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Molecular Oncology, Markers, Clinical Correlates |
Division of Gastroenterology and Hepatology, Department of Internal Medicine [Y. K., T. C.] and Department of Surgery and Surgical Basic Science [Y. K., S. A., A. M., M. F., M. I.], Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
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ABSTRACT |
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INTRODUCTION |
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Recently, several studies suggested that VEGF is the angiogenic factor associated most closely with induction and maintenance of the neovasculature in human tumors (7 , 17, 18, 19) , and clinical studies have implicated VEGF expression in tumor progression and metastasis. Furthermore, using a murine model system for colon cancer, Warren et al. (20) demonstrated that a monoclonal antibody against VEGF inhibits s.c. tumor formation in a dose- and time-dependent manner and reduces the number and size of liver metastases. There have also been a few studies focusing on the suppression of tumor growth using VEGF antisense or c-src antisense expression vectors (20, 21, 22, 23) . These studies have thus been aimed at blocking VEGF, and they have resulted in the fairly general agreement that VEGF is one of the molecules that can be targeted to stop tumor growth and metastasis (24, 25, 26, 27, 28) .
To our knowledge, however, there have been no studies that attempted to analyze the changes in malignant potential induced by high expression of VEGF. Considering this background, the current study was designed to investigate the effect of high constitutive expression of VEGF on tumor growth and metastasis, thereby establishing the role of VEGF in colon cancer, based on our previous reports that VEGF mRNA expression was closely correlated with highly malignant potential in the human colon cancer.
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MATERIALS AND METHODS |
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Subcloning of VEGF121 into pCAG-BSD and DNA
Transfection.
The full-length cDNA for VEGF121 was made from
reverse transcription-PCR products of human colon cancer specimens. We
used the following PCR primers, which were based on the human VEGF cDNA
sequence: (a) sense primer, 5'-CCTCCGAAACCATGAACTTT-3'; and
(b) antisense primer, 5'-AGAGATCTGGTTCCCGAAAC-3'. The human
VEGF121 cDNA was subcloned into pBluescript II
SK(-), and we confirmed the sequence using an ALF red DNA
Sequencer (Pharmacia Biotech, Uppsala, Sweden). A eukaryotic expression
vector, pCAG-BSD, was newly constructed with two parts of pCAGGS (a
kind gift from Dr. J. Miyazaki, Osaka University, Osaka, Japan;
Ref. 29
) and pMAM2-BSD (Kaken, Tokyo, Japan). The human
VEGF121 cDNA was cloned into the XhoI
restriction enzyme site of pCAG-BSD to create the plasmid
pCAG-BSD-VEGF, in which the transcription of VEGF was constitutively
driven by the CAG enhancer promoter, and the drug-resistant selection
gene BSD was present (30)
. The orientation of the VEGF
insert was confirmed by restriction mapping.
The cloned wild-type LoVo cells were cultured in 6-well plates. DNA transfections were performed using 4 µl of LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) and 1 µg of pCAG-BSD-VEGF or 1 µg of pCAG-BSD vector alone (as a control) per well. The cells were cultured in 1 ml of serum-free OPTI-MEM (Life Technologies, Inc.) medium containing the DNA/LipofectAMINE complex for 4 h and then supplemented with 1 ml of RPMI 1640 containing 20% FBS. After 48 h, the cells in each well were trypsinized and replated 1:20 into two 10-cm culture dishes in complete culture medium containing 10 µg/ml blasticidin S (Kaken) to select transfectants. Some cell death was observed after 3–4 days in culture, and discrete colonies were apparent by 7 days after selection. Twenty-two individual colonies of sense VEGF transfectants and 10 individual colonies of vector transfectants were picked and pooled, and cells were maintained in complete culture medium with 10 µg/ml BSD to obtain stable transfectants. These transfectants were individual clones.
RNA Isolation and Northern Blot Analysis.
