Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway
ABSTRACT
Angiogenesis is a significant prognostic factor in melanoma, but the
angiogenic factors controlling the neovascularization are not well defined.
The purpose of this study was to investigate whether the angiogenesis and
metastasis of melanoma are promoted by vascular endothelial growth
factor (VEGF), interleukin 8 (IL-8), platelet-derived endothelial cell
growth factor (PD-ECGF), and/or basic fibroblast growth factor (bFGF).
Cells from human melanoma lines (A-07, D-12, R-18, and U-25) transplanted to BALB/c nu/nu mice were used as tumor models. Expression of
angiogenic factors was studied by ELISA, Western blotting, and immunohistochemistry. Angiogenesis was assessed by using an intradermal
angiogenesis assay. Lung colonization and spontaneous lung metastasis
were determined after i.v. and intradermal inoculation of tumor cells,
respectively. The specific roles of VEGF, IL-8, PD-ECGF, and bFGF in
tumor angiogenesis, lung colonization, and spontaneous metastasis were
assessed in mice treated with neutralizing antibody. The melanoma lines
expressed multiple angiogenic factors, and each line showed a unique
expression pattern. Multiple angiogenic factors promoted angiogenesis in
the most angiogenic melanoma lines, whereas angiogenesis in the least
angiogenic melanoma lines was possibly promoted solely by VEGF. Tumor growth, lung colonization, and spontaneous metastasis were controlled by the rate of angiogenesis and hence by the angiogenic factors
promoting the angiogenesis. Lung colonization and spontaneous metastasis in A-07 were inhibited by treatment with neutralizing antibody against
VEGF, IL-8, PD-ECGF, or bFGF. Each of these angiogenic factors may
promote metastasis in melanoma, because inhibition of one of them could
not be compensated for by the others. Our observations suggest that
efficient antiangiogenic treatment of melanoma may require identification
and blocking of common functional features of several angiogenic factors.
INTRODUCTION
Malignant melanoma is a highly metastatic tumor type of increasing incidence, poor prognosis, and high resistance to treatment (1–3).
The progression of melanoma is characterized by distinct sequential
steps, from common acquired melanocytic nevus via dysplastic nevus,
radial growth phase primary melanoma, and vertical growth phase
primary melanoma to melanoma metastasis (4, 5). There is increasing
evidence that melanoma progression requires induction of new capillary blood vessels, a process called angiogenesis (6 –12). Thus, the
transition from the radial to the vertical growth phase, which represents a significant worsening of the prognosis, has been shown to be
dependent on neovascularization (7–9). Moreover, the probability of
metastasis has been found to increase with increasing microvessel
density of the primary tumor (10 –12).
Melanoma cells have been shown to produce and secrete a wide
Received 12/29/99; accepted 7/6/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by grants from The Norwegian Cancer Society.
2
To whom requests for reprints should be addressed, at Department of Biophysics,
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310
Oslo, Norway. Phone: 47-2293-4279; Fax: 47-2293-4270; E-mail: e.k.rofstad@labmed.
uio.no.
variety of angiogenic factors, including VEGF3 (13–16), IL-8 (17–
19), PD-ECGF (20, 21), and bFGF (22–24). VEGF, also known as
vascular permeability factor, is a strong specific mitogen for endothelial cells and may also stimulate endothelial cell migration and
reorganization (25, 26). IL-8, which belongs to the superfamily of
CXC chemokines, is a multifunctional cytokine that exhibits potent
angiogenic activities both in vitro and in vivo (27, 28). PD-ECGF, also
known as thymidine phosphorylase and gliostatin, stimulates endothelial cell mitogenesis and chemotaxis in vitro and is strongly angiogenic in vivo, possibly through modulation of nucleotide metabolism (29). bFGF, which belongs to the family of heparin-binding
growth factors, is a multifunctional protein having a well-established
key role in tumor angiogenesis (30 –32).
