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Regular Articles |
From the Departments of Medical Oncology,*
Pathology,
and Cell Biology andImmunology,
Vrije Universiteit MedicalCentre, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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VEGF seems to play an important role in the process of angiogenesis in many types of cancer, including ovarian cancer. Expression of VEGF121 and VEGF165 mRNAs has been detected in normal ovaries,12,13 whereas transcripts of VEGF145 and VEGF189 have also been observed in ovarian cancer cell lines.4,14 VEGF mRNA levels can be increased in ovarian cancer tissue of patients.15,16 In addition, high VEGF expression and microvessel density have been correlated with poor disease-free survival and overall survival in patients with early or advanced stage ovarian cancer.15-17 VEGF also seems to be involved in the pathogenesis of ascitic fluid accumulation associated with peritoneal metastases by increasing the vascular permeability of the microvessels in the peritoneal wall.18,19
In addition to its role in tumor growth, VEGF may also be of negative influence on the response of tumors to conventional anti-cancer therapy. Node-positive breast cancer patients seem to have the highest likelihood of favorable outcome after treatment with adjuvant chemotherapy or hormone therapy, when VEGF levels in the primary tumor are low.20 Moreover, high pretreatment serum VEGF concentrations are associated with poor response to combination therapy and unfavorable survival in patients with small-cell lung cancer.21 So far, the exact mechanisms that explain the above-mentioned phenomena are primarily unknown and have hardly been explored in in vivo tumor systems.
We have developed a human ovarian cancer xenograft model in which OVCAR-3 ovarian cancer cells were stably transfected with VEGF165. Here, we report on the histology, the immunohistochemistry, and on the in vitro and in vivo growth properties of parental and weakly, moderately, and highly VEGF165-overexpressing OVCAR-3 xenografts.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Isolation of human umbilical vein endothelial cells (HUVECs) was performed according to the protocol of Van Hinsbergh and colleagues.22 HUVECs were grown in M199 medium (Gibco/Life Technologies, Breda, The Netherlands) supplemented with 10% heat-inactivated fetal calf serum, 10% human serum, 2 mmol/L glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml endothelial cell growth factor. OVCAR-3 human ovarian cancer cells23 and stably transfected derivatives were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco/Life Technologies) supplemented with 10% heat-inactivated fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin.
Plasmid Constructs and Transfection
pCMVVEGF165 was constructed by cloning the KpnI-XbaI fragment of pBSVEGF16524 into the KpnI and XbaI polylinker sites of pcDNA3 (Invitrogen, Groningen, The Netherlands).
OVCAR-3 cells were transfected with pCMVVEGF165 or with the empty pcDNA3 vector by the calcium-phosphate precipitation method.25 After a 6-hour incubation period, the cells were washed, refed with medium, and left for 16 hours. Subsequently, the medium was replaced with medium containing 400 µg/ml of geneticin (G418, Gibco/Life Technologies) to select for geneticin-resistant cells. After 10 days, the concentration of geneticin was increased to 600 µg/ml. Two weeks later, isolated colonies were expanded and characterized. Transfected cell lines were grown in medium containing 300 µg/ml of geneticin for maintenance.
RNase Protection
pBSVEGF165 was linearized with Styl and
161-nucleotide sense and 301-nucleotide anti-sense probes were
generated with T3 and T7 polymerase, respectively. The sense probe
included 106 nucleotides from the VEGF165 cDNA
(nucleotide positions -23 to 83) and 55 nucleotides of vector
sequence. The anti-sense probe included 252 nucleotides from the
VEGF165 cDNA (nucleotide positions +341 to +593)
and 49 nucleotides of vector sequence. In both cases, +1 was the first
nucleotide of the first codon. Because of alternative splicing of VEGF
transcripts, hybridization with the anti-sense probe allowed the
possible detection of fragments of different sizes.
VEGF165 mRNA is expected to protect a fragment of
252 nucleotides, whereas VEGF206 and
VEGF189 mRNAs may protect fragments of 82
nucleotides and 170 nucleotides. Furthermore,
VEGF145 and VEGF121 mRNAs
may give rise to protected fragments of 82 nucleotides and 38
nucleotides as shown in Figure 1
.3,4
An anti-sense
-actin probe was used as an internal control and has been described
elsewhere.26
RNase protection assays were performed as
described.27
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Conditioned media of OVCAR-3 cells and stably transfected derivatives for ELISA and Western blotting were obtained by seeding 2 x 106 cells in a 96-mm culture dish. After 16 hours, the medium was replaced by serum-free medium. Seventy-two hours later, the conditioned media were collected and the cells were trypsinized and counted.