All transfectants were grown to confluence in RPMI 1640 in
10-cm2 Petri dishes, and total cellular RNA was
extracted using Trizol regent (Life Technologies, Inc.). RNA samples
were resolved by electrophoresis through 1% agarose-formaldehyde gels
and transferred to Hybond-N+ nylon membrane
filters (Amersham, Buckinghamshire, United Kingdom). The probes were
labeled with [
-32P]deoxycytidine
5'-triphosphate using the Megaprime Random Primer DNA Labeling Kit
(Amersham). Hybridization was performed at 65°C for 2 h in Rapid
Hybridization Solution (Amersham) with a labeled probe. After
hybridization, the filters were washed twice with 2x SSC (1x SSC = 0.15 M NaCl plus 0.015 M sodium citrate) and
0.1% SDS at room temperature for 10 min, washed once with 1x SSC and
0.1% SDS at 65°C for 15 min, and then washed twice with 0.1x SSC
and 0.1% SDS at 65°C for 15 min. Autoradiographs were made using
X-ray film and an imaging plate and analyzed with a BAS 2000 Image
Analyzer (Fuji, Tokyo, Japan). The membranes were rehybridized with a
human glyceraldehyde-3- phosphate dehydrogenase probe.
Western Blot Analysis.
Nearly confluent cells were washed twice with PBS and replenished with
serum-free medium. After 24 h, the medium was collected,
centrifuged for 10 min at 3000 rpm to remove cell debris, and
concentrated with acetonic sulfate. The proteins were separated by
electrophoresis on 12% polyacrylamide gels and transferred to
Immobilon-P membranes (Millipore Corp., Bedford, MA) by
electrotransfer. After blocking with 5% milk in 0.1% Tween 20 in PBS
solution, the membrane-bound proteins were probed with the primary
antibody (1:5000 dilution of polyclonal rabbit antihuman VEGF; Santa
Cruz Biochemicals, Santa Cruz, CA). The membrane was washed, and the
secondary antibody labeled with horseradish peroxidase (1:2000 dilution
of goat antirabbit immunoglobulin; Zymed) was applied. After
washing, the bound antibody complexes were detected using an enhanced
chemiluminescence reagent (Amersham) and X-ray film, as described by
the manufacturers.
Determination of in Vitro Cell Proliferation.
Into each well of a 6-well plate, 1 x 105
stably transfected LoVo cells were seeded in RPMI 1640–10% FBS
containing 10 µg/ml blasticidin S. Cells were fed with fresh medium
every other day. On the days indicated, cells were released by exposure
to trypsin, pelleted, resuspended in the same medium, and counted using
a hemocytometer.
Detection of Neovascularization with Dorsal Air Sac Method.
The dorsal air sac method was used as described previously
(28)
. Sense transfectant (S17) and vector transfectant
(V7) cells were washed once with PBS and suspended in PBS at a
concentration of 2 x 106 cells/0.2 µl. A
Millipore chamber (diameter, 10 mm; filter pore size, 0.45 µm) was
filled with 0.2 ml of the cells and implanted s.c. into the dorsal side
of the mice. At 5 days after implantation, the mice were anesthetized
and fixed in the prone position. A wide, rectangular incision was made
in the skin on the dorsal side, and the skin was carefully ablated. To
locate the chamber-contacting region, a ring (Millipore) of the same
shape as the chamber was placed onto the s.c. tissues adjacent to the
chamber region, and the area was photographed. For histological
analysis, all samples were taken from this skin area, embedded in 10%
formaldehyde, and sliced into 4-µm-thick vertical sections, which
were then stained with H&E.
s.c. Tumors.
Confluent cultures of sense VEGF-transfected LoVo cells (S17) and
vector-transfected LoVo cells (V7) grown in
10-cm2 Petri dishes were harvested by a brief
trypsinization (0.05% trypsin/0.02% EDTA in
Ca2+/Mg2+-free PBS), washed
several times in
Ca2+/Mg2+-free PBS, and
resuspended at a final concentration of 3 x
106 cells/0.2 ml in PBS. Athymic BALB/c nude mice
(4-week-old males obtained from Charles River, Yokohama, Japan) were
housed in sterilized cages and injected s.c. with 3 x
106 viable tumor cells. Animals were observed
daily for tumor growth, and s.c. tumors were measured every 7 days.
Tumor volume was calculated as 1/2 x length x
width2 (length > width).
Liver Metastases.
Sense VEGF transfectants (S17) and vector transfectants (V7) were grown
to confluence and harvested as described above for s.c. injection and
resuspended in PBS at a concentration of 2 x
106 cells/0.2 ml. Athymic mice were anesthetized
by ether inhalation and wiped with antiseptic solution, and the spleen
was exteriorized through a left-flank incision. Two million cells were
slowly injected into the splenic pulp through a 30-gauge needle. All
animals were killed on day 28. The spleens and livers were excised, and
the metastases were checked by H&E staining.
i.p. Injection.