There is significant experimental evidence that these angiogenic
factors are involved in the growth and metastasis of malignant melanoma. Inoculation of human melanoma cells transfected with the
gene encoding VEGF into immune-deficient mice results in tumors
with increased vascularization (13–15), microvessel permeability (14,
15, 33), and volumetric growth rate (14, 33). The constitutive expression of IL-8 in human melanoma cells is correlated with their lung
colonization efficiency after i.v. inoculation in immune-deficient mice
(17). Human melanoma cells show increased lung colonization efficiency after transfection with the gene encoding VEGF (15) or IL-8
(18). Moreover, xenografted tumors initiated from human melanoma
cell lines transfected with antisense-VEGF (14, 15) or antisense-bFGF
(24) show reduced vascularization and growth.
A panel of angiogenic factors may thus be involved in the angiogenesis, growth, and metastasis of melanomas, and the panel may
differ among tumors differing in angiogenic activity (34 –36). However, angiogenic factors that show sufficiently high constitutive expression to promote spontaneous metastasis have not been identified
conclusively thus far. The work reported here was aimed at investigating whether the angiogenesis and metastasis of melanoma may be
promoted by VEGF, IL-8, PD-ECGF, and/or bFGF. Four human
melanoma xenograft lines showing widely different constitutive expression of these angiogenic factors were included in the study. The
specific role of each of the factors was investigated by assessing
tumor angiogenesis and metastasis in mice treated with neutralizing
antibody.
MATERIALS AND METHODS
Mice. Adult (8 –10 weeks of age) female BALB/c nu/nu mice were used to
assess tumor angiogenesis, lung colonization, and spontaneous metastasis. The
mice were bred at our institute and maintained under specific pathogen-free
conditions at constant temperature (24 –26°C) and humidity (30 –50%). Sterilized food and tap water were given ad libitum.
Cell Lines. Four human melanoma cell lines (A-07, D-12, R-18, and U-25)
were included in the study (37). They were maintained in monolayer culture in
RPMI 1640 (25 mM HEPES and L-glutamine) supplemented with 13% bovine
calf serum, 250 mg/l penicillin, and 50 mg/l streptomycin. The cultures were
3
The abbreviations used are: VEGF, vascular endothelial growth factor; bFGF, basic
fibroblast growth factor; IL, interleukin; PD-ECGF, platelet-derived endothelial cell
growth factor.
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incubated at 37°C in a humidified atmosphere of 5% CO2 in air and subcultured twice a week. The cell lines were verified to be free from Mycoplasma
contamination.
ELISA. Commercial ELISA kits (Quantikine; R&D Systems, Abingdon,
United Kingdom) were used according to the instructions of the manufacturer
to measure VEGF, IL-8, and bFGF concentrations in culture medium. Medium
samples from cultures in exponential growth were collected 24 h after change
of medium. Cell numbers at the time of medium change and at the time of
medium collection were determined by using a hemocytometer and a phase
contrast microscope. The medium samples were centrifuged to remove particles, diluted to appropriate concentrations, and analyzed in duplicate at several
dilutions. Absorbances were determined at 450 nm. Readings at 570 nm were
subtracted from the readings at 450 nm to correct for optical imperfections. A
standard curve was obtained by linear regression analysis of protein concentration versus absorbance in a double logarithmic plot. The rate of protein
secretion (Rsec) was calculated as:
R sec ⫽
V⌬C
N i⌬t
⫻
ln共 N f/N i兲
共 N f/N i ⫺ 1)
where ⌬C is the increase in protein concentration during the time interval ⌬t.
Here, ⌬t was 24 h. Ni and Nf are the initial and final cell numbers, and V is the
volume of medium. Replicate cultures were used to determine Ni. The second
factor of the product is based on the assumption that the cell number increased
exponentially with time during ⌬t, an assumption that was verified to be valid.
Western Blotting. Cells from cultures in exponential growth were washed
in PBS and boiled in Laemmli lysis buffer for 5 min (38). Proteins were
separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were incubated with antihuman VEGF rabbit polyclonal
antibody (Santa Cruz Biotechnology, Santa Cruz, CA), antihuman IL-8 mouse
monoclonal antibody (R&D Systems), antihuman PD-ECGF goat polyclonal
antibody (R&D Systems), or antihuman bFGF rabbit polyclonal antibody
(Oncogene Science, Cambridge, MA) for 60 min. Bound antibody was detected by using a biotin-streptavidin alkaline phosphatase staining procedure.
