Cancer Therapy: Preclinical

Pigment Epithelium ^ Derived Factor Gene Therapy Inhibits

Human Pancreatic Cancer in Mice
Ryunosuke Hase, Masaki Miyamoto, Hirofumi Uehara, Masatoshi Kadoya, Yuma Ebihara,
Yoshihiro Murakami, Ryo Takahashi, Seiji Mega, Li Li, Toshiaki Shichinohe,
Yo Kawarada, and Satoshi Kondo

Abstract Purpose: Pigment epithelium ^ derived factor (PEDF), which has recently been shown to be the
most potent inhibitor of angiogenesis in the mammalian eye, is also expressed in the pancreas.
Previously, we have screened the expression of PEDF by immunohistochemical analysis and
showed that low expression of PEDF is associated with increased risk of hepatic metastasis and
short survival. The purpose of this study was to investigate whether PEDF gene is a potent tumor
suppressor and a potential candidate for cancer gene therapy.
Experimental Design:We investigated both in vitro and in vivo growth characteristics of human
pancreatic adenocarcinoma cell lines that were stably transfected to overexpress human PEDF
and therapeutic effects of lentivirus-based vectors expressing PEDF on tumor growth in murine
s.c. tumor model.
Results: We discovered that cells secreted PEDF protein in the media and this exhibited strong
inhibitory effects on proliferation and migration of human umbilical vein endothelial cells. The
size of PEDF-overexpressing pancreatic adenocarcinoma tumors was significantly smaller than
that of control tumors in s.c. tumor models. Moreover, the growth of PEDF-overexpressing pan-
creatic adenocarcinoma cells was significantly suppressed in comparison with control cells in
peritoneal metastasis models. In gene transfer models, intratumoral injection of a lentivirus vec-
tor encoding PEDF (LV-PEDF) caused significant inhibition of tumor growth. The antitumor ef-
fect observed after treatment with LV-PEDF was associated with decreased microvessel density
in tumors.
Conclusion: Our data suggest that PEDF may exert a biological effect on tumor angiogenesis and
PEDF gene therapy may provide a new approach for treatment of pancreatic adenocarcinoma.

Pancreatic cancer, which has an overall 5-year survival rate of
0.4% to 4%, has a poor prognosis and is one of the most
common malignancies worldwide (1 3). Surgical resection
improves the prognosis although only f10% of patients
with pancreatic cancer are eligible for the procedure (4 8).
Most treatment failures are due to local recurrence, hepatic
metastases, or both and occur within 1 to 2 years after surgery
(9 11).
The dependency of tumor growth on the ability to induce
a neovascular response is supported by significant experi-
mental and clinical data (12, 13). Angiogenesis is regulated
by complex signaling pathways acting on endothelial cells
Authors Affiliation: Department of Surgical Oncology, Division of Cancer
Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido,
Japan
Received 6/21/05; revised 9/3/05; accepted 9/21/05.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Ryunosuke Hase, Department of Surgical Oncology,
Division of Cancer Medicine, Hokkaido University Graduate School of Medicine,
North 15, West 7, Kita-ku, Sapporo, Hokkaido 060-8648, Japan. Phone: 81-11-
706-7714; Fax: 81-11-706-7158; E-mail: ryu-hase@ med.hokudai.ac.jp.
F 2005 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-1323
and these pathways stimulate cell proliferation, migration,
and subsequent tube formation (14). The level of angiogenic
activity within a tissue is determined by the balance
between stimulatory and inhibitory molecular regulators of
endothelial cell activation. As tumors progress, they must
acquire the ability to switch from a physiologically inhibitory
environment to one that promotes new blood vessel deve-
lopment.
