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3 The abbreviations used are: VEGF, vascular endothelial growth factor:PDGF, platelet-derived growth factor: TGF, transforming growth factor.
Vol. 3, 1309-1316, August 1997 Clinical Cancer Research 1309
Enhanced Expression of Vascular Endothelial Growth Factor in
Human Pancreatic Cancer Correlates with Local
Disease Progression1
Jun Itakura, Toshiyuki Ishiwata, Helmut Friess,
Hideki Fujii, Yoshiro Matsumoto,
Markus W. B#{252}chler, and Murray Korc2
Division of Endocrinology, Diabetes, and Metabolism, Departments
of Medicine, Biological Chemistry, and Pharmacology, University ofCalifornia, Irvine, California 92697 [J. I., T. I., M. K.1: Department of
Visceral and Transplantation-Surgery, University of Bern, Inselspital,
CH-3010 Bern. Switzerland IH. Fr., M. W. B.l: and The FirstDepartment of Surgery, Yamanashi Medical University. Yamanashi
409-38, Japan [H. Fu., Y. M.J
ABSTRACT
Vascular endothelial growth factor (VEGF) is an an-
giogenic polypeptide that has been implicated in cancer
growth. In the present study, we characterized VEGF ex-
pression in cultured human pancreatic cancer cell lines and
determined whether the presence VEGF in human pancre-
atic cancers is associated with enhanced neovascularization
or altered clinicopathological characteristics. VEGF mRNA
transcripts were present in all six tested cell lines (ASPC-1,
CAPAN-1, MIA-PaCa-2, PANC-1, COLO-357, and T3M4).
Immunoblotting with a highly specific anti-VEGF antibody
revealed the presence of VEGF protein in all of the cell lines.
Northern blot analysis of total RNA revealed a 5.2-fold
increase in VEGF mRNA transcript in the cancer samples in
comparison with the normal pancreas. Immunohistochemi-
cal and in situ hybridization analysis confirmed the expres-
sion of VEGF in the cancer cells within the tumor mass.
Immunohistochemical analysis of 75 pancreatic cancer tis-
sues revealed the presence of strong VEGF immunoreactiv-
ity in the cancer cells in 64% of the cancer tissues. The
presence of VEGF in these cells was associated with in-
creased blood vessel number, larger tumor size, and en-
hanced local spread but not with decreased patient survival.
These findings indicate that VEGF is commonly overex-
pressed in human pancreatic cancers and that this factor
Received 9/16/96: revised 3/18/97: accepted 4/24/97.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 1 8 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported by Public Health Service Grant CA-40l62
from the National Cancer Institute (to M. K.). J. I. was the recipient of
an award from the Overseas Research Scholars of the Japanese Ministryof Education.2 To whom requests for reprints should be addressed. at Division of
Endocrinology, Diabetes and Metabolism, Medical Sciences I, C240.
University of California, Irvine. CA 92697. Phone: (714) 824-6887:
Fax: (714) 824-2200.
may contribute to the angiogenic process and tumor growth
in this disorder.
INTRODUCTION
Pancreatic ductal adenocarcinoma is the fifth leading cause
of cancer death in the United States ( I ). In recent years, there
has been a decrease in mortality rates and improvement in
survival rates of pancreatic cancer patients following surgery
such as pancreatico-duodenectomy (2-5). Nonetheless, the
overall 1 year survival rate after diagnosis of pancreatic cancer
is less than 20%, and the overall 5-year survival rate is only 3%
(6). One reason for this poor prognosis is the propensity of
pancreatic cancers to invade adjacent blood vessels and form
hematogeneous metastasis in the early phase of the disease,
independently of primary tumor growth (7).
The growth and metastasis of cancers has been shown to be
angiogenesis dependent (8), and considerable interest has de-
veloped in the possible participation of VEGF3 in angiogenesis.
VEGF is a homodimeric glycoprotein with an approximate
molecular weight of Mr 46,000. It consists of four isoforms
having 121, 165, 189 or 206 amino acid residues in the mature
monomer. These monomers are generated by differential splic-
ing of mRNA derived from a single gene (9, 10). All four forms
are mitogenic to vascular endothelial cells and induce vascular
permeabilization. Only VEGF 121 does not bind heparin, and
each isoform exhibits a different affinity for heparan sulfate
proteoglycans ( 1 1). Two related transmembrane receptors bind
VEGF with high affinity. Both are class III transmembrane
protein tyrosine kinases. VEGF receptor- I was originally named
the fms-like tyrosine kinase, and is also known as fit ( I 2).
VEGF receptor-2, also known as KDR, is the human homologue
offlk-1 (13).