Recombinant human VEGF, IL-8, PD-ECGF, or bFGF (R&D Systems) was
used as positive control. The specificity of the antibody-antigen interactions
was confirmed by peptide competition studies and by incubation of membranes
in solutions without primary antibody. Protein molecular weights were estimated by using broad range or high range prestained standards according to the
instructions of the manufacturer (SDS-PAGE standards; Bio-Rad Laboratories,
Hercules, CA).
Immunohistochemistry. Immunohistochemical staining of tumor tissue
was performed by using an indirect immunoperoxidase method. Tumors were
fixed in phosphate-buffered 4% paraformaldehyde or snap-frozen in liquid
nitrogen. Antihuman VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology), antihuman IL-8 rabbit polyclonal antibody (Endogen, Woburn, MA),
antihuman PD-ECGF goat polyclonal antibody (R&D Systems), or antihuman
bFGF rabbit polyclonal antibody (Santa Cruz Biotechnology) was used as
primary antibody. Controls included omission of the primary antibody, incubation with normal rabbit immunoglobulin or normal rabbit serum, and incubation with blocking peptides before staining. The sections were counterstained with hematoxylin.
Angiogenesis Assay. Tumor angiogenesis was assessed by using an intradermal angiogenesis assay (39). A 100-␮l Hamilton syringe was used to
inoculate aliquots of 10 ␮l of cell suspension into the flanks of mice. The
inoculation point lay above the s.c. muscle tissue in the deeper part of the
dermis. The number of cells per inoculum was 1.0 ⫻ 106. The mice were killed
on day 7 after the inoculation. Small vascularized tumors had developed in the
inoculation sites at that time. The skin around the inoculation sites was
removed, and the tumors were located with a dissecting microscope. The
capillaries in the dermis oriented toward the tumors were counted, and the diameters of the tumors were measured, using an ocular with a scale. The
number of capillaries was corrected for the background, determined after
the injection of 10 ␮l of HBSS. Angiogenesis was quantified as number of
capillaries per tumor or number of capillaries per mm of tumor circumference.
Lung Colonization Assay. Aliquots of 3.0 ⫻ 106 A-07 cells or 3.0 ⫻ 105
D-12 cells suspended in 0.2 ml of HBSS were inoculated into the lateral tail
vein of mice by using a tuberculin syringe with a 26-gauge needle. The mice
were killed and autopsied 5 weeks after the inoculation. The lungs were
removed, rinsed in HBSS, and fixed in Bouin’s solution for 24 h to facilitate
the scoring of colonies. The number of surface colonies was determined by
using a stereomicroscope.
Spontaneous Metastasis Assay. Aliquots of 3.5 ⫻ 105 A-07 or D-12 cells
suspended in 10 ␮l of HBSS were inoculated intradermally in the left flank of
mice, using the same procedure as used in the angiogenesis assay described
above. The inoculations were performed 24 h after the mice had been immunosuppressed by 5.0 Gy of whole-body irradiation. The whole-body irradiation
was performed by using a Siemens Stabilipan X-ray unit, operated at 220 kV,
19 –20 mA, and with 0.5-mm copper filtration (40). The tumors were removed
surgically when the largest diameter had attained ⬃10 mm, and the wounds
were closed with surgical clips. The mice were examined for clinical signs of
metastases twice a week. They were killed and autopsied 3 months after the
primary tumor was removed or when they were moribund. The lungs were
examined for the presence of macroscopic metastases as described above.
Treatment with Neutralizing Antibody in Vivo. The specific roles of
VEGF, IL-8, PD-ECGF, and bFGF in tumor angiogenesis, lung colonization,
and spontaneous metastasis were investigated by treating host mice with
neutralizing antibody against these angiogenic factors. The antibodies used for
treatment were antihuman VEGF mouse monoclonal antibody (R&D Systems), antihuman IL-8 mouse monoclonal antibody (R&D Systems), antihuman PD-ECGF goat polyclonal antibody (R&D Systems), and antihuman
bFGF goat polyclonal antibody (R&D Systems). The antibody solutions were
diluted in PBS and administered to the mice in volumes of 0.25 ml by i.p.
injection. In the angiogenesis and lung colonization experiments, the treatments consisted of four doses of 25 ␮g (VEGF and bFGF) or 100 ␮g (IL-8 and
PD-ECGF) of antibody given in intervals of 24 h. The first dose was given 1 h
before the tumor cell inoculation. In the spontaneous metastasis experiments,
the treatments consisted of eight doses of antibody given daily the last 8 days
before the primary tumor was removed.
