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
Research. on June 3, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1310 VEGF and Prognosis in Pancreatic Cancer
penicillin streptomycin solution from Irvine Scientific (Santa
Ana, CA); gene screen membranes from New England Nuclear
(Cleveland OH); random-primed labeling kit, digoxigenin RNA
labeling kit, and digoxigenin nucleic acid detection kit from
Boehringer-Mannheim (Indianapolis, IN); [a-32p]dCTP (3000
Ci/mmol) from Amersham Corp. (Arlington Heights, IL); a
highly specific VEGF polyclonal antibody from Santa Cruz
Biotech (Santa Cruz, CA); a highly specific anti-factor VIII
mouse monoclonal antibody from DAKO Corp. (Carpinteria,
CA); leupeptin from United States Biochemical Corp. (Cleve-
land, Ohio); Immobilon P membrane from Millipore Intertech
(Bedford, MA); and Hyperfilm-ECL from Amersham. The
VEGF 165 cDNA (19) was a gift from Dr. Judith A. Abraham
(Mountainview, CA). All other chemicals and reagents were
purchased from Sigma Chemical Corp. (St. Louis, MO).
Cell Culture. ASPC- I , CAPAN- 1 , MIA-PaCa-2, and
PANC- I human pancreatic cancer cells were obtained from the
American Type Culture Collection (Rockville, MD). T3M4 and
COLO-357 human pancreatic cancer cells were a gift from R. S.
Metzger at Duke University. Cells were grown in monolayer
culture in a humidified 5% CO2 and 95% air atmosphere at 37#{176}C.
ASPC- I , CAPAN- I , and T3M4 cells were grown in RPMI
1640. COLO-357, MIA-PaCa-2, and PANC-l cells were grown
in DMEM. Media contained antibiotics and 10% fetal bovine
serum, 100 units/ml penicillin, and 100 mg/ml streptomycin.
Tissues for Northern Blot Analysis. Normal pancreatic
tissue samples were obtained from 10 healthy individuals (7
males and 3 females; mean age, 40.5 ± 15.6 years; range,
22-64 years) through an organ donor program. Pancreatic can-
cer tissues were obtained from 15 patients (7 males and 8
females; mean age, 60.2 ± I 2.3 years; range, 32-78 years) who
underwent surgery for pancreatic cancer. Tissue samples were
frozen in liquid nitrogen and held at -80#{176}Cuntil use for RNA
extraction.
Tissues for Immunohistochemistry. Normal pancreatic
tissue samples were obtained from four male and one female
donors, through an organ donor program. The age of the organ
donors ranged from 47 to 65 years, with a mean age of 55.6 ±
6.6 years. Pancreatic carcinoma tissue samples were obtained
from 75 patients (45 males and 30 females; mean age, 62.4 ±
9.6 years; range. 3 1-77 years) undergoing surgery for pancreatic
cancer. The vast majority of these patients (66 individuals) did
not have gross evidence for metastatic disease at the time of
clinical presentation. The tumors were classified according to
the Tumor-Node-Metastasis classification for pancreatic cancer
(20). Histologically, there were 1 3 grade 1 , 44 grade 2, 17 grade
3, and I grade 4 ductal adenocarcinomas. There were 15 stage
I, 9 stage II, 42 stage III, and 9 stage IV tumors. Tissues were
fixed in Bouin’s solution or 10% paraformaldehyde solution for
I 8 -20 h and embedded in paraffin. All studies were approved
by the Human Subjects Committees of the University of Cali-
fornia (Irvine, CA), the University of Bern (Bern, Switzerland),
and the Ethics Committee of the Yamanashi Medical University
(Yamanashi, Japan).