Pigment epithelium derived factor (PEDF) was first iden-
tified in a conditioned medium of cultured fetal retinal
pigment epithelial cells (15). It is a 50-kDa secreted
glycoprotein and is a member of the serpin superfamily of
serine protease inhibitors (16). Preliminary studies have
shown that the biological activity of PEDF is preserved in
peptide fragments although complete epitope mapping of the
molecule has not yet been carried out (17). In addition to its
neurotrophic activity on the nervous system and retina, PEDF
was recently found to be a strong inhibitor of angiogenesis in
the eye and it might be responsible for maintenance of the
avascular status of corneal tissue (18). The antiangiogenic
efficiency of PEDF is more potent than that of other
endogenous angiogenic inhibitors, including angiostatin,
thrombospondin-1, and endostatin (19). A major component
of PEDF action is the induction of apoptosis in proliferating
endothelial cells. The finding that blood vessel growth

www.aacrjournals.org 8737 Clin Cancer Res 2005;11(24) December 15, 2005

Cancer Therapy: Preclinical
increases in PEDF knockout mice is one of the several lines of
evidence that support an inhibitory role for PEDF in
angiogenesis (20). Previously, we have creened the expression
of PEDF by immunohistochemical analysis and showed that
low expression of PEDF is associated with increased risk of
hepatic metastasis and short survival (21). Based on this
background, we hypothesized that loss of PEDF expression
could be associated with the progression of pancreatic cancer
toward a metastatic phenotype.
In this study, we investigated both in vitro and in vivo growth
characteristics of human pancreatic adenocarcinoma cell lines
that were stably transfected to overexpress human PEDF. The
data presented here show that lentivirus-mediated gene transfer
of PEDF could significantly reduce tumoral neoangiogenesis
and tumor growth in animal models with human pancreatic
adenocarcinoma.
Materials and Methods
Cells and mice. Human pancreatic cancer cell lines of the PCI series
were established from surgically resected primary carcinoma tissues
(22). PCI43P5 was established by peritoneal dissemination-prone
subcultures using nude mice by repetitive in vivo selection of i.p.
inoculated PCI43 cells. Two human embryonic pancreas-derived cell
lines, 1C3D3 and 1B2C6, were purchased from Riken (Tokyo, Japan).
These were maintained in RPMI 1640 supplemented with 10% FCS and
1% penicillin/streptomycin. Human umbilical vein endothelial cells
(HUVEC) were purchased from Kurabo (Osaka, Japan) and maintained
in HuMedia-EG2. In addition, 293FT human kidney cells (Invitrogen
Corp., Carlsbad, CA) were maintained in DMEM containing 10% FCS,
2 mmol/L L-glutamine, 0.1 mmol/L MEM nonessential amino acids,
and 1% penicillin/streptomycin. These cell lines were maintained at
37jC in a humidified atmosphere containing 5% CO2.
Clin Cancer Res 2005;11(24) December 15, 2005
Female BALB/c-nu/nu mice, ages 4 to 6 weeks, were purchased from
Japan Charles River Laboratory (Tokyo, Japan) and maintained under
specific pathogen-free conditions. All animal procedures were conducted
according to the guidelines of the Hokkaido University Institutional
Animal Care and Use Committee using an approved protocol.
Construction of recombinant lentiviral vector containing the PEDF
gene. Lentiviral expression vectors were constructed using the Vira-
Power Lentiviral System (Invitrogen). The human PEDF cDNA was
originally cloned from a human splenic cDNA library (Clontech, Palo
Alto, CA) and inserted into an entry vector, pENTR2B (Invitrogen). The
pLenti-PEDF-IRES-GFP vector was generated by perfoming an LR
recombination reaction between the pENTR2B-PEDF-IRES-GFP vector
and a pLenti6/V5-DEST vector (Invitrogen).
Defective lentiviruses were generated through transient transfection
of 293FT cells with packaging and lentiviral vector plasmids.
Lentiviral transduction of pancreatic cancer cells. For lentiviral
transduction, PCI24 and PCI43P5 cells were plated in six-well plates
at a density of 1  105 per well and allowed to attach overnight. The
medium was replaced with 1 mL fresh complete medium, 100 AL
lentiviral supernatant (LV-PEDF and LV-GFP), and 8 Ag/mL hexadi-
methrine bromide (Sigma, St. Louis, MO) for assisting the uptake of
viral particles. Twenty-four hours after transduction, the cells were
maintained in full growth medium containing 10 Ag/mL blasticidin
(Invitrogen) for selection of transductants. Stably transduced clones
were expanded and the clones were characterized for PEDF production.
Both LV-PEDF transduced cells (PCI24-PEDF and PCI43P5-PEDF) and
LV-GFP transduced cells (PCI24-GFP and PCI43P5-GFP) were collect-
ed 2 weeks after selection and the number of GFP-positive cells
determined by fluorescence-activated cell sorting analysis was used as a
variable to estimate the genetically modified cell fraction, which was
>80%.
Vector titration and determination of relative transduction efficiencies.