Treatment with Neutralizing Antibody in Vitro. Possible cytotoxic or
antiproliferative effects of the neutralizing antibodies described above were
investigated in vitro. A-07, D-12, R-18, or U-25 cells were cultured in RPMI
1640 (25 mM HEPES and L-glutamine) supplemented with 13% bovine calf
serum, 250 mg/l penicillin, and 50 mg/l streptomycin in the absence or
presence of 5 ␮g/ml of antibody for up to 8 days. The number of cells in the
cultures was determined 2, 4, 6, or 8 days after the cultures were initiated by
counting cells in a hemocytometer.
Statistical Analysis. Results are presented as arithmetic mean ⫾ SE.
Statistical comparisons of data were performed by one-way ANOVA. When
significant differences were found, the Dunnett’s method was used to identify
the data sets that differed from the control data. Probability values of P ⬍ 0.05
were considered significant. All Ps were determined from two-sided tests. The
statistical analysis was performed by using SigmaStat statistical software
(Jandel Scientific GmbH, Erkrath, Germany).
RESULTS
Significant secretion of VEGF and IL-8 was seen in all cell lines,
whereas A-07 was the only line that showed detectable secretion of
bFGF (Table 1). A-07 showed higher VEGF secretion than U-25
(P ⬍ 0.05), which in turn showed higher VEGF secretion than D-12
and R-18 (P ⬍ 0.05). The IL-8 secretion was also higher in A-07 than
in the other lines (P ⬍ 0.05). Moreover, D-12 showed higher IL-8
secretion than U-25 (P ⬍ 0.05), which in turn showed higher IL-8
secretion than R-18 (P ⬍ 0.05).
All cell lines showed significant expression of VEGF, IL-8, and
bFGF, whereas A-07 was the only line that showed significant exTable 1 Rate of secretion of angiogenesis factors of human melanoma cell lines
Secretion rate (pg/106 cells•h)a
a
Melanoma
VEGF
IL-8
bFGF
A-07
D-12
R-18
U-25
714 ⫾ 140
18 ⫾ 1
16 ⫾ 2
85 ⫾ 8
571 ⫾ 153
291 ⫾ 86
4⫾1
109 ⫾ 27
1.9 ⫾ 0.8
0
0
0
Mean ⫾ SE of five independent experiments.
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pression of PD-ECGF, as revealed by Western blot analysis (Fig. 1).
A-07 showed substantially higher VEGF, IL-8, and bFGF expression
than the other lines. Moreover, six specific bFGF bands were seen in
A-07, whereas D-12, R-18, and U-25 showed only 2– 4 specific bFGF
bands.
A-07 tumors showed significant expression of VEGF, IL-8, PDECGF, and bFGF, as revealed by immunohistochemistry (Fig. 2).
Also D-12, R-18, and U-25 stained positive for VEGF, IL-8, and
bFGF in vivo, but the staining was weaker than that in A-07 tumors for
all proteins. Significant expression of PD-ECGF could not be detected
in D-12, R-18, or U-25 tumors.
Intradermal inoculation of A-07, D-12, R-18, or U-25 cells in mice
evoked a strong angiogenic response (Fig. 3). The angiogenic response, measured as number of tumor-oriented capillaries per tumor at
day 7 after the inoculation of 1.0 ⫻ 106 cells, differed substantially
among the lines. A-07 evoked the strongest angiogenic response,
followed by D-12, R-18, and U-25. The differences in number of
tumor-oriented capillaries between A-07 and D-12, D-12 and R-18,
and R-18 and U-25 were all statistically significant (P ⬍ 0.05). The
angiogenic response evoked by A-07 was inhibited in mice treated
with neutralizing antibody against VEGF, IL-8, PD-ECGF, or bFGF
(P ⬍ 0.05). Treatment with neutralizing antibody against VEGF or
IL-8 also reduced the angiogenic response evoked by D-12 (P ⬍ 0.05)
but not treatment with neutralizing antibody against PD-ECGF or
bFGF (P ⬎ 0.05). The angiogenic response evoked by R-18 and U-25
was inhibited in mice treated with neutralizing antibody against
VEGF (P ⬍ 0.05) but not in mice treated with neutralizing antibody
against IL-8, PD-ECGF, or bFGF (P ⬎ 0.05). Similar results and
identical conclusions were achieved by expressing angiogenic response as number of tumor-oriented capillaries per mm of tumor
circumference (data not shown).
