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10.1245/ASO.2005.03.081
Annals of Surgical Oncology 12:1025-1036 (2005)
© 2005 Society of Surgical Oncology
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Original Article

Herpes Simplex Virus Amplicon Delivery of a Hypoxia-Inducible Soluble Vascular Endothelial Growth Factor Receptor (sFlk-1) Inhibits Angiogenesis and Tumor Growth in Pancreatic Adenocarcinoma

Maura Reinblatt, MD1, Richard H. Pin, MD1, William J. Bowers, PhD2, Howard J. Federoff, MD, PhD2 and Yuman Fong, MD1

1 Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021
2 Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Correspondence: Address correspondence and reprint requests to: Yuman Fong, MD; E-mail: fongy{at}mskcc.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Tumor hypoxia induces vascular endothelial growth factor (VEGF) expression, which stimulates angiogenesis and tumor proliferation. The VEGF signaling pathway is inhibited by soluble VEGF receptors (soluble fetal liver kinase 1; sFlk-1), which bind VEGF and block its interaction with endothelial cells. Herpes simplex virus (HSV) amplicons are replication-incompetent viruses used for gene delivery. We attempted to attenuate angiogenesis and inhibit pancreatic tumor growth through HSV amplicon–mediated expression of sFlk-1 under hypoxic control.

Methods: A multimerized hypoxia-responsive enhancer (10 x HRE) was cloned upstream of the sFlk-1 gene (10 x HRE/sFlk-1). A novel HSV amplicon expressing 10 x HRE/sFlk-1 was genetically engineered (HSV10 x HRE/sFlk-1). Human pancreatic adenocarcinoma cells (AsPC1) were transduced with HSV10 x HRE/sFlk-1 and incubated in normoxia (21% oxygen) or hypoxia (1% oxygen). Capillary inhibition was evaluated by human umbilical vein endothelial cell assay. Western blot assessed sFlk-1 expression. AsPC1 flank tumor xenografts (n = 24) were transduced with HSV10 x HRE/sFlk-1.

Results: Media from normoxic AsPC1 transduced with HSV10 x HRE/sFlk-1 yielded a 36% reduction in capillary formation versus controls (P < .05), whereas hypoxic AsPC1 yielded a 76% reduction (P < .005). Western blot of AsPC1 transduced with HSV10 x HRE/sFlk-1 demonstrated greater sFlk-1 expression in hypoxia versus normoxia. AsPC1 flank tumors treated with HSV10 x HRE/sFlk-1 exhibited a 59% reduction in volume versus controls (P < .000001).

Conclusions: HSV amplicon delivery of a hypoxia-inducible soluble VEGF receptor significantly reduces new vessel formation and tumor growth. Tumor hypoxia can thus be used to direct antiangiogenic therapy to pancreatic adenocarcinoma.

Key Words: Amplicon • Hypoxia • Flk-1 • Herpes simplex virus • Pancreatic cancer • Vascular endothelial growth factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The selective delivery of therapeutic genes to tumor cells remains a primary obstacle in cancer gene therapy. However, lower tumor oxygen levels compared with surrounding normal tissues have recently been used to successfully target cancer.1 Hypoxia, a common solid-tumor condition, develops as tumors proliferate and outgrow their vasculature. Pancreatic tumors are hypoxic, with median oxygen tensions between 0 and 5.3 mm Hg and with significantly greater oxygen tensions in adjacent normal pancreatic tissue (24–93 mm Hg).2 Tumor hypoxia has been correlated with metastatic spread and resistance to standard chemoradiation. Hypoxic tumors therefore behave more aggressively and are associated with a poor prognosis irrespective of treatment modality.3–5 Hypoxia, an innate tumor characteristic that promotes a more malignant phenotype, may be used to target gene therapy in pancreatic adenocarcinoma.