ELISA was performed using the reagents and protocol supplied with the Quantikine Human VEGF Immunoassay kit (R&D Systems/ITK Diagnostics, Uithoorn, The Netherlands). The ELISA data were normalized for the number of cells counted at the time the conditioned media were collected.
For Western blot analysis, 2 ml of the conditioned media were incubated overnight at 4°C with 20 µl of heparin-agarose beads (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands). Protein-heparin complexes were washed, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride-membrane (Immobilon; Millipore, Etten-Leur, The Netherlands). The membrane was blocked for 1 hour in TBST (10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, and 0.025% Tween-20)/5% milk and incubated overnight at 4°C with rabbit polyclonal human VEGF-directed antiserum (1:1000 dilution, catalog no. sc-152; Sanver Tech/Santa Cruz Biotechnology, Heerhugowaard, The Netherlands) in the same solution. After washing with TBST, the membrane was incubated for 1 hour at room temperature with horseradish peroxidase-conjugated pork anti-rabbit antiserum (DAKO/ITK Diagnostics) in TBST/5% milk. The membranes were washed again with TBST and VEGF proteins were visualized by electro-chemiluminescence.
HUVEC Proliferation Assay
Conditioned media of OVCAR-3 cells and transfected derivatives were obtained as described above. Three thousand HUVECs per well were plated in 100 µl of medium in 96-well tissue culture plates. After 24 hours, 100 µl of nondiluted and 2, 4, 8, 16, and 32 times in serum-free medium diluted samples of the different conditioned media were added in three replicate wells. Seventy-two hours later, the number of living cells was estimated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,6-dimethyl-morpholino)-2,5-diphenyl-tetrazolium bromide] viability assay.28
For the proliferation-inhibition assays, 100 µl of the conditioned media samples of the VEGF-overexpressing cell lines inducing maximal proliferation of HUVECs (clone 19, nondiluted; clone 26, 16 times diluted; clone 31, 4 times diluted) was added to HUVECs in the absence or presence of 1 µg/ml of normal goat serum or 1 µg/ml of a neutralizing goat polyclonal human VEGF165-directed antiserum in duplicate wells. As a control, 100 µl of serum-free Dulbecco’s modified Eagle’s medium with or without 40 ng/ml of recombinant VEGF or 20 ng/ml of recombinant basic fibroblast growth factor (bFGF) was also tested on HUVECs in the absence or presence of 1 µg/ml of normal goat serum or 1 µg/ml of a neutralizing goat polyclonal human VEGF165-directed antiserum in duplicate wells. Recombinant VEGF, bFGF, normal goat serum, and the VEGF165-directed antiserum were all purchased from R&D Systems/ITK Diagnostics.
Cell-Cycle Distribution
Exponentionally growing OVCAR-3 cells and transfected derivatives were washed twice with phosphate-buffered saline (PBS) and were detached from the culture flasks by incubation with PBS/0.2% ethylenediaminetetraacetic acid. Cells were collected, centrifuged, and resuspended in 1 ml of 70% ethanol. After centrifugation and removal of the ethanol, the cells were resuspended in PBS and centrifuged again. Subsequently, the cells were resuspended in 30 µl of PBS and 200 µl of RNase A (250 µg/ml in 0.1% Triton X-100) was added. After a 20-minute incubation period at room temperature, 200 µl of propidium iodide (100 µg/ml) was added and the samples were incubated for another 20 minutes at 4°C in the dark. Cells were analyzed by using a FACScan flow cytometer. Results were expressed as the percentage of cells of each sample in the G0/G1, S, and G2/M phase.
In Vitro Cell Growth
For each cell line a standard curve was obtained by plating
100,000, 50,000, 25,000, 12,500, 6250, 3125, 1562, and 781 cells per
well in 100 µl of medium in three replicate wells in a 96-well
microtiter plate. After 3 hours, the MTT assay was
performed.28
Mean absorbances measured were plotted
against the number of cells using regression analysis. In addition,
5000 cells per well of each cell line were plated in 12 replicate wells
of 96-well microtiter plates. These cells were grown for 24, 48, 72,
and 96 hours, after which the MTT assay was performed. Mean
absorbances measured at each time point were related to the cell number
from the standard curves. Doubling times were calculated by the
following formula: Nt =
Nte.e
.t where is the
growth constant and Nt and
Nte are the number of cells on the last day and
the first day that the cells are in exponential growth phase,
respectively. Except from the first day after plating, the cells were
growing exponentially during the whole test period.