Northern Blot Analysis. Total RNA was extracted by
the acid guanidinium thiocyanate method, and poly(A)� mRNA
was prepared by oligodeoxythymidine column chromatography
(21). RNA was fractionated on 0.8% agarose/2.2% formalde-
hyde gels, electrotransferred onto nylon membranes, and cross-
linked by UV irradiation (22). The blots were prehybridized and
hybridized with the indicated a-32P-labeled cDNA probes and
washed under high stringency conditions as described previ-
ously (22). For use in hybridizations, the VEGF cDNA was
subcloned into the pLen eukaryotic expression vector using a
BarnHI restriction site (19). Equivalent loading of RNA in each
lane was confirmed by hybridizing the total RNA filters with a
mouse 75 cDNA that cross-hybridizes with human cytoplasmic
75 RNA. Equivalent loading of poly(A)� mRNA filters was
confirmed by hybridization with a glyceraldehyde phosphate
dehydrogenase cDNA (23). All cDNAs were labeled with
[a32pldCTP using a random primer labeling system (24). Blots
were exposed at -80#{176}Cto Kodak XAR-5 film using intensify-
ing screens. Densitometric analysis of the autoradiograms was
performed with a LKB Ultrascan XL enhanced laser densitom-
eter (Uppsala, Sweden).
Immunoblotting. Human pancreatic cancer cells were
solubilized in lysis buffer containing 50 msi Tris, 150 msi NaC1,
1 mr�i EGTA, 1% NP4O, 1% sodium deoxycholate, 1 m�i sodium
vanadate, 50 mM sodium fluoride, 2 msi EDTA (pH 8.0), 100�g/ml benzamidine, 50 �i.g/ml aprotinin, 10 pg/ml leupeptin, 10
p.g!ml pepstatin A, and 1 ms� phenylmethylsulfonyl fluoride.
Proteins were subjected to SDS-PAGE and transferred to Im-
mobilon P membranes. Membranes were incubated for 90 mm
with a highly specific anti-VEGF polyclonal antibody that red-
ognizes VEGF12I, VEGF165, and VEGF189. This antibody
was raised against a glutathione S-transferase fusion protein
construct containing human VEGF sequences corresponding to
amino acids 1-191 with 44 amino acid detection from amino
acids 142-185 (25). Membranes were washed and incubated
with a horseradish peroxidase-coupled secondary goat anti-rab-
bit antibody for 60 mm. After washing, antibodies were visual-
ized by enhanced chemiluminescence.
Immunohistochemistry. The same anti-VEGF poly-
clonal antibody that was used for immunoblotting was also used
for immunohistochemical analysis. This antibody was shown
previously to be highly specific in immunohistochemical read-
lions of human gastric and lung cancers (26, 27). Paraffin-
embedded sections (4 jim) from pancreatic cancer and normal
pancreatic tissues were subjected to immunostaining using the
streptavidin-peroxidase technique. After deparaffinization, en-
dogenous peroxidase activity was blocked by incubation for 30
mm with 0.3% hydrogen peroxide in methanol. Tissue sections
were incubated for 15 mm (23#{176}C)with 10% normal goat serum
and then incubated with polyclonal VEGF antibody (0.03 mg/mI
in PBS containing 1% BSA) for 16 h at 4#{176}C.Bound antibody
was detected with biotinylated goat anti-rabbit IgG secondary
antibody and streptavidin-peroxidase complex (Kirkegaard &
Perry Laboratories, Inc., Gaithersburg, MD), using diaminoben-
zidine tetrahydrochloride as the substrate. Sections were coun-
terstained with Mayer’s hematoxylin. Some sections were incu-
bated with nonimmunized rabbit IgG or without primary
antibodies, which did not yield positive immunoreactivity. Scor-
ing was carried out by two independent observers blinded to the
patient’s status. Positive staining was defined as the presence of
VEGF immunoreactivity in at least 10% of the cancer cells. A
factor VIII mouse monoclonal antibody (1 : 100 dilution) was
used to stain endothelial cells. The number of capillaries and
microvessels adjacent to the foci of cancer cells was determined
Research. on June 3, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
I,
Clinical Cancer Research 1311
at X 100 after identifying highly vascular areas at X4O (27).