i.v. inoculation of A-07 or D-12 cells in mice resulted in significant
lung colonization (Fig. 4). The lung colonization efficiency of A-07
was reduced in mice treated with neutralizing antibody against VEGF,
Fig. 1. Western blots showing the expression of VEGF, IL-8, PD-ECGF, or bFGF in
exponentially growing A-07, D-12, R-18, and U-25 monolayer cell cultures. Proteins from
5.0 ⫻ 105 cells were loaded into each lane. Specific bands are indicated by the arrows to
the left. The other bands were attributable to nonspecific staining, as shown by peptide
competition studies and in blots where incubation with primary antibody was omitted. The
arrows to the right indicate the positions of standards with known molecular weights.
IL-8, PD-ECGF, or bFGF (P ⬍ 0.05). Treatment with neutralizing
antibody against VEGF or IL-8 also reduced the lung colonization
efficiency of D-12 (P ⬍ 0.05) but not treatment with neutralizing
antibody against PD-ECGF or bFGF (P ⬎ 0.05). R-18 and U-25 cells
do not develop lung colonies after i.v. inoculation in mice (41).
Intradermal A-07 and D-12 tumors developed spontaneous lung
metastases in ⬃50% of the mice (Fig. 5). The metastatic efficiency of
A-07 tumors was reduced in mice treated with neutralizing antibody
against VEGF, IL-8, PD-ECGF, or bFGF (P ⬍ 0.05). Treatment with
neutralizing antibody against VEGF or IL-8 also reduced the metastatic efficiency of D-12 tumors (P ⬍ 0.05) but not treatment with
neutralizing antibody against PD-ECGF or bFGF (P ⬎ 0.05).
Treatment with neutralizing antibody against VEGF, IL-8, PDECGF, or bFGF in vitro had no cytotoxic or antiproliferative effects
on A-07, D-12, R-18, or U-25 cells; the number of cells in antibodytreated cultures was not significantly different from that in control
cultures (P ⬎ 0.05), irrespective of cell line, treatment time, and
neutralizing antibody (data not shown).
DISCUSSION
A-07, D-12, R-18, and U-25 melanoma cells expressed multiple
angiogenic factors in vitro, and each cell line showed a unique
expression pattern. Thus, A-07 showed high expression and secretion
of VEGF, IL-8, and bFGF and significant expression of PD-ECGF.
D-12, R-18, and U-25 showed significant expression of VEGF, IL-8,
and bFGF, but the expression of each of these factors was lower than
that in A-07. D-12, R-18, and U-25 differed from A-07 also by
showing no expression of PD-ECGF and no secretion of bFGF. D-12
differed from R-18 and U-25 by showing substantially higher secretion of IL-8. Finally, U-25 differed from R-18 by showing significantly higher secretion of VEGF and IL-8. These observations are
consistent with previous observations suggesting that melanoma cell
lines may differ substantially in the expression of angiogenic factors
(34 –36).
The differences among A-07, D-12, R-18, and U-25 cells in the
expression of VEGF and IL-8 were more pronounced when the
expression was measured by ELISA than when measured by Western
blotting. Western blot analysis gives information on intracellular
protein concentrations, whereas ELISA analysis provides information
on the rate of protein secretion. There is no a priori reason to believe
that the rate of protein secretion should be correlated to the intracellular concentration of protein. In fact, the present study suggests that
the rate of protein secretion cannot be predicted from the intracellular
protein concentration as determined by Western blot analysis. The rate
of protein secretion is probably more relevant for the rate of tumor
neovascularization than is the intracellular concentration of protein,
because angiogenesis is induced by binding of protein to endothelial
cell receptors.