Xenografts
Female nude mice (Hsd, athymic nude-nu) were purchased at the age of 6 weeks (Harlan, Horst, The Netherlands). The animals were maintained in cages with paper filter covers under controlled atmospheric conditions. Cages, bedding, food, and water were changed and sterilized weekly. The OVCAR-3 human ovarian cancer xenograft is a poorly differentiated serous adenocarcinoma and has been described previously.29 VEGF165-overexpressing OVCAR-3 xenografts were established as follows: VEGF165-overexpressing OVCAR-3 cells were grown for 2 days in medium without G418 and were in the exponential growth phase at the time of harvesting. Cells were harvested by brief trypsinization, washed twice in PBS, and resuspended in PBS. The presence of single cells was confirmed by light microscopy and cell viability was confirmed by trypan blue exclusion. Cells ( 0.5 x 107) were injected subcutaneously in both flanks of the mice. The solid tumors arising at the inoculation site (passage 1) were transferred as tissue fragments with a diameter of 2 to 3 mm through a small skin incision into both flanks of 8- to 10-week-old mice.
Immunohistochemistry
Indirect immunoperoxidase staining was performed on 4-µm frozen xenograft tissue sections stored at -70°C. On thawing, sections were fixed with acetone (ice-cold), incubated for 10 minutes at 4°C, and air-dried. Sections were preincubated for 15 minutes at room temperature with 10% normal serum of the animal species from which the primary antibody, as used in the next step, was obtained. The serum was decanted and the sections were incubated for 60 minutes at room temperature with PBS/0.2% bovine serum albumin (negative control), a rabbit polyclonal human VEGF-directed antiserum (1:40 dilution, catalog no. sc-152; Santa Cruz Biotechnology), a goat polyclonal antiserum directed against the endothelial cell marker CD31 (1:40 dilution, catalog no. SC-1506; Santa Cruz Biotechnology), or rat monoclonal antibodies directed against monocytes and macrophages (MOMA-2, 1:10 dilution and F4/80 1:1 dilution, catalogue nos. MCA519 and MCA497, respectively; Serotec, UK). In all staining series, appropriate negative control antisera were used, which were irrelevant same species polyclonal antisera for VEGF and CD31 and an irrelevant isotype (IgG2b) rat monoclonal antibody for MOMA-2 and F4/80. Subsequently, the sections were washed three times for 5 minutes at room temperature with PBS. These washing conditions were repeated after all incubation periods thereafter. To inactivate endogenous peroxidase activity, sections were incubated for 10 minutes at room temperature with methanol/0.006% H2O2. Incubations with biotin rabbit anti-goat serum, biotin goat anti-rabbit serum, and biotin rabbit anti-rat streptavidin-horseradish peroxidase conjugate and substrate mixture were performed as described by the manufacturer’s protocol (Zymed/Sanbio, Uden, The Netherlands). Counterstaining was performed by incubation of the sections with hematoxylin for 5 minutes at room temperature followed by extensive rinsing with tap water. The sections were enclosed by a coverslip and a drop of aquamount.
Description of histopathological features and scoring of immunohistochemistry was performed by an experienced pathologist in a blinded manner in two to three separate tumors of parental OVCAR-3 xenografts and xenografts from transfected derivatives (size <500 mm3). Histopathology of xenograft tissue included description of tissue compactness, cyst formation, and the presence of dilated capillaries. Intensity of VEGF staining was semiquantitatively expressed as negative (-), weakly positive (+), positive (++), or strongly positive (+++). Vascular density of the CD31-stained slides was difficult to score because of vascular rim formation around cysts and tumor cell islands, and was therefore limited to noting the presence of vascular rimming (as previously described by Guidi et al30 ) and dilated capillaries. The number of tumor-associated monocytes/macrophages was semiquantitatively expressed as low (+), moderate (++), or high (+++).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Expression of VEGF165 and
VEGF121 mRNAs in the human ovarian cancer cell
line OVCAR-3 has been demonstrated previously.12
We
further examined the expression of VEGF mRNAs in the OVCAR-3 cell line
and in subcutaneous OVCAR-3 tumor tissue by the RNase protection assay.