Both evaluators were blinded with respect to patient’s history
and immunohistochemical results.
In Situ Hybridization Analysis. To carry out in situ
hybridization analysis, tissue sections (4-p..m thick) were placed
on 3-aminopropyl-methoxysilane-coated slides, deparaffinized,
and incubated at 23#{176}Cfor 20 mm with 0.2 N HC1 and at 37#{176}Cfor
15 mm with 20 �i.g/ml proteinase K. The sections were then
postfixed for S mm in PBS containing 4% paraformaldehyde,
incubated briefly twice with PBS containing 2 mg/ml glycine
and once for 1 h in 50% (v/v) formamide/2X SSC. (1 X SSC =
150 mr�i NaC1, 15 mrvi sodium citrate, pH 7.0.) The hybridization
buffer contained 0.6 M NaCl, 1 mr�i EDTA, 10 msi Tris-HC1 (pH
7.6), 0.25% SDS, 200 p.g/ml yeast tRNA, 1 X Denhart’s solu-
lion, 10% dextran sulfate, 40% formamide, and 500 ng/ml of the
indicated digoxigenin-labeled riboprobe. Hybridization was per-
formed in a moist chamber for 16 h at 42#{176}C.The sections were
then washed sequentially with 50% formamide/2X SSC for 30
mm at 50#{176}C,2X SSC for 20 mm at 50#{176}C,and digested with
RNase A (1 p�g!ml) in TNE solution (10 msi Tris-HC1, 500 msi
NaCI, and 1 mM EDTA, pH 7.6) for 15 mm at 37#{176}C.The
sections were washed with TNE solution for 10 mm at 37#{176}Cand
0.2X SSC for 20 mm at 50#{176}C.
For immunological detection, the Genius 3 nonradioactive
nucleic acid detection kit was used. The sections were washed
briefly with buffer 1 solution (100 m�i Tris-HC1 and 150 msi
NaCI, pH 7.5) and incubated for 60 mm at 23#{176}Cwith 1% (w/v)
blocking reagents in buffer 1 . The sections were then incubated
for 30 mm at 23#{176}Cwith a 1 :2000 dilution of alkaline phos-
phatase-conjugated polyclonal sheep anti-digoxigenin Fab frag-
ment containing 0.2% Tween 20. The sections were then
washed twice for 15 mm at 23#{176}Cin buffer I solution containing
0.2% Tween 20 and equilibrated with buffer 3 (100 msi Tris-
HC1, 100 mM NaCl, and 50 msi MgC12, pH 9.5) for 2 mm. The
sections were then incubated with color solution containing
nitroblue tetrazolium and X-phosphate in a dark box for 2-3 h.
After the reaction was stopped with TE buffer (10 msi Tris-HCI
and 1 mM EDTA, pH 8.0), the sections were mounted in aque-
ous mounting medium.
Statistical Analysis. Differences in distribution of
VEGF were determined using the x2 exact test. Kaplan-Meier
survival analysis was used to estimate survival time, and log-
rank test was used to compare differences in survival time
between VEGF-positive and VEGF-negative groups (28, 29).
For all tests, P < 0.05 was considered to be significant.
RESULTS
Analysis of poly(A)� RNA isolated from human pancre-
atic cancer cell lines indicated that COLO-357, T3M4, ASPC- 1,
and CAPAN-l cells expressed the 4.1-kb VEGF transcript,
whereas MIA-PaCa-2 and PANC-1 cells expressed the 3.3-kb
VEGF transcript. In this cell line, there was also a faint 1.8-kb
transcript (Fig. 1). Immunoblotting with a highly specific anti-
VEGF-antibody revealed an approximately Mr 43,000 band in
all of the cell lines and occasionally also a Mr 41,000 band (Fig.