A-07 cells, which evoked the strongest angiogenic response in vivo,
showed substantially higher expression of bFGF than did D-12, R-18,
and U-25 cells. bFGF may promote tumor angiogenesis directly by
acting synergistically with VEGF (42– 44) and indirectly by upregulating the synthesis and secretion of VEGF (45, 46).
The expression of VEGF, IL-8, PD-ECGF, and bFGF of A-07,
D-12, R-18, and U-25 tumors, determined by immunohistochemical
analysis, was in agreement with that of the corresponding cell lines in
vitro, i.e., A-07 tumors showed substantially stronger staining for
VEGF, IL-8, and bFGF than did D-12, R-18, and U-25 tumors and
were the only tumors that stained positive for PD-ECGF, consistent
with the in vitro data in Table 1 and Fig. 1. This observation was not
expected, because A-07, D-12, R-18, and U-25 tumors differ substantially in the fraction of hypoxic cells (47), and hypoxia may
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Fig. 2. Immunohistochemical preparations of A-07 tumors stained with anti-VEGF, anti-IL-8, anti-PD-ECGF, and anti-bFGF by using the avidin-biotin immunoperoxidase method.
The sections were counterstained with hematoxylin. Bars, 50 ␮m.
up-regulate the expression of VEGF (48, 49), IL-8 (19, 50), and
PD-ECGF (51). The expression of VEGF has been shown to be
up-regulated in A-07, D-12, R-18, and U-25 cells exposed to hypoxia
in vitro (16, 52). However, hypoxia-induced up-regulation of VEGF,
IL-8, or PD-ECGF was not detected in the immunohistochemical
analysis. All tissue sections stained homogeneously for VEGF, IL-8,
and PD-ECGF, i.e., the staining was not more intense in regions
adjacent to necrosis than in regions close to functional capillaries,
possibly because VEGF, IL-8, and PD-ECGF were secreted and
diffused from hypoxic tissue into surrounding normoxic tissue. The
staining patterns of VEGF, IL-8, and PD-ECGF differed substantially
from the staining pattern of the hypoxia marker pimonidazole in A-07,
D-12, R-18, and U-25 tumors (53).
The angiogenic factors expressed in vivo were produced primarily
by the parenchymal melanoma cells and were thus of human origin.
The immunohistochemical analysis showed that the angiogenic factors were mainly localized intracellularly in melanoma cells and not in
stromal host cells. Moreover, host cells, i.e., macrophages, leukocytes,
and fibroblasts, have been shown to constitute ⬍1% of the total
number of cells in A-07, D-12, R-18, and U-25 tumors (37). Our
conclusion is also supported by the observation that the expression of
angiogenic factors in vivo was similar to that in vitro.
The angiogenic response after intradermal cell inoculation differed
significantly among the melanoma lines. The sequence of the lines
from high to low angiogenic response, measured as number of tumororiented capillaries per tumor or number of tumor-oriented capillaries
per mm of tumor circumference, was A-07 ⬎ D-12 ⬎ R-18 ⬎ U-25.
The tumor growth rate has also been shown to differ significantly
among the melanoma lines (39). The sequence of the lines from high
to low volumetric growth rate is identical to that for angiogenic
response, i.e., A-07 ⬎ D-12 ⬎ R-18 ⬎ U-25. The close association
between volumetric growth rate and angiogenic response suggests that
the growth of A-07, D-12, R-18, and U-25 tumors is governed
primarily by the rate of angiogenesis.