Hybridization of total RNA to the VEGF165
anti-sense probe resulted in the protection of a 252-nucleotide
fragment and confirmed the expression of VEGF165
mRNA in the OVCAR-3 cell line and in OVCAR-3 tumor tissue (Figure 1
and
Figure 2A
, lanes 5 and 7). Fragments of
170 nucleotides and 82 nucleotides were also detected, which may be
because of protection by mRNAs of
VEGF206/VEGF189 and
VEGF145/VEGF121,
respectively (Figure 1
and Figure 2A
, lanes 5 and 7). Another protected
fragment of 
78 nucleotides was also observed. This fragment was most
likely also protected by mRNAs of
VEGF145/VEGF121, but the
size of this fragment could not be explained on a theoretical basis.
All of the protected fragments were not detectable when the
VEGF165 sense control probe was used (Figure 2A
,
lanes 4 and 6) and were not observed after hybridization of the VEGF
anti-sense probe with control tRNA (Figure 2A
, lane 9). These data are
indicative for mRNA expression of human VEGF165
and of longer and shorter human VEGF isoforms in the OVCAR-3 cell line
and in OVCAR-3 tumor tissue.
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OVCAR-3 cells were stably transfected with
pCMVVEGF165 and with the empty control vector.
G418-resistant clones from each transfection were analyzed for
VEGF165 expression by the RNase protection assay.
It should be noted that the pCMVVEGF165 was
cloned by insertion of a VEGF165 cDNA fragment
isolated from pBSVEGF165. The
VEGF165 anti-sense RNA probe was transcribed from
linearized pBSVEGF165. Because of the presence of
complementary pBS vector sequences, the fragment protected by
overexpressed VEGF165 mRNAs is expected to be 11
nucleotides longer than that protected by endogenous
VEGF165 mRNA. Figure 2B
shows the results of a
subset of the transfected clones analyzed. The intensity of the
252-nucleotide fragment relative to the -actin-protected fragment
was similar in all cell lines, indicating that the endogenous
VEGF165 mRNA levels in the parental cell line and
the stably transfected sublines were comparable (lanes 3 to 8). A
protected fragment of 263 nucleotides was indeed present in the case of
VEGF165-transfected clones 19, 26, and 31 (lanes
6 to 8), but not in the case of the parental cells (lane 3) or the
control-transfected clones (lanes 4 and 5). As judged by the intensity
of the 263-nucleotide fragments, overexpression of
VEGF165 mRNA was weak in clone 19, moderate in
clone 31, and high in clone 26.
Secretion of VEGF165 Protein
The parental OVCAR-3 cells and the subset of the control- and
VEGF165-transfected clones were analyzed for the
secretion of VEGF165 protein by Western blotting.
As can be seen in Figure 3
, only a short
exposure time of the Western blot was required to detect the
glycosylated (23 kd) and nonglycosylated (21 kd) monomeric forms of the
VEGF165 protein31
isolated from the
conditioned media of the VEGF165-transfected
clones 26 and 31 (top panel, lanes 5 and 6). Endogenous
VEGF165 protein secreted by the parental cells
and control-transfected cells and overexpressed
VEGF165 protein produced by
VEGF165-transfected clone 19 were only detectable
after long exposure (bottom panel, lanes 1 to 4).
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shows that the
VEGF165-overexpressing clones 19, 26, and 31
secreted 50, 225, and 5000 times more
VEGF165 than the parental cell line. The
control-transfected clones 7 and 15 also secreted slightly more
VEGF165, but it is questionable if this small
increase is significant. In summary, these data demonstrated that the
VEGF165-transfected clones 19, 26, and 31
overexpressed VEGF165 protein of the correct
size.
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To assess whether the overexpressed VEGF165
proteins were functional, we compared the potency of different diluted
samples of conditioned media from the parental cell line, the
control-transfected clones, and the
VEGF165-overexpressing clones in a HUVEC
proliferation assay (Figure 4)
. Although
the parental cells and the control-transfected cells secreted
VEGF165 protein (Figure 3
, Table 1
), the amount
was not sufficient to induce proliferation of HUVECs (Figure 4A)
. In
contrast, stimulation of proliferation was observed by the conditioned
media of the VEGF165-overexpressing clones 19,
26, and 31 (Figure 4A)
. Maximal stimulation was induced with 12, 4, and
2 times diluted samples of clones 26, 31, and 19, respectively.