FACScan (Becton Dickinson, San Jose, CA) was used to determine
reference titers on HeLa cells by fluorescence-activated cell sorting
analysis for GFP expression. Approximately 2  105 HeLa cells were
plated in six-well clusters and infected with serial 10-fold dilutions of
Fig. 1. PEDF expression in human embryonic
pancreas-derived cell lines 1C3D3 and 1B2C6 and pancreatic
cancer cell lines PCI6, PCI10, PCI24, PCI35, PCI43, PCI43P5,
PCI55, and PCI66. A, PEDF mRNAs from human embryonic
pancreas-derived cell lines and pancreatic cancer cell lines
were subjected to reverse transcription-PCR analysis. GAPDH
was used as the control. 1C3D3, 1B2C6, PCI10, PCI35,
PCI43, PCI43P5, and PCI66 cells were found to express
PEDF mRNA. B, the conditioned medium and lysate from
human embryonic pancreas-derived cell lines and pancreatic
cancer cell lines were subjected to Western blotting.
Recombinant human PEDF protein (10 ng) was used as the
positive control. PEDF was detected at 50 kDa in the
conditioned medium from 1C3D3, 1B2C6, PCI43, and 43P5
cells and in the lane loaded with recombinant PEDF.
8738 www.aacrjournals.org

Fig. 2. A, construction of LV-PEDF and LV-GFP. B, Western blot analysis of the conditioned medium and protein from PCI24 and PCI43P5 cells, not transduced or stably
transduced with the IRES-GFP expression lentivirus alone or with the human PEDF-IRES-GFP expression lentivirus. Recombinant human PEDF proteins (10 ng) were used as
the PEDF-positive control. C, GFP and PEDF immunohistochemical staining of tumor-implanted severe combined immunodeficient mice.
viral vectors (10 1-10 5) in a 2-mL volume of culture medium. In
the case of lentiviral vectors, 8 Ag/mL hexadimethrine bromide
(Sigma) was added to the culture media. The actual cell number at
the time of infection was determined by trypsinizing and counting
uninfected wells that had been plated in parallel. The cells were
collected 72 hours after infection and the number of GFP-positive
cells within the linear range of FACScan detection was used to
quantify the titer based on the following formula: transduction units
per milliliter (TU/mL) = (cell number at the time of infection) 
(percentage of GFP-positive cells)  (dilution factor). To assess the
relative infectivity of lentivirus vectors on the different types of cells
used for these experiments, 2  105 HeLa, PCI24, and PCI43P5 cells
were infected with serial dilutions of the LV-PEDF or LV-GFP vector
preparations and the respective titers were determined after 72 hours
by fluorescence-activated cell sorting analysis analysis as described
above.
Western blot analysis. Briefly, samples were resolved using 10%
SDS-PAGE and were then transferred to a nitrocellulose membrane
(Amersham, Aylesbury, United Kingdom) for Western blot analysis. A
monoclonal mouse anti-human PEDF antibody (Trans Genic, Kuma-
moto, Japan) was used as the primary antibody and a goat anti-mouse
immunoglobulin G (Jackson ImmunoResearch Laboratories, West
Grove, PA) was used as the secondary antibody. Immunoreactivity
was detected by an enhanced chemiluminescence detection system
(Amersham). Recombinant human PEDF peptide was used as the
positive control (Upstate, Lake Placid, NY).
www.aacrjournals.org
Antitumor Activities of PEDF on Human Pancreatic Cancer
Reverse transcription-PCR. Total cellular RNA was isolated from
each cell line using the Trizol reagent (Invitrogen). Each 20-AL cDNA
synthesis reaction mixture contained 1 Ag total RNA, 4 AL First
Strand Buffer [250 mmol/L Tris-HCl (pH 8.3), 375 mmol/L KCl, and
15 mmol/L MgCl2; Invitrogen], 10 mmol/L of each deoxynucleotide
triphosphate, 1 AL (200 units) SuperScript II (Invitrogen), 2 AL of
0.1 mol/L DTT (Invitrogen), and 1 AL Oligo(dT) (Invitrogen). The
reverse transcription reaction was carried out for 50 minutes at
42jC and inactivated by heating at 70jC for 15 minutes. Multiplex
PCR was done as previously described (23). Briefly, each 30-AL
reaction mixture contained 1 AL reverse transcription reaction
products, 0.1 AL Taq DNA polymerase (Promega, Madison, WI),
3 AL reaction buffer (Promega), 160 mmol/L of each deoxynucleo-
tide, and 20 pmol of each of the 3V and 5V primers specific for
PEDF (sense, 5V-ATGAAGAGAGGACCGTGAGGG-3V; antisense,
5V-CCCATCCTCGTTCCACTCA-3V) and glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH; sense, 5V-ACCCCTTC-ATTGACCTCAACT-3V;
antisense, 5V-TGAGTCCTTCCACGATACCAA-3V). PEDF cDNA was
amplified for 30, 35, and 40 cycles. GAPDH cDNA was amplified
for 35 cycles. Conditions for PEDF PCR were as follows: 94jC for
40 seconds, 58jC for 40 seconds, and then 72jC for 40 seconds.
