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Pigment Epithelium–Derived Factor Gene Therapy Inhibits Human Pancreatic Cancer in Mice

Authors' Affiliation: Department of Surgical Oncology, Division of Cancer Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan

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{at}med.hokudai.ac.jp.

	Abstract

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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 pancreatic adenocarcinoma cells was significantly suppressed in comparison with control cells in peritoneal metastasis models. In gene transfer models, intratumoral injection of a lentivirus vector encoding PEDF (LV-PEDF) caused significant inhibition of tumor growth. The antitumor effect 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.

The dependency of tumor growth on the ability to induce a neovascular response is supported by significant experimental and clinical data (12, 13). Angiogenesis is regulated by complex signaling pathways acting on endothelial cells 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 development.

Pigment epithelium–derived factor (PEDF) was first identified 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 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

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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 37°C in a humidified atmosphere containing 5% CO₂.

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 ViraPower 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 x 10⁵ per well and allowed to attach overnight. The medium was replaced with 1 mL fresh complete medium, 100 µL lentiviral supernatant (LV-PEDF and LV-GFP), and 8 µg/mL hexadimethrine 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 µg/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 collected 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 x 10⁵ HeLa cells were plated in six-well clusters and infected with serial 10-fold dilutions of viral vectors (10^–1-10^–5) in a 2-mL volume of culture medium. In the case of lentiviral vectors, 8 µg/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) x (percentage of GFP-positive cells) x (dilution factor). To assess the relative infectivity of lentivirus vectors on the different types of cells used for these experiments, 2 x 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, Kumamoto, 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).

Reverse transcription-PCR. Total cellular RNA was isolated from each cell line using the Trizol reagent (Invitrogen). Each 20-µL cDNA synthesis reaction mixture contained 1 µg total RNA, 4 µL 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 µL (200 units) SuperScript II (Invitrogen), 2 µL of 0.1 mol/L DTT (Invitrogen), and 1 µL Oligo(dT) (Invitrogen). The reverse transcription reaction was carried out for 50 minutes at 42°C and inactivated by heating at 70°C for 15 minutes. Multiplex PCR was done as previously described (23). Briefly, each 30-µL reaction mixture contained 1 µL reverse transcription reaction products, 0.1 µL Taq DNA polymerase (Promega, Madison, WI), 3 µL reaction buffer (Promega), 160 mmol/L of each deoxynucleotide, and 20 pmol of each of the 3' and 5' primers specific for PEDF (sense, 5'-ATGAAGAGAGGACCGTGAGGG-3'; antisense, 5'-CCCATCCTCGTTCCACTCA-3') and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sense, 5'-ACCCCTTCATTGACCTCAACT-3'; antisense, 5'-TGAGTCCTTCCACGATACCAA-3'). PEDF cDNA was amplified for 30, 35, and 40 cycles. GAPDH cDNA was amplified for 35 cycles. Conditions for PEDF PCR were as follows: 94°C for 40 seconds, 58°C for 40 seconds, and then 72°C for 40 seconds. Conditions for GAPDH PCR were as follows: 94°C for 30 seconds, 56°C for 30 seconds, and then 72°C 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 full-length PEDF cDNA derived from the human splenic cDNA library (Clontech) and cloning into the NheI and EcoRI sites of pcDNA3.1(+) (Invitrogen).

Preparation of conditioned media derived from pancreatic cancer cell lines. PCI series and human embryonic pancreas-derived cell lines (2 x 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-µm Millipore Ultrafree centrifugal filters (Millipore, Bedford, MA).

In vitro proliferation assay. Five thousand HUVECs were resuspended in 100 µL culture medium, dispensed in each well of a 96-well culture plate, and preincubated. Then, 100 µL 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 µL 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-µm pores; Costar) for 24-well culture plates were coated with 100 µg/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 µL 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 chambers. These chambers were incubated for 5 hours at 37°C 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 x400 magnification.

In vivo growth of PEDF-overexpressing cells in subcutaneous tumor models. PCI24 and PCI43P5 cells (2 x 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 x width² / 2.

In vivo growth of PEDF-overexpressing cells in peritoneal metastasis models. PCI24 and PCI43P5 cells (1 x 10⁷ UT, GFP, and PEDF cells) resuspended in 1,000 µL 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 x 10⁶) were harvested, resuspended in 100 µL 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 x 10⁷ TU in 100 µL 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 carried 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 immunoglobulin secondary antibody, diaminobenzidine substrate, and hematoxylin counterstain. Unstained sections were incubated for 32 minutes at 37°C with a PEDF antibody (monoclonal mouse anti-human PEDF antibody; Chemicon International, Temula, CA; 1:200 dilution) 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 3,3'-diaminobenzidine (Ventana 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 (DakoCytomation, Carpinteria, CA). After the area of highest neovascularization (hotspots) was located by light microscopy at a total magnification of x40, the microvessel counts were determined at a total magnification of x100. 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.

ELISA. Concentrations of PEDF in serum and conditioned medium were determined using a commercial PEDF ELISA kit (Chemicon International) according to the instructions of the manufacturer. Briefly, the serum and supernatants were treated with 8 mol/L urea on ice for 1 hour. The urea-treated samples were diluted 1:100 in assay diluent, immediately added to the antibody-coated wells, and incubated at 37°C for 1 hour. After four washes, 100-µL diluted biotinylated mouse anti-human PEDF monoclonal antibody was added to each well and the wells were incubated at 37°C for 1 hour. Then, 100-µL diluted streptavidin-peroxidase conjugate was added and incubated at 37°C for 1 hour. Following addition of tetramethylbenzidine in a proprietary buffer with enhancer for 5 to 10 minutes, 100-µL 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 ± SE. Statistical significance was evaluated using the Mann–Whitney U test; P < 0.05 was considered significant.

	Results

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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).

View larger version (59K): [in this window] [in a new window]   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.

View larger version (39K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Tumor volumes of 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. A and B, the sizes of PCI24-PEDF and 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 (PCI43P5) mice in each group; bars, SE. *, P < 0.05. C, representative photomicrographs showing the effect of PEDF on the growth of s.c. models.

View larger version (27K): [in this window] [in a new window]   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.

View larger version (34K): [in this window] [in a new window]   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, x 200). C, columns, mean tumor microvessel densities in sections from lentivirus-injected tumors; bars, SE.

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).

View larger version (9K): [in this window] [in a new window]   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.

	Discussion

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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 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 investigated 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.

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 containing 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 nondividing 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 40% 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.

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.

	Acknowledgments

 
We thank Hiraku Shida and Rika Osanai for their technical support in immunohistochemical analyses and Dr. Jiro Arikawa and Tsutomu Osanai for their advice on animal experiments.

	Footnotes

 
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.

Received 6/21/05; revised 9/ 3/05; accepted 9/21/05.

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