Clearly, the media of clone 26 was most potent, followed by that of
clones 31 and 19, which was likely related to the amount of secreted
VEGF165 protein. Complete inhibition of
proliferation occurred by addition of a VEGF-neutralizing antiserum,
but not by addition of a control serum (Figure 4B)
. This was not
because of a general toxic effect of the VEGF-neutralizing
antiserum, because this antiserum did not inhibit proliferation induced
by the potent angiogenic factor bFGF (Figure 4C)
. These data
demonstrated that induction of HUVEC proliferation by the media of
clones 19, 26, and 31 was dependent on the presence of overexpressed
VEGF165 protein and confirmed the biological
activity of these proteins.
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To examine whether overexpression of VEGF165
affected the growth of OVCAR-3 cells in vitro, we determined
the doubling times and the cell-cycle distribution of the parental
OVCAR-3 cells, the control-transfected cells, and the
VEGF165-overexpressing cells. As can be seen in
Table 2
, the doubling times of the
different cell lines were comparable and no major differences were
observed between the percentages of cells in
G0/G1, S, and
G2/M phase. From these results we concluded that
VEGF165 overexpression had no influence on the
growth rate of OVCAR-3 cells in vitro.
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To analyze whether VEGF165 overexpression
had an effect on the angiogenic phenotype of OVCAR-3 xenografts when
grown subcutaneously in nude mice, we visualized the vasculature of
highly, moderately, and weakly
VEGF165-overexpressing tumors by
immunohistochemical staining. Semiquantitative scoring of VEGF
expression and the presence of
CD31-positive microvessels is presented in Table 3
. Figure 5A
shows the CD31 staining of the
vasculature of the parental OVCAR-3 xenografts. The constitutive
expression of VEGF in tumor cells and a relatively intense staining of
VEGF in the tumor stroma are demonstrated in Figure 5B
. Appropriate
control antisera confirmed specific staining for VEGF and CD31.
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. Some dilated capillaries were
detected. The structure of the xenografts appeared to be less compact
than that of the parental xenografts. VEGF staining of the tumor cells
was also slightly more intense than that of the parental xenografts
(Figure 5D)
.
The moderately and highly VEGF165-overexpressing
xenografts (derived from clones 31 and 26, respectively) had a less
compact structure than the parental and the weakly
VEGF165-overexpressing xenografts (Figure 5
,
compare E and G with A and C) and contained round to oval cysts as
often seen in human serous papillary ovarian adenocarcinomas. These
cysts were lined with tumor cells, sometimes forming papillary
structures into the lumina. The cysts in xenografts <500
mm3
varied in size of 1 to 10 mm and were
separated by varying amounts of stroma. Around the cysts within the
stroma there was extensive rimming of CD31-positive capillary
structures (Figure 5, E and G)
. Some of these capillary structures were
larger in diameter than those detected in the parental xenografts. As
expected, VEGF was highly expressed in the tumor cells that lined the
cysts and in areas of solid tumor tissue (Figure 5, F and H)
. On growth
of the xenografts >500 mm3
the cysts gradually
increased in size becoming macroscopically visible and containing
hemorrhagic fluid.
These data demonstrated that VEGF165 overexpression induces an increase of the angiogenic response in OVCAR-3 xenografts and that VEGF165 expression levels may be rate-limiting in the normal process of angiogenesis in these xenografts. In addition, these results are indicative for a role of VEGF165 in cyst formation.
Immunohistochemistry for Monocytes and Macrophages
On more detailed examination of the histology of the parental and VEGF165-overexpressing OVCAR-3 tumors, it was observed that a significant number of small mononuclear cells was present at the periphery and in the stroma of the tumors. These cells were clearly smaller in size than the tumor cells and were nonendothelial because no staining was observed with the CD31-specific antiserum. Staining with an antiserum directed against the cell marker CD45 confirmed the hematopoietic origin of the cells (data not shown). To further identify the mononuclear cells, stainings were performed with antibodies directed against granulocytes and B lymphocytes. Although staining of a limited number of granulocytes and B lymphocytes was detected in the tumor stroma of both parental and VEGF165-overexpressing xenografts, no staining was observed of the small mononuclear cells (data not shown).