VEGF and its receptors play an important role in angio-
genesis during embryonic development and wound healing (14,
15). A number of studies have suggested that VEGF may have
an important role in tumor growth and metastasis (16-18). It is
not known, however, whether VEGF has a role in human
pancreatic cancer. Therefore, in the present study, we examined
the expression of VEGF in cultured pancreatic cancer cell lines
and in surgical specimens from patients with pancreatic cancer.
We now report that VEGF is expressed in pancreatic cancer cell
lines and in human pancreatic cancers.
MATERIALS AND METHODS
Materials. The following were purchased: DMEM,
RPMI 1640, fetal bovine serum, trypsin-EDTA solution, and
Fig. 2 Immunoblot analysis of VEGF expression in human pancreaticcancer cell lines. Cell lysates (50 p.g/lane) were prepared from COLO-357 (Lane 1), MIA-PaCa-2 (Lane 2), PANC- I (Lane 3), T3M4 (Lane 4),
ASPC- I (Lane 5), and CAPAN- 1 (Lane 6) cells, transferred to polyvi-nylidene difluoride membranes, and analyzed by immunoblotting with ahighly specific anti-VEGF antibody. Left, migration positions of molec-
ular weight markers (in thousands).
I 2 3 4 5 6 7 8 9 10 11
28S - � �4.. � � I � V VEGF
18S- -
w�
7S -
Fig. 3 Northern blot analysis of VEGF expression in human pancreatictissues. Total RNA (20 p.gllane) was isolated from four normal (Lanes
1-4) and seven pancreatic cancer tissues (Lanes 5-11), size-fraction-ated, and hybridized with 32P-labeled VEGF cDNA (700,000 cpm/ml; 7
days of exposure) and 7S cDNA (30,000 cpm/ml; 6 h of exposure). Left,
migration positions of 285 and 185 ribosomal subunits.
To confirm the immunostaining results, in situ hybridiza-
tion analysis was next carried out in the cancer tissues. Foci of
cancer cells that were positive for VEGF inimunoreactivity (Fig.
5, A and B) also exhibited a specific mRNA in situ hybridization
signal (Fig. 5C). A faint to moderate VEGF in situ hybridization
signal was also present in some of the fibroblasts around cancer
cells (Fig. SC), as well as in the islet cells surrounding the
cancer areas (data not shown). Treatment of the sections with
excess RNase abolished these in situ hybridization signals (Fig.
SD).
We next sought to determine whether there was a correla-
lion between VEGF expression and tumor neovascularization,
tumor stage and grade, and patient survival. The blood vessel
numbers in VEGF-negative and VEGF-positive groups were
50.1 ± 7.6 and 77.7 ± 5.6, respectively. This difference was
statistically significant, indicating that there was greater neovas-
cularization in the VEGF-overexpressing cancers (Fig. 6). Fur-
thermore, �2 analysis indicated that the presence of VEGF in the
#{149}#{149}S#{149}S�#{149}��#{149}“AS
-.�- �
.:‘.-�
. �
� �9:-:--.
‘�
I � �
�
- -. - - ,
.�
Fig. 4 VEGF immunoreactivity in the human pancreas. In the normalpancreas (A), VEGF immunoreadtivity was always present in the islets(arrowheads) and occasionally present in the ductal cells of smallductules and in a few acinar cells. In the pancreatic cancers (B), the
ductal-like cancer cells exhibited abundant VEGF immunoreactivity inthe cytoplasm. Occasionally, these cells exhibited apical VEGF immu-noreactivity (arrows) in addition to cytoplasmic immunoreactivity. A
and B, X500.
cancer cells was associated with a statistically significant in-
crease in tumor size and local extension (T category). However,
there was no correlation between the presence of VEGF in the
cancer cells and the histological grade or tumor stage of the
cancers (Table 1). The 30-month survival of the VEGF-negative
and VEGF-positive groups were 7.4 and 2.1%, respectively, and
the mean survival duration of these two groups was 14.8 ± 13.4
and 10.8 ± 10.3 months, respectively. Thus, there was a tend-
ency for shorter survival in the patients with VEGF-positive
tumors. However, Kaplan-Meier analysis (Fig. 7) and the log-
rank test indicated that there was no significant difference in
survival between these two groups.
DISCUSSION
Due to alternative splicing of mRNA, VEGF has four
different mature isoforms: VEGF1 2 1 , VEGF16S, VEGF1 89,
and VEGF2O6 (10, 19). VEGF16S is the most abundant form in
the majority of cells and tissues (10, 19). All four isoforms are
mitogenic toward endothelial cells (30). The two larger iso-
forms, VEGF1 89 and VEGF2O6, have a high affinity toward
factor, and TGF43 (22, S 1-54). Additional studies are, therefore,
necessary to determine whether inclusion of a larger number of
patients or consideration of the role of other angiogenic factors
may reveal a significant correlation between VEGF expression,
the expression of other angiogenic factors, and decreased sur-
vival.
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