Conditions for GAPDH PCR were as follows: 94jC for 30 seconds,
56jC for 30 seconds, and then 72jC for 30 seconds. All PCR
products were electrophoresed in a 2.0% agarose gel and visualized
by ethidium bromide staining. As positive controls, plasmids
expressing PEDF were generated by PCR amplification of the
8739 Clin Cancer Res 2005;11(24) December 15, 2005
Cancer Therapy: Preclinical
Fig. 3. Biological activity of PEDF produced by transduced human pancreatic
cancer cells. A, inhibition of HUVEC proliferation by PEDF expressed by lentiviral
vector. B, inhibition of HUVEC migration by PEDF expressed by lentiviral vector.
Columns, mean of six independent experiments; bars, SE. *, P < 0.05, compared
with PCI24 or PCI24GFP.
full-length PEDF cDNA derived from the human splenic cDNA
library (Clontech) and cloning into the Nhe I and Eco RI sites of
pcDNA3.1(+) (Invitrogen).
Preparation of conditioned media derived from pancreatic cancer
cell lines. PCI series and human embryonic pancreas-derived cell lines
(f2  106 cells) were plated on 100-mm cell culture dishes. After 24
hours, the cells were briefly rinsed twice with PBS and then with serum-
free RPMI for 4 hours. The cells were then incubated in 10 mL of fresh,
serum-free RPMI for 48 hours and the media were collected, centrifuged
to remove cell debris, concentrated, and filtrated using 0.45-Am
Millipore Ultrafree centrifugal filters (Millipore, Bedford, MA).
In vitro proliferation assay. Five thousand HUVECs were resus-
pended in 100 AL culture medium, dispensed in each well of a 96-well
culture plate, and preincubated. Then, 100 AL of conditioned medium
from PCI24-untransduced (PCI24-UT), PCI24-infected LV-GFP (PCI24-
GFP), PCI24-infected LV-PEDF (PCI24-PEDF), and PCI43P5 (UT, GFP,
and PEDF) cells and 100 AL of HuMedia-EG2 were added. Cell culture
was continued for 72 hours and assessed using the Cell Counting Kit
8 (Wako Chemicals, Osaka, Japan). The absorbance value of each well
was determined at 450 and 600 nm using a microplate reader
(Molecular Devices, Tokyo, Japan).
In vitro migration assay. Inserts (8-Am pores; Costar) for 24-well
culture plates were coated with 100 Ag/mL rat tail collagen type I
(Becton Dickinson, Franklin Lakes, NJ). HUVECs at passages 4 to 6
were resuspended in HuMedia-EG2 (Sigma) and 10,000 cells in 250 AL
were seeded into the upper chamber. The lower chamber was filled with
HuMedia-EG2. HUVECs were preincubated with conditioned medium
for 30 minutes before human vascular endothelial growth factor
(Kurabo) was added to a final concentration of 5 ng/mL to the lower
Clin Cancer Res 2005;11(24) December 15, 2005
chambers. These chambers were incubated for 5 hours at 37jC with
5% CO2 to allow the cells to migrate through the collagen-coated
membranes. The nonmigrated cells were scraped from the upper surface
of the membrane using cotton swabs. After one rinse with PBS, the
membrane was stained with Diff-Quick Solution (Sysmex, Kobe,
Japan). The number of migrated cells was determined by microscopic
counting of the cells in four representative fields in each well under
a microscope at 400 magnification.
In vivo growth of PEDF-overexpressing cells in subcutaneous tumor
models. PCI24 and PCI43P5 cells (2  106 UT, GFP, and PEDF cells)
were implanted s.c. into the left flanks of BALB/c nude mice. The tumors
were monitored every day and measured after a 7-day interval; the tumor
volume was calculated as follows: tumor volume = length  width2 / 2.
