enhanced expression of vascular endothelial growth...

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

http://clincancerres.aacrjournals.org/

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



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).



- ‘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 � �

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- -. - - ,

.�

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


- .‘�.. . - ‘-

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-,� ..� �1 � , � � -� .. - -y..#{176}:.�t%.. �.

� t.rc �

� --�



.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.


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-



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.


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




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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