The angiogenic factors promoting angiogenesis in A-07, D-12,
R-18, and U-25 tumors, identified by measuring tumor-induced angiogenic response in mice treated with neutralizing antibody, differed
among the lines. The angiogenesis of A-07 tumors, which showed the
highest rate of angiogenesis, was promoted by VEGF, IL-8, and
PD-ECGF, as well as bFGF. VEGF and IL-8, but probably not
PD-ECGF or bFGF, promoted angiogenesis in D-12 tumors, which
showed the second highest rate of angiogenesis. The angiogenesis of
R-18 and U-25 tumors was promoted by VEGF but probably not by
IL-8, PD-ECGF, or bFGF. All lines expressed bFGF, whereas A-07
was the only line secreting bFGF, suggesting that promotion of tumor
angiogenesis by bFGF may require bFGF secretion. A-07 and D-12
showed significantly higher secretion of IL-8 than did R-18 and U-25,
indicating that promotion of tumor angiogenesis by IL-8 may require
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Fig. 3. A-07, D-12, R-18, and U-25 cells were harvested from
exponentially growing monolayer cultures and inoculated intradermally in BALB/c nu/nu mice for evocation of angiogenesis and tumor
formation. Angiogenesis was quantified by counting the capillaries in
the dermis oriented toward the tumors at day 7 after the inoculation
of 1.0 ⫻ 106 cells. The plots show the number of tumor-oriented
capillaries/tumor in control mice and in mice treated with neutralizing
antibody against VEGF, IL-8, PD-ECGF, or bFGF. Mean values
(columns) and SEs (bars) of 20 tumors are shown.
high IL-8 concentrations. Our observations suggest that multiple
angiogenic factors are involved in the angiogenesis of highly angiogenic melanomas, whereas the angiogenesis in poorly angiogenic
melanomas may be promoted solely by VEGF. Angiogenic factors
other than those studied here may also be involved in the angiogenesis
of melanoma, including angiogenin (54) and angiopoietin-1 (55).
The angiogenic factors that were found to promote tumor angiogenesis were also found to be essential for tumor metastasis. Lung
colonization and spontaneous metastasis in A-07 were inhibited by
treatment with neutralizing antibody against VEGF, IL-8, PD-ECGF,
or bFGF, as was A-07-induced angiogenesis. Treatment with neutralizing antibody against VEGF or IL-8 inhibited lung colonization and
spontaneous metastasis as well as angiogenesis in D-12, whereas none
of these biological phenomena was inhibited by treatment with neutralizing antibody against PD-ECGF or bFGF. These observations
suggest that spontaneous lung metastasis in A-07 and D-12 tumors is
governed by the angiogenic potential of the tumor cells and hence
controlled by the angiogenic factors controlling the angiogenesis.
The metastatic process is composed of a cascade of linked, sequential, and highly selective steps involving multiple host-tumor interactions. These steps include invasion of tumor cells into blood vessels,
survival in the blood circulation, arrest in the capillary bed of a
secondary organ, extravasation into the secondary organ interstitium
and parenchyma, and tumor cell proliferation and angiogenesis in the
secondary organ. The metastatic propensity of a tumor may be influenced by the angiogenic potential of the tumor cells by two independent mechanisms: high microvessel density in the primary tumor may
enhance the opportunity of tumor cells to gain access to the blood
circulation; and elevated capacity to induce neovascularization may
increase the probability of tumor cells trapped in secondary organ
capillary beds to give rise to macroscopic tumor growth (56). The
probability of spontaneous lung metastasis in A-07 and D-12 tumors
was probably limited by the capacity of the tumor cells to induce
Fig. 4. A-07 and D-12 cells were harvested from exponentially growing monolayer
cultures and inoculated i.v. in BALB/c nu/nu mice for formation of lung colonies. Lung
colonization was quantified by counting the surface colonies at day 35 after the inoculation of 3.0 ⫻ 106 A-07 cells or 3.0 ⫻ 105 D-12 cells. The plots show the number of lung
colonies in control mice and in mice treated with neutralizing antibody against VEGF,
IL-8, PD-ECGF, or bFGF. Mean values (columns) and SEs (bars) of 20 mice are shown.
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Fig. 5. The percentage of BALB/c nu/nu mice that developed spontaneous lung
metastases after intradermal inoculation of 3.5 ⫻ 105 A-07 or D-12 cells. The metastatic
efficiency was assessed in control mice and in mice treated with neutralizing antibody
against VEGF, IL-8, PD-ECGF, or bFGF. Points, single experiments involving 10 mice
each.
angiogenesis in the lungs, because spontaneous lung metastasis and
lung colonization were inhibited by treatment with neutralizing antibody against the same angiogenic factors. However, it cannot be
excluded that the probability of spontaneous lung metastasis was
influenced by the angiogenic potential of the tumor cells also via the
microvessel density of the primary tumor. Interestingly, the microvessel density is ⬃2.5-fold higher in A-07 tumors than in D-12 tumors
(57).