In vivo growth of PEDF-overexpressing cells in peritoneal metastasis
models. PCI24 and PCI43P5 cells (1  107 UT, GFP, and PEDF cells)
resuspended in 1,000 AL PBS were implanted into the peritoneal cavity
of BALB/c nude mice to study peritoneal metastasis. The animals were
sacrificed 20 days after the injection and the mesenteric nodules were
counted.
In vivo gene transfer in subcutaneous tumor models. PCI24 cells (2 
106) were harvested, resuspended in 100 AL PBS, and implanted s.c. into
the left flanks of BALB/c nude mice. After 9 days, when the tumors
measured 3 to 5 mm in diameter, the animals were administered an
intratumoral injection of PBS, LV-PEDF, or LV-GFP at a titer of 3  107
TU in 100 AL PBS. Three days after the first injection, the animals were
administered a second injection in the same manner. The tumors were
monitored every day and measured after a 7-day interval. The animals
were sacrificed 3 weeks after the injection and the tumors were analyzed.
Immunohistochemistry. Immunohistochemical reactions were car-
ried out by the streptavidin-biotin-peroxidase method. Each slide was
deparaffinized with xylene, rehydrated through a graded series of
ethanol/water, and treated in a pressure cooker for 10 minutes.
The slides were immunostained using the Ventana ES automated
immunohistochemistry system (Ventana Medical Systems Japan,
Yokohama, Japan). The automated protocol is based on an indirect
biotin-avidin system and uses a universal biotinylated immunoglob-
ulin secondary antibody, diaminobenzidine substrate, and hematox-
ylin counterstain. Unstained sections were incubated for 32 minutes
at 37jC with a PEDF antibody (monoclonal mouse anti-human
PEDF antibody; Chemicon International, Temula, CA; 1:200 dilu-
tion) or GFP antibody (monoclonal rabbit anti-GFP antibody;
Chemicon International; 1:500 dilution). The anti-PEDF or anti-
GFP antibodies were detected by adding biotinylated goat anti-mouse
antibody, avidin-biotin complex, and 3,3V-diaminobenzidine (Ven-
tana DAB Universal Kit, Ventana-Bio Tek Solutions, Tucson, AZ).
The sections were then counterstained in hematoxylin for 1 minute
and mounted in Permount (Microslides, Muto-Glass, Tokyo, Japan).
Retinal pigment epithelial cells, which are known to react strongly
to PEDF, were used as a positive control. We used 10% normal mouse
serum as the primary antibody for negative controls.
Microvessel staining and counting. The staining process was similar
to that used for PEDF. Intratumoral microvessels were detected using a
monoclonal antibody against the CD34 antigen (monoclonal rat anti-
mouse CD34 antibody; HyCult Biotechnology, Uden, the Netherlands).
The antibody was used at a 1:10 dilution in antibody diluent (Dako-
Cytomation, Carpinteria, CA). After the area of highest neovasculariza-
tion (hotspots) was located by light microscopy at a total magnification
of 40, the microvessel counts were determined at a total magnification
of 100. In all samples, the mean value of the number of microvessels
was calculated from four vascular hotspots.
Sera from healthy volunteers and patients with pancreatic cancer. We
collected sera from healthy volunteers (n = 8) and from patients with
pancreatic cancer before the operation (n = 11; stage I, 1; stage III, 1;
stage IVA, 3; and stage IVB, 6; at this Department from January to
November 2004). Informed consent was obtained from each patient
before enrollment in this study in accordance with the guidelines of the
Ethics Committee of Hokkaido University.