A single-cell suspension of 2 x 106 cells
with a viability of >95% was injected into the peritoneal cavity of
the mice. The mice were monitored daily for evidence of disease
(abdominal swelling, hunched posture, and listlessness) and were killed
when moribund or approximately 42 days after the i.p. injection. All
mice were necropsied, and the pattern (discrete solid lesions,
carcinomatosis) and extent of abdominal disease (size and number of
lesions, volume of ascites) were determined.
Orthotopic Implantation to the Cecum.
Athymic BALB/c nude mice (4–5-week-old males) were anesthetized by
ether inhalation, and the abdomen was prepared for sterile surgery. A
small abdominal incision was made, and the cecum was identified. Tissue
blocks of about 3 mm in diameter of each s.c. tumor were implanted to
the serosal side. The abdomen was closed with continuous nylon sutures.
The animals were killed 12 weeks later, and abdominal organs and the
thorax were examined for the presence of macroscopic "primary"
cecal tumors and metastasis. Organs including the cecum, liver,
mesenteric lymph nodes, and lungs were removed and examined
histologically as described below.
Histological Examination of Metastases.
Specimens for histological examination were fixed in 4%
paraformaldehyde for 24 h. Representative sections of cecum, lymph
nodes, lung, and liver were also cut and embedded in paraffin.
Four-µm sections were then cut and stained with H&E.
Microvessel Staining and Counting.
The streptavidin-biotin-peroxidase complex method was performed using a
streptavidin-biotin kit. Antihuman von Willebrand factor (factor
VIII) rabbit serum (DAKO, Copenhagen, Denmark) was used at a dilution
of about 1:100 in BSA. As negative controls for immunohistochemical
staining, tissue sections were treated with normal rabbit serum instead
of primary antibodies. Microvessel density was assessed in tumor areas
showing the highest density of staining, as determined by an initial
scan with low magnification (x40). For vessel counting, one field
magnified 200-fold (high-power field, i.e., x20 objective
and x10 ocular, 0.739 per field) in each of three vascularized areas
was counted, and the average counts were recorded (31)
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Statistical Analysis.
Microvessel densities in the s.c. tumors and cecal primary tumors were
compared by the Wilcoxon-Mann-Whitney test. The statistical
analysis was done using JMP 3.1 software (SAS Institute, Cary,
NC), and a P value of less than 0.05 based on a
two-tailed test was considered to indicate statistical significance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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revealed a high
level of VEGF mRNA expression in sense VEGF-transfected LoVo (S17)
cells and very low levels of expression in two cell lines, parent LoVo
cells and vector-transfected LoVo (V7 cells). Two cell lines, S17 and
V7, were chosen for further study. Western blot analysis revealed the
presence of VEGF protein only in the conditioned medium of S17 cells
(Fig. 1B)
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. On the fifth
day after implantation of the S17 cells, the blood vessels had expanded
and branched out from the large blood vessels. In some of the mice
injected with S17 cells, severe hemorrhaging from these vessels was
observed. Fig. 3
shows representative
views of H&E staining of skin sections 5 days after implantation. The
surface with S17 cells attached showed the development of small vessels
and large hemorrhages. On the other hand, neither markedly increased
vessels nor hemorrhages were seen among the mice injected with V7
cells.
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and 5)
.
The sizes of tumors at 90 days were 5215 ± 1612.04
(n = 10) and 528 ± 181.78
mm3 (n = 10) for S17 and V7
cells, respectively (Table 1)
. VEGF mRNA expression in the s.c. tumors
is shown in Fig. 1C
. s.c. tumors derived from S17 cells had
high levels of VEGF mRNA expression for as long as 90 days after the
inoculation.
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Hepatic Metastases after Splenic Portal Injection.
Among 10 mice injected with S17 cells, macroscopic metastasis and
microscopic metastasis in the liver were seen in 5 mice (50%), and the
percentage of mice with tumor growth in the spleen was 80%, whereas no
mice injected with V7 cells had even microscopic metastasis in the
liver or growth in the spleen (Fig. 6
;
Table 1
).