2), both of which correspond to the VEGF165 isoform ho-
modimer. In addition, Mr 32,000 and Mr 31 ,000 bands corre-
sponding to the VEGF121 isoform homodimer were seen in
123456
VEGF
GAP
Fig. 1 Northern blot analysis of VEGF expression in human pancreatic
cancer cell lines. Poly(A)� RNA (5 �i.g/lane) was isolated from COLO-
357 (Lane 1), MIA-PaCa-2 (Lane 2), PANC:I.(Lane3),T3M4(Lane 4),ASPC-l (Lane 5), and CAPAN-l (Lane 6) cells. RNA was size-frac-
tionated and hybridized with 32P-labeled VEGF cDNA (900,000 cpm/ml; 4.5 h of exposure) and glyceraldehyde-3-phosphate dehydrogenase
(GAP) cDNA (50,000 cpm/ml: 24 h of exposure).
MIA-PaCa-2 cells and, to a lesser extent on the original immu-
noblot, in PANC-l cells (Fig. 2). Highest levels of VEGF
mRNA and protein were observed in T3M4 and ASPC- 1 cells
(Figs. I and 2).
Northern blot analysis of total RNA isolated from human
pancreas revealed the presence of the 4. 1-kb VEGF transcript in
all normal pancreatic samples and the cancer samples (Fig. 3).
Densitometric analysis of the autoradiograms indicated that the
levels of this 4. 1-kb transcript were 5.2-fold higher in the
pancreatic cancers by comparison with the normal pancreas, and
this difference was statistically significant (P < 0.05). In two
cancer samples (Fig. 3, Lanes 7 and 8), a 3.3-kb VEGF tran-
script was also visible. In addition, in one cancer sample an
approximately 4.6-kb transcript was also evident (data not
shown).
The same highly specific anti-VEGF antibody that was
used in the immunoblotting studies was used next to localize
VEGF immunohistochemically. In the normal pancreas, mod-
crate to strong VEGF immunoreadtivity was present in the
cytoplasm of endocrine islet cells, in some ductal cells within
the small ductules, and in a few acinar cells (Fig. 4A). In the
pancreatic cancers, moderate to strong VEGF immunoreactivity
was present in the cytoplasm of many of the cancer cells (Fig.
4B) in 48 (64%) of the 75 cancers. Occasionally, the cancer cells
also exhibited strong apical VEGF immunoreadtivity (Fig. 48).
Faint VEGF immunoreadtivity was also present in some of the
fibroblasts within the connective tissue around cancer cells (Fig.
4B) and moderately intense VEGF immunoreactivity was also
seen in vascular smooth muscle cells of blood vessels (data not
shown).
Research. on June 3, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
- ‘p� ‘-6F;�,�.
....�
-‘4- -.
, � � � --
� ** �10� -
w :�-- � ; - � - , - � ., --� - -
4. ,.
- :� A
123456
�: � I
29-
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
1312 VEGF and Prognosis in Pancreatic Cancer
- .‘�.. . - ‘-
4�J.. 2c:T�5� 4� �P�; � � �
-,� ..� �1 � , � � -� .. - -y..#{176}:.�t%.. �.
� t.rc �
� --�
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.3
. .. � �4i - ‘:-. . � -
. ,::��2P�;’ �
�r�
� -.‘.-- -. .� �
� � � �.
. - .. � � �‘
2 d��< %4�
�‘/� � . ‘
#{182}�, I �
� � � :‘ �
� dp’� ;��r .�, - �. -
4� :� -“-i-
�
“- -. I
:� . -3+’Y� .. ‘�‘4I�,
� � C-,� - ;� . - -
#. -, . -,..�. �.
� -. --I :-
41 ;�- �
I.
. . �
; -
�,. .. -t�--.
D- 1_ -
p<0.05200
I..
E
�100S
S
S
-.-
I
I
1-(+)
VEGF
Fig. 6 Relationship between VEGF expression and blood vessel num-
her. Ten areas of four VEGF-negative cases ( - ) and 39 areas of 13
VEGF-positive cases (+) were analyzed following staining with anti-factor VIII antibody. Blood vessel numbers were 50.1 ± 7.6 in the
VEGF-negative group and 77.7 ± 5.6 (means ± SE) in the VEGF-positive group (P < 0.05). Horizontal bars, blood vessel numbers.