8740 www.aacrjournals.org
 Antitumor Activities of PEDF on Human Pancreatic Cancer

ELISA. Concentrations of PEDF in serum and conditioned medium LV-PEDF or control vector LV-GFP, we incubated HUVECs
were determined using a commercial PEDF ELISA kit (Chemicon with conditioned medium at a final concentration of 50%. We
International) according to the instructions of the manufacturer. observed that HUVECs incubated with the conditioned
Briefly, the serum and supernatants were treated with 8 mol/L urea
medium from PCI24-PEDF and PCI43P5-PEDF cells inhibited
on ice for 1 hour. The urea-treated samples were diluted 1:100 in assay
proliferation by 25% and 28%, respectively, when compared
diluent, immediately added to the antibody-coated wells, and
incubated at 37jC for 1 hour. After four washes, 100-AL diluted bio-
with conditioned medium from untransduced and GFP
tinylated mouse anti-human PEDF monoclonal antibody was added to cells (Fig. 3A). The conditioned medium from PCI24-GFP
each well and the wells were incubated at 37jC for 1 hour. Then, and PCI43P5-GFP cells had no inhibitory effect on the
100-AL diluted streptavidin-peroxidase conjugate was added and proliferation of HUVECs. The PEDF-mediated inhibitory effect
incubated at 37jC for 1 hour. Following addition of tetramethylbenzi- on proliferation is specific to HUVECs because we found that
dine in a proprietary buffer with enhancer for 5 to 10 minutes, 100-AL
stop solution was added and the absorbance was immediately
measured at 450 nm using a microplate reader. For standardization,
the PEDF concentration was normalized to the protein concentration
in the samples.
Statistical analysis. All values were presented as mean F SE.
Statistical significance was evaluated using the Mann Whitney U test;
P < 0.05 was considered significant.

Results
PEDF expression by pancreatic adenocarcinoma cells and
embryonic pancreatic tissue. To determine the endogenous
expression of PEDF, we examined PEDF mRNA and protein
expression levels in the human embryonic pancreas-derived cell
lines 1C3D3 and 1B2C6 and in the human pancreatic cancer
cell lines of the PCI series. PEDF mRNAs were detected in two
of two human embryonic pancreas-derived cell lines and in five
of eight human pancreatic adenocarcinoma cell lines (Fig. 1A).
Western blotting revealed PEDF protein expression in two of
two human embryonic pancreas-derived cell lines and in two
of eight human pancreatic adenocarcinoma cell lines in the
conditioned media (Fig. 1B).
Expression of PEDF in pancreatic cancer cells after lentivirus-
mediated gene transfer in vitro. The bicistronic lentiviral
construct included human PEDF cDNA followed by an
internal ribosomal entry site (IRES) coupled to GFP cDNA
(Fig. 2A). Control cells were transduced with the same
lentiviral construct lacking the PEDF cDNA. To determine
whether PEDF could be secreted in lentivirus-infected cells,
Western blotting using a monoclonal antibody against human
PEDF was used to analyze the conditioned medium and lysate
from PCI24 and PCI43P5 cells that were infected in vitro with
either LV-PEDF or the control vector LV-GFP. As shown in
Fig. 2B, in the conditioned medium and lysate from LV-
PEDF infected cells, we observed only one band of MW 50
kDa; this band coincided with the one present in the lane
loaded with recombinant human PEDF protein. No PEDF
was expressed in the conditioned medium and lysates from
PCI24-UT and PCI24-GFP whereas little PEDF was expressed
in the conditioned medium and lysates from PCI43P5-UT and
PCI43P5-GFP. To confirm the expression of PEDF in vivo, we
immunostained PCI24-UT , PCI24-GFP , and PCI24-PEDF
implanted severe combined immunodeficient mice. The
transgene expressions of tumors were clearly observed in the
s.c. tumor sections of severe combined immunodeficient mice,
Fig. 4. Tumor volumes of PCI24 and PCI43P5 cells not transduced or stably
which were immunostained with anti-PEDF and anti-GFP transduced with the IRES-GFP expression lentivirus alone or with the human
antibodies (Fig. 2C). PEDF-IRES-GFP expression lentivirus. A and B, the sizes of PCI24-PEDF and
Biological activity of PEDF produced by LV-PEDF in vitro. PCI43P5-PEDF were significantly smaller than those of PCI24-GFP and
PCI43P5-GFP in s.c. injection models. Points, mean from eight (PCI24) and five
To evaluate the antiproliferative effect of the conditioned (PCI43P5) mice in each group; bars, SE. *, P < 0.05. C, representative
medium from PCI24 and PCI43P5 cells infected with photomicrographs showing the effect of PEDF on the growth of s.c. models.

www.aacrjournals.org 8741 Clin Cancer Res 2005;11(24) December 15, 2005

Cancer Therapy: Preclinical
the conditioned medium from PCI24-PEDF and PCI43P5-
PEDF cells had no effect on the growth rates of PCI24 and
PCI43P5 tumor cells (data not shown). We also tested the
effect of the conditioned medium from PCI24-PEDF and
PCI43P5-PEDF cells on the migration of HUVECs by using a
modified double chamber migration assay. We found that in
comparison with the control group, the percentage of cell
migration of HUVECs was reduced by 55% and 61% when
using the conditioned medium from PCI24-PEDF and
PCI43P5-PEDF cells (Fig. 3B). The conditioned medium from
PCI24-GFP and PCI43P5-GFP cells did not affect cell
migration.