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). Both types of tumors showed
microvessel metastasis. However, no obvious metastasis in lung, liver,
or mesenteric lymph nodes was observed in either group, except for
lymph node and lung metastasis in one of seven mice of the
S17-inoculated group (Fig. 6)
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Peritoneal Dissemination.
Macroscopic observation 28 days after inoculation revealed numerous
tumors (up to 10 mm in diameter) in the peritoneal cavity in 9 of 10
mice injected with S17 cells (Fig. 7)
. A
large number of small white tumor nodules were formed in the greater
omentum, mesenterium, and gonadal fat. Occasionally, smaller nodules
were seen at the surface of the stomach and intestine. On the other
hand, neither metastasis nor ascites were seen in the mice injected
with V7 cells (Table 1)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found that sense VEGF transfectants have a growth advantage in vivo but not in vitro. The rapid in vivo growth of sense VEGF transfectants was due to vigorous tumor angiogenesis, which was confirmed using the dorsal air sac method. Namely, the constitutive expression of VEGF led to expansion of the blood vessels, the branching out of newly formed blood vessels from large blood vessels, and occasional severe hemorrhaging, possibly due to fragile neovasculature. In addition, we also evaluated the microvessel density of the s.c. tumors and concluded that the growth advantage of S17 cells appeared to arise from increased tumor vascularization induced by VEGF. With regard to the role of VEGF in metastasis, a number of clinical studies have shown a positive correlation between VEGF expression and metastatic potential. We also demonstrated that colon cancers with high levels of expression of VEGF mRNA metastasized to the liver and lymph nodes at significantly higher rates than those without high levels of VEGF mRNA expression (32) .
However, those studies did not prove a causative role of VEGF in metastasis but rather showed an association of VEGF with metastasis. In the present study, we obtained direct evidence that VEGF plays a crucial role in hepatic metastasis in the intrasplenic portal inoculation model system, although it is true that the hepatic metastasis in this experimental model system occurs by the forced introduction of the cancer cells into the blood vessels and thus does not completely mimic spontaneous metastasis. We also checked whether these cells have the ability to complete all stages of the metastasis cascade after orthotopic inoculation, and we found that even S17 cells formed no apparent metastases, despite the rapid tumor growth. Taken together, these findings suggested that VEGF contributes predominantly to tumor growth, probably due to the induction of angiogenesis, and thereby leads to visible foci rather than dormant tumors in either the primary site or metastatic lesions. However, it appears that VEGF alone cannot overcome all of the barriers that must be surmounted for achieving metastasis.
Peritoneal dissemination is also a critical event affecting patient prognosis. Over the past few years, a considerable number of studies have been performed on the correlation between VEGF and peritoneal dissemination (39, 40, 41) . The present study showed clearly that VEGF transfectants had a greater potential to induce peritoneal dissemination than did the controls. Considering that peritoneal dissemination seems to require lower neovascularization than solid tumor growth, we have not yet clarified why VEGF transfectants showed massive peritoneal dissemination. One putative mechanism is that VEGF transfectants can be supplied more efficiently than the control transfectants with a large quantity of nutrients by diffusion because VEGF is also able to function as vascular permeability factor, which may, in turn, occasionally cause ascites. Indeed, Kraft et al. (42) reported that VEGF might play an important role in tumor progression and the formation of malignant effusions (43) . Mesiano et al. (44) demonstrated that neutralization of VEGF activity may have clinical application in inhibiting malignant ascites formation in ovarian cancer.
In conclusion, the present study provided clear evidence that VEGF plays an important role in cancer growth due to stimulation of neovascularization and subsequent metastasis, which is probably mediated by preventing tumors from entering a dormant state.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Surgery and Surgical Basic Science,
Graduate School of Medicine, Kyoto University of Medicine, Kyoto
University, 54-Shogoin Kawara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Phone: 81-75-751-3445; Fax: 81-75-751-3219; E-mail: charly{at}kuhp.kyoto-u.ac.jp 
2 The abbreviations used are: VEGF, vascular
endothelial growth factor; BSD, blasticidin S deaminase.
Received 8/ 9/99; revised 11/ 1/99; accepted 11/15/99.
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REFERENCES |
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, S17 (n = 10); •, V7
(n = 10). 