Clinical Cancer Research 1313
Fig. 5 In situ hybridization
analysis of VEGF expression in
cancer tissues. VEGF immuno-reactivity (A and B) and in situ
hybridization (C and D) analy-sis was performed in serial
tissue sections. Regions of can-
cer cells that were positive forVEGF immunoreadtivity (A
and B) also exhibited a moder-ate to strong in situ hybridiza-tion signal following hybridi-
zation with antisense ribo-probe (C). Islet cells were alsopositive for VEGF immunore-
activity (arrowheads, A) and in
situ hybridization signal (datanot shown). Treatment of serialsections with excess RNase
abolished the in situ hybridiza-
tion signal of the sense ribo-probe (D). A, X250: B-D,
X500.
:�. .�.� �
� - - ‘4;;’� .
-S
�� , ��
� �
-?- �
heparin and bind to the extracellular matrix following their
release from their cells of origin (9, 31). In contrast, the two
shorter isoforms, VEGF12I and VEGF16S, have low or no
affinity to heparin and are secreted as diffusible molecules (9,
31). Following incorporation into the extracellular matrix,
VEGF165, VEGF189, and VEGF2O6 are released from their
bound states by proteolysis, and the released dimers have same
activity as VEGF12I (9, 31). Therefore, it has been suggested
that the bioavailability of VEGF may be regulated by mecha-
nisms that control alternative splicing, which than dictates
whether VEGF will be soluble or incorporated into a biological
reservoir (30).
Previous studies have demonstrated that colorectal and
breast cancer cell lines express relatively high levels of VEGF
mRNA (16, 32, 33) and that ovarian cancer cell lines express
high levels of VEGF as determined by immunoblotting (18). In
the present study, we determined that cultured human pancreatic
cancer cell lines also express relatively high levels of VEGF.
Four of the six tested cell lines expressed the 4.1-kb VEGF
transcript that encodes VEGF189, whereas the remaining two
cell lines (MIA-PaCa-2 and PANC-1) expressed the 3.3-kb
VEGF transcript that encodes VEGF165. T3M4 cells also cx-
pressed a 1 .8-kb transcript of unknown significance. In general,
there was a good correlation between VEGF mRNA expression
in these cell lines and VEGF protein levels, as determined by
immunoblotting. All of the cell lines expressed the Mr 43,000
VEGF proteins corresponding to the VEGF165 isoform ho-
Research. on June 3, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
a
- - - VEGF(-)- VEGF(#{247})
10 20
Follow up (Months)
“ The rates of VEGF immunoreactivity (positive or negative) indifferent patient subgroups were compared by the x2 two-sided exact
trend test. NS, not significant.
F, TNM, Tumor-Node-Metastasis.
Fig. 7 Survival curve. Cumulative survival (Kaplan-Meier) plot of thepostoperative survival period of patients whose pancreatic tumors cx-
hibited cytoplasmic VEGF immunostaining (-) versus those whose
tumors were negative for VEGF ( ). Log-rank test indicated nosignificant difference between the two groups.
1314 VEGF and Prognosis in Pancreatic Cancer
Table I VEGF expression in relation to the clinicopathologicalcharacteristics of 75 patients with pancreatic cancer”
VEGF VEGF
Factor negative positive P
Age 61.9 ± 10.7 62.4 ± 9.6 NSGender
Male 20 26Female 7 22
TNMh categories P < 0.01
T 4 0T2 11 17
T3 12 31NS
N0 10 18
N 17 30
NS
M0 25 41M 2 7
Stage NSI 8 7II 2 7
III 15 27
IV 2 7
Grade NS1 4 92 16 28
3 7 10
4 0 1
modimer. The two cell lines that expressed the shorter 3.3-kb
VEGF transcript also exhibited the smaller Mr 32,000 and Mr
31,000 protein bands corresponding to the VEGF121 isoform
homodimer. These observations confirm the specificity of the
anti-VEGF antibody used in the present study and indicate that
human pancreatic cancer cells express VEGF at the IIIRNA and
protein levels, and that in some instances they express more than
one VEGF isoform.