In vivo growth of PEDF-overexpressing pancreatic cancer cells
in subcutaneous tumor models. We examined whether PEDF
overexpression influences tumor growth in PCI24 and PCI43P5
cells in s.c. tumor models in vivo. As shown in Fig. 4, untra-
nsduced and control vector-transduced cells formed rapidly
growing tumors. In contrast, PEDF overexpression inhibited
in vivo tumor growth of PCI24 and PCI43P5 cells to a great
extent throughout this experiment (Fig. 4).
In vivo growth of PEDF-overexpressing pancreatic cancer cells
in peritoneal dissemination models. We subsequently evaluated
the growth of PEDF-overexpressing peritoneal tumors. PCI43P5
cells were implanted into the peritoneal cavity and tumors were
established in athymic mice because these cells could be trans-
planted with greater efficiency than other cell lines. As shown
in Fig. 5, the growth of PCI43P5-PEDF was significantly
suppressed in comparison with that of PCI43P5-UT and
PCI43P5-GFP (P < 0.05) in models used for the study of
peritoneal metastasis.
In vivo gene transfer studies in subcutaneous tumor models.
The ability of PEDF gene transfer to inhibit tumor growth
in vivo was evaluated by injecting established s.c. flank tumors
with LV-PEDF and following their growth over time. Nine days
after the inoculation of PCI24 cells, the mice were administered
an intratumoral injection of LV-PEDF or control vector LV-GFP
at a dose of 3  107 TU/mL or were administered PBS. As
shown in Fig. 6A, LV-PEDF treated mice showed a significant
inhibition of tumor growth when compared with mice treated
with the control vector LV-GFP or PBS. Fourteen days after the
intratumoral injection, the tumor volume of the LV-PEDF
treated mice was significantly smaller than that of animals
from control groups; a 44% and 55% reduction in tumor
volume was observed in LV-PDEF treated mice when com-
Fig. 5. A, representative photomicrographs showing the effect of PEDF on the growth of peritoneal metastasis. B, counts of mesenteric nodules in mice that were
administered PCI43P5, PCI43P5-GFP, or PCI43P5-PEDF cells (n = 5). *, P < 0.05, compared with PCI43P5 or PCI43P5GFP.
Clin Cancer Res 2005;11(24) December 15, 2005
pared with mice treated with the control vector LV-GFP or PBS,
respectively.
To confirm the transgene expressions, we evaluated the
tumors treated with LV-PEDF, LV-GFP, and PBS by immuno-
histochemical analysis for detection of the anti-GFP antibody.
PCI24 cells treated with LV-PEDF and LV-GFP were clearly
stained in the tumors (Fig. 6B).
The ability of PEDF gene transfer to inhibit angiogenesis
was evaluated by immunohistochemical analysis of treated
flank tumors through blinded quantification of microvessel
density. As shown in Fig. 6B, tumors from animals receiving
PBS or the control vector LV-GFP showed intense staining
for CD34, indicating the presence of extensive angiogenesis in
this type of tumor. Tumors from LV-PEDF treated mice
showed a marked reduction in microvessel density (Fig. 6C).
Quantitative analysis showed 70% and 75% reduction in
the intratumoral microvessel density in LV-PEDF treated
animals when compared with control mice treated with
LV-GFP or PBS, respectively. Treatment with the control vector
LV-GFP did not alter the intratumoral microvessel density.
PEDF expression in human serum by ELISA. We examined
whether PEDF expression is altered in the sera of healthy
volunteers or patients with pancreatic cancer. ELISA revealed
that the levels of PEDF protein in the sera of patients with
pancreatic cancer were significantly lower than those in healthy
volunteers (Fig. 7).
Discussion
In this study, we have shown that in vivo transfer of PEDF
mediated by the lentiviral vector exerts dramatic inhibition of
tumor growth in athymic nude mice implanted with the
human pancreatic adenocarcinoma cell lines. The suppression
of tumor growth was associated with decreased microvessel
density in tumors treated with LV-PEDF.