As tumors are known to be often infiltrated by monocytes and
macrophages, we performed stainings with two different antibodies
recognizing both monocytes and macrophages (MOMA-2 and F4/80). A
similar staining pattern was observed for both antibodies identifying
the small mononuclear cells as cells of the monocyte/macrophage
lineage. Results obtained with the MOMA-2 antibody are shown in Figure 6
. Semiquantitative scoring of the number
of monocytes/macrophages is presented in Table 3
. The control rat
monoclonal antisera of the IgG2b isotype indicated the specificity of
MOMA-2 and F4/80 staining. Areas of monocytes/macrophages were detected
at the periphery of parental and
VEGF165-overexpressing OVCAR-3 xenografts. In
parental xenografts, a relatively small number of infiltrated
monocytes/macrophages was also detected in the stroma between tumor
islets (Figure 6A)
. A similar pattern of monocyte/macrophage staining
was observed in weakly VEGF165-overexpressing
xenografts (Figure 6B)
.
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. The overall staining of monocytes and macrophages in the former
xenografts was much more pronounced and large areas of
monocyte/macrophage infiltrates were not only observed at the tumor
periphery, but also within the tumor stroma. Infiltrating monocytes and
macrophages were particularly observed in areas where CD31 staining was
relatively intense. Note that in all xenografts, monocytes and/or
macrophages were also stained with the VEGF-directed antiserum (Figure 5; B, D, F, and H
). These data suggest that
VEGF165 overexpression stimulates the
infiltration of monocytes and macrophages in OVCAR-3 xenografts. Growth Rates of VEGF165-Overexpressing OVCAR-3 Xenografts
The higher number of CD31-positive cells would suggest increased vascularization in the VEGF165-overexpressing xenografts. This did not result, however, in increased tumor growth when compared to the growth of parental xenografts. The mean volume doubling time (±SEM) of parental tumors was 7.9 ± 2.1 days, whereas that of the moderately and highly VEGF165-overexpressing tumors was 12.9 ± 1.4 days and 7.5 ± 1.5 days, respectively. Weakly VEGF165-overexpressing tumors grew slightly slower than the parental tumors with a mean volume doubling time of 8.8 ± 0.7 days. These data suggest that additional parameters, such as monocyte/macrophage content or cyst formation, determine the tumor growth rate of VEGF165-overexpressing xenografts.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The histological pattern of the VEGF165-overexpressing tumors was different from that of the parental tumors. All VEGF165-overexpressing tumors contained an increased number of capillaries, some of which were wider in diameter than those observed in the parental tumors. In contrast to the weakly VEGF165-overexpressing tumors, the moderately and highly overexpressing VEGF165-overexpressing tumors also contained large cysts that were lined with tumor cells and surrounded by CD31-positive cells. The formation of cysts in the moderately and highly VEGF165-overexpressing OVCAR-3 xenografts is intriguing because cysts are often present in human serous papillary ovarian adenocarcinomas. In fact, high levels of VEGF protein have been measured in ovarian cyst fluid and were shown to be associated with ovarian malignancy.32 It has been suggested previously that VEGF may contribute to the accumulation of cyst fluid in ovarian cancer by increasing the permeability of the tumor vasculature.32 High concentrations of VEGF have also been measured in fluid of other types of tumors.33 Our study provides functional evidence that VEGF165 indeed plays a role in the formation of cysts.