The antibody treatments that inhibited lung colonization and spontaneous metastasis in A-07 and D-12 melanomas lasted 4 and 8 days,
respectively. The reduced lung colonization and spontaneous metastasis in antibody-treated mice were most likely a consequence of
inhibited angiogenesis rather than of cytotoxic or antiproliferative
effects of the antibodies, because the growth of melanoma cells in
vitro was not inhibited by antibody treatment. This interpretation is
based on the assumption that neovascularization and macroscopic
growth of lung colonies can be inhibited by treatment with neutralizing antibodies against angiogenic factors also when the antibody
treatment is given to lung colonies consisting of less than ⬃50 cells,
which is the maximum size of 4-day-old A-07 and D-12 lung colonies.
This assumption is not consistent with the general current thinking
that angiogenesis is initiated when the tumor mass reaches a diameter
of ⬃1 mm. However, the validity of our assumption is strongly
supported by recent observations by Li et al. (58) studying the initial
phases of tumor cell-induced angiogenesis in skin window chambers.
They observed modifications of the host vasculature when the tumor
mass reached 60 – 80 cells and saw functional new blood vessels when
the tumor mass reached 100 –300 cells. Moreover, they found that
ex-flk 1, a truncated soluble VEGF receptor protein known to be
antiangiogenic and nontoxic, prevented angiogenesis and tumor
growth in four of six window chambers when administered as a single
dose at the same time as 40 –50 tumor cells were inoculated into the
window chambers. This observation led the authors to conclude that
individual tumor cells are dependent on early angiogenic activities for
both survival and proliferation in vivo (58).
Previous studies have shown that lung colonization of human
melanoma cells in immune-deficient mice can be inhibited by treatment with neutralizing antibody against VEGF (52) or by treatment
with agents blocking the receptor of IL-8 (59). The present study
confirms these observations and shows further for the first time that
treatment with neutralizing antibody against VEGF or IL-8 can inhibit
spontaneous lung metastasis of human melanoma xenografts. It has
been shown by others that treatment with neutralizing antibody
against VEGF can inhibit spontaneous metastasis of human epidermoid carcinoma xenografts (60), human prostate carcinoma xenografts (61), and human colon and gastric carcinoma xenografts (62),
and treatment with neutralizing antibody against IL-8 can inhibit
spontaneous metastasis of non-small cell lung carcinoma xenografts
(63). Moreover, the study reported here also shows for the first time
that spontaneous lung metastasis of human melanoma xenografts can
be inhibited by treatment with neutralizing antibody against PDECGF or bFGF.
The most important finding reported here is that the angiogenesis
and spontaneous lung metastasis of A-07 melanoma xenografts can be
inhibited by treatment with neutralizing antibody against VEGF, IL-8,
PD-ECGF, or bFGF. Each of these angiogenic factors was probably
essential for the angiogenesis and metastasis of A-07 tumors, because
inhibition of one of them could not be compensated for by the others.
This observation suggests that inhibition of a single angiogenic factor
pathway may be beneficial in the treatment of malignant melanoma.
However, it is unlikely that complete inhibition of tumor growth and
metastasis of highly angiogenic melanomas would be possible by this
strategy, because multiple angiogenic factors are involved. Efficient
antiangiogenic treatment of malignant melanoma may require identification and blocking of common functional features of several angiogenic factors. Treatment strategies that target the common signaling cascades in the end organ of angiogenesis, i.e., the endothelial cell,
may be more universally effective than those targeting specific angiogenic factors or their receptors.
ACKNOWLEDGMENTS
We thank Berit Mathiesen and Kanthi Galappathi for excellent technical
assistance.
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Research.
Vascular Endothelial Growth Factor, Interleukin 8,
Platelet-derived Endothelial Cell Growth Factor, and Basic
Fibroblast Growth Factor Promote Angiogenesis and
Metastasis in Human Melanoma Xenografts
Einar K. Rofstad and Ellen F. Halsør
Cancer Res 2000;60:4932-4938.
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