These data suggest that decreased PEDF expression may
contribute to tumor progression, possibly through increased
tumor cell proliferation and increased angiogenesis. Because
cancer cells acquire their proliferative ability through the
accumulation of several genetic changes, effective therapy must
be developed based on the biological feature of individual
tumors. In pancreatic tissues, although normal tissue requires
PEDF expression to maintain itself, down-regulation of PEDF
expression is one factor that results in abnormalities and
8742 www.aacrjournals.org

Fig. 6. Inhibition of tumor growth and angiogenesis by treatment with LV-PEDF.
A, columns, mean tumor volume of PCI24 cells after intratumoral administration of
lentivirus; bars, SE. Intratumoral administration of LV-PEDF inhibits the growth of
established murine tumors. B, immunohistochemical analysis of the growing
tumors 30 days after administration of lentivirus (original magnification,  200).
C, columns, mean tumor microvessel densities in sections from lentivirus-injected
tumors; bars, SE.
malignant features that lead to the formation of cancer cells.
Indeed, knockout of the PEDF gene causes epithelial cell
hyperplasia in the pancreas in mice (20) and reduced PEDF
expression is one of the factors responsible for liver metastasis
(21). In respect to this, we investigated whether PEDF secretion
is down-regulated in pancreatic adenocarcinoma and investi-
gated the serum PEDF protein concentration in patients with
pancreatic adenocarcinoma. In the present study, the PEDF
concentration in the sera of pancreatic cancer patients was
significantly lower than that of healthy volunteers although it is
unclear whether the total amount of PEDF was secreted from
the pancreas. These data suggest that PEDF is a key molecule in
patients with pancreatic cancer and may be responsible for the
high malignancy of this disease.
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Antitumor Activities of PEDF on Human Pancreatic Cancer
Fig. 7. PEDF protein expression in serum of patients with pancreatic
adenocarcinoma. Serum samples were analyzed by ELISA. The levels of PEDF
protein in the serum of patients with pancreatic adenocarcinoma were lower than
those in healthy volunteers. P < 0.05, compared with healthy volunteers.
Data presented in this article show for the first time that
using lentiviral vectors, the antiangiogenic properties of PEDF
could be exploited to inhibit tumor angiogenesis and growth
in pancreatic adenocarcinoma. Previous studies on a murine
model of lung cancer and hepatocellular carcinoma have
shown that tumor growth could be inhibited by systemic
administration or intratumoral injection of adenovirus con-
taining PEDF (24, 25). Although adenoviruses are also useful
as vectors for gene transfer in a variety of cell types, they result
in short-term expression. However, long-term expression of
antiangiogenic factor is necessary to inhibit tumor growth.
Lentiviral vectors are attractive tools for human cancer gene
therapy (26 29) and, based on their ability to integrate into
the genome, they have the potential to achieve long-term
stable expression and maintain therapeutic levels of secreted
peptides. Therefore, lentivirus-mediated gene delivery of
antiangiogenic factors seems to be a promising approach for
cancer gene therapy. In addition to their ability to achieve
stable integration into the chromosomes and their relatively
large cloning capacity, lentiviruses offer the advantage of
transducing nondividing cells. This feature is of great
advantage for gene transfer in cancer cells because nondivid-
ing cancer cells are usually concentrated in the hypoxic cores
of tumors (30) and represent a chemoresistant population
(31). About the present data on tumor volume, suppression of
tumor growth by LV-PEDF was only f40% because gene
therapy was started after the tumors had grown >30 mm3. Few
studies on gene therapies have reported notable therapeutic
efficacy for established tumor. However, in respect to cell
viability, abundant necrotic cells were observed in the tumor
infected with LV-PEDF than in the control tumors and a small
number of viable cells were found in only the growing edge of
tumors.
In the clinical trial of LV-PEDF for human pancreatic cancer,
LV-PEDF should be administered by i.m. injection because we
believe that it should be used as an adjuvant therapy for
micrometastasis of a few cancer cells after surgical resection. In
the present study, LV-PEDF was injected directly into tumors;
however, it can be used even when the location of the tumor is
unclear because PEDF is secreted from LV-PEDF infected
normal tissues.
8743 Clin Cancer Res 2005;11(24) December 15, 2005
Cancer Therapy: Preclinical
In conclusion, our data suggest that PEDF may exert a
biological effect on tumor angiogenesis and PEDF gene therapy
may provide a new approach for treatment of pancreatic
adenocarcinoma.
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