Another interesting finding in this study is that the moderately and highly VEGF165-overexpressing tumors had a much higher content of monocytes and macrophages than parental and weakly VEGF165-overexpressing tumors. Although numerous monocytes and macrophages were detected at the periphery of all tumors, the intratumoral density of these cells was significantly higher in moderately and highly VEGF165-overexpressing tumors. These data show an interesting parallel with clinical studies of Leek and colleagues34,35 demonstrating a positive correlation between macrophage infiltration and VEGF expression levels or the vascular grade in breast cancer. As VEGF is reported to be a chemotactic agent for monocytes,9 it is interesting to speculate that high VEGF overexpression by tumor cells directly leads to an increased recruitment of these cells from the peripheral circulation. VEGF165 overexpression in OVCAR-3 tumor cells may also indirectly stimulate macrophage/monocyte infiltration through induction of the expression of other chemoattractants. An example is macrophage chemoattractant protein (MCP)-1, which can be induced in endothelial cells on VEGF treatment.36 In this respect, it is interesting to mention that MCP-1 concentrations have been found to be positively correlated with VEGF expression levels and macrophage accumulation in primary breast cancer.37
The growth rate of the highly VEGF165-overexpressing tumors was comparable with that of the parental tumors, whereas weakly and moderately VEGF165-overexpressing xenografts grew slower. This is in contrast to other cancer xenograft models in which an increase in vascularization because of VEGF165 overexpression resulted in accelerated tumor growth.38-40 It should be noted that tumor growth was measured by volume measurements. Moderately and highly VEGF165-overexpressing xenografts developed large cysts on growth and had a less compact structure. As mentioned above, the intratumoral density of monocytes/macrophages was also considerably increased. Hence, the absolute number of the tumor cells in these xenografts was lower, suggesting that the proliferation rate of these cells was reduced.
The in vitro growth rate and cell-cycle distribution of VEGF165-overexpressing and parental OVCAR-3 cells was comparable. Interestingly, VEGF has been demonstrated to act as a growth factor and as a survival factor for some tumor cell types expressing KDR or flt-1 receptors in vitro.10,11 We have also detected KDR mRNA expression in OVCAR-3 parental and VEGF165-overexpressing cell lines and the respective xenografts by reverse transcriptase-polymerase chain reaction (data not shown). The fact that VEGF165 overexpression has no effect on OVCAR-3 cell growth in vitro, does not exclude a role for KDR receptor signaling in this process. Autocrine stimulation of KDR receptor activity by VEGF165 overexpression may influence the expression levels of other angiogenic regulators in OVCAR-3 cells. Changes in the levels of these regulators may not be manifested in altered growth properties of OVCAR-3 cells in vitro, but may contribute to the process of angiogenesis and thereby influence tumor growth in vivo. Alternatively, maximal activation of KDR receptors may already be induced by endogenous levels of VEGF165 protein. To further explore the role of the KDR receptor in OVCAR-3 cells and OVCAR-3 xenografts, we are currently performing experiments that are aimed at inhibition of KDR receptor activity.
The role of monocytes and macrophages in the growth and angiogenesis in OVCAR-3 xenografts is unclear. Tumor-associated monocytes/macrophages can both negatively and positively regulate tumor growth.41 On the one hand, these cells can negatively influence tumor growth by eliciting a cytostatic effect against tumor cells. On the other hand, they are believed to contribute to angiogenesis and tumor growth by producing angiogenic growth factors on activation within the tumor environment.42 One such a factor is VEGF, which is expressed in activated, tumor-associated macrophages.43 The latter hypothesis is supported by clinical studies in breast cancer and endometrial cancer in which macrophage infiltration has been reported to be positively correlated with angiogenesis and/or reduced survival.34,44 So far, the effect of monocytes and macrophages on tumor growth and angiogenesis has not been extensively studied in in vivo model systems. In a human melanoma xenograft model, however, it was described that overexpression of interleukin-10 led to a decrease in tumor growth rate and angiogenesis, presumably by inhibiting angiogenic cytokine production in macrophages.45
In the VEGF165-overexpressing OVCAR-3 xenograft models we present here, there is no clear relationship between monocyte/macrophage content and tumor growth. Although the density of infiltrated monocytes/macrophages was strongly increased within the tumor stroma of highly and moderately VEGF165-overexpressing OVCAR-3 xenografts, this did not result in an increase in the tumor growth rate. In fact, the number of tumor cells in these xenografts was even reduced (see above). A possible explanation may be that the tumoricidal capacity of the infiltrating macrophages dominates their tumor growth-promoting effects in these xenografts. To further analyze the contribution of macrophages to tumor growth and angiogenesis in parental and weakly VEGF165-overexpressing tumors, it would be interesting to assess effects of agents that inhibit macrophage function.45,46
The xenograft model we have presented is an excellent tool to further determine the function of VEGF in monocyte/macrophage infiltration and offers the possibility to study whether VEGF165 expression levels or/and the degree of vascularization influences the efficacy of anticancer agents.
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Acknowledgements |
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Footnotes |
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Supported by the "Spinoza grant," awarded to Prof. H. M. Pinedo in 1997.
Accepted for publication October 26, 2001.
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References |
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