Several lines of evidence in the present study point toward
aberrant VEGF expression in pancreatic cancers in vivo: (a)
Northern blot analysis revealed the presence of a single 4. 1-kb
VEGF mRNA transcript in all normal pancreatic tissues and in
all pancreatic cancer samples. However, 2 of the 15 cancer
samples expressed a 3.3-kb VEGF mRNA transcript, and 1
cancer sample exhibited an approximately 4.6-kb VEGF mRNA
transcript. The presence of a multiple VEGF mRNA transcripts
is consistent with the propensity of cancer tissues to express
different VEGF isoforms (16, 32, 33, 34-37) and with our
findings in the pancreatic cancer cell lines; (b) Northern blot
analysis revealed a 5.2-fold increase in the 4.1-kb VEGF mRNA
transcript in the cancer samples by comparison with the normal
samples, indicating that VEGF is overexpressed in pancreatic
cancers; and (c) the distribution of VEGF in the normal and
cancerous tissues was different. Thus, in the normal pancreas,
VEGF was relatively abundant in the endocrine islets, less
frequently present in the ductal cells, and only occasionally
present in acinar cells. In contrast, in many of the pancreatic
cancers, VEGF was abundant in the ductal-like cancer cells.
Inasmuch as VEGF is a specific mitogen toward endothelial
cells, these observations suggest that various isoforms of cancer
cell-derived VEGF have the potential to act in a paracrine
manner on endothelial cells within the pancreatic tumor mass,
thereby leading to enhanced angiogenesis and greater tumor
growth.
Overexpression of VEGF has been reported in brain, mam-
mary, colorectal, renal, liver, ovarian, and gastric malignancies
(16, 17, 32, 33, 34-36, 38, 39). In the present study, 64% of the
pancreatic cancer samples exhibited VEGF immunoreactivity in
the cancer cells within the tumor mass. The reasons for this
overexpression are not known. However, it has been demon-
strated that mutations in the H-ras or K-ras oncogene are
associated with marked up-regulation of VEGF (33), and K-ras
mutations are frequent in pancreatic cancer (40-42). Further-
more, VEGF expression is enhanced by epidermal growth fac-
tor, PDGF-BB, and TGF-�3 isoforms (43-46), and the epidermal
growth factor family of ligands, TGF43 isoforms, and PDGFand its receptors are overexpressed in this malignancy (22,
47-50). Together, these alterations may combine to enhance
VEGF expression in pancreatic cancer.
Univariant analysis of the immunohistochemical data dem-
onstrated that the presence of VEGF in the pancreatic cancer
cells was associated with enhanced tumor size and extension
and greater neovascularization, indicating that VEGF has the
potential to contribute to pancreatic tumor growth in vivo. Al-
though there was a tendency for shorter survival in the group
with VEGF-positive tumors, this correlation was not statistically
significant. Because the vast majority of the patients in the
present study did not have detectable metastatic disease at the
time of presentation for surgery, it cannot be determined from
the limited number of cases with metastases whether VEGF
expression correlates with enhanced propensity of the pancreatic
cancer to metastasize. In addition, the present study did not take
Research. on June 3, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1315
into account the angiogenic potential of other growth factors
that are overexpressed in pancreatic cancer, including basic
fibroblast growth factor, PDGF-BB, TGF-a, hepatocyte growth
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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1997;3:1309-1316. Clin Cancer Res J Itakura, T Ishiwata, H Friess, et al. progression.human pancreatic cancer correlates with local disease Enhanced expression of vascular endothelial growth factor in
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