Adenocarcinoma of the pancreas carries a dismal overall 5-year survival of <5%, with adjacent tissue invasion, early metastases, and resistance to conventional chemoradiotherapy.6,7 The aggressive nature of pancreatic cancer has been correlated with tumor hypoxia. Low tumor oxygen levels stimulate the production of angiogenic factors, such as vascular endothelial growth factor (VEGF), which promote tumor growth and spread.8 Hypoxia upregulates VEGF expression by inducing the transcriptional regulator hypoxia-inducible factor (HIF)-1, which binds to the hypoxia-responsive element (HRE) in the VEGF promoter. HIF-1 is a heterodimer composed of the hypoxia-inducible {alpha} subunit (HIF-1{alpha}) and the constitutively expressed ß subunit (HIF-1ß).9 The interaction between HIF-1 and HRE is the main stimulus for VEGF expression in pancreatic cancer cells.10

VEGF is a key regulator of tumor angiogenesis and is the most potent endothelial cell mitogen.11,12 Binding of VEGF to its receptor on the endothelial cell membrane stimulates the VEGF signaling pathway. VEGF receptor 2 (fetal liver kinase 1; Flk-1) is the primary VEGF receptor on endothelial cells.13 After VEGF binds to Flk-1, the receptor dimerizes and transduces an intracellular signal through associated receptor tyrosine kinase activity.14 This process stimulates endothelial cell differentiation and survival, capillary tube formation, and vascular permeability.15,16 Conversely, blocking the interaction between VEGF and Flk-1 has been shown to inhibit human tumor xenograft growth in athymic mice.17 Soluble forms of VEGF receptors, lacking their membrane spanning domains, have been used to interrupt the VEGF signaling pathway.18 These soluble receptors, such as soluble Flk-1 (sFlk-1), bind VEGF and prevent its interaction with membrane-bound endothelial cell receptors.

Directing sFlk-1 expression to tumor cells can be accomplished through viral vectors. Replication-incompetent herpes simplex viruses (HSV amplicons) are highly efficient gene-transfer vehicles.19 Genetically engineered to lack viral sequences necessary for replication, HSV amplicons function as transducing vectors for therapeutic genes but elicit minimal cytotoxicity themselves.20 They have been used successfully to deliver immunomodulatory cytokines for cancer gene therapy.21,22 HSV amplicons are able to transduce a wide array of cell types and have the capacity to carry large transgenes (up to 150 kilobases).23 In addition, they transduce actively dividing, quiescent, and postmitotic cells.19 These amplicons are therefore attractive vectors for directing antiangiogenic therapy to pancreatic tumors.

This study explored the potential application of a hypoxia-inducible soluble VEGF receptor in treating pancreatic adenocarcinoma. We set out to attenuate angiogenesis and inhibit pancreatic tumor growth through HSV amplicon–mediated delivery of sFlk-1 under hypoxic control.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Hypoxic Treatment
AsPC1 are a well-described human pancreatic adenocarcinoma cell line. AsPC1 cells were obtained (American Type Culture Collection, Manassas, VA), maintained in a 5% carbon dioxide humidified incubator at 37°C, and subcultured twice a week. Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM of L-glutamine, 1.5 g/L of sodium bicarbonate, 4.5 g/L of glucose, and 1 mM of sodium pyruvate.

Human umbilical vein endothelial cells (HUVECs) were obtained (Cambrex, East Rutherford, NJ), maintained in a 5% carbon dioxide humidified incubator at 37°C, and subcultured twice a week. HU-VECs were grown in EGM-2 media (Cambrex). All HUVEC culture surfaces were treated with .2% gelatin.

Hypoxic conditions were created by displacement of oxygen with nitrogen in a triple-gas incubator (NuAire, Plymouth, MN). Oxygen concentrations as low as 1% could be maintained. After plating, cells were routinely incubated for 12 hours in 21% oxygen to allow for attachment and stabilization before hypoxic exposure.

VEGF Expression
AsPC1 were plated at 5 x 106 cells per dish in 100-mm culture dishes (Costar, Corning Inc., Corning, NY) and incubated in either normoxia (21% oxygen) or hypoxia (1% oxygen). After a 24-hour incubation, cell lysates were collected (Cell Signaling Technology, Beverly, MA), and protein concentrations were determined according to the Bradford method (Bio-Rad Protein Assay Reagent; Bio-Rad, Hercules, CA) by measuring absorbance at 595 nm (DU 640 spectrophotometer; Beckman, Fullerton, CA). VEGF enzyme-linked immunosorbent assay (ELI-SA; R&D Systems Inc., Minneapolis, MN) was performed in quadruplicate on 30 µg of protein from each sample.

HIF-1{alpha} Expression
AsPC1 cells were plated at 5 x 106 cells per dish in 100-mm culture dishes (Costar, Corning Inc.). Cells were incubated in either 21% oxygen or 1% oxygen for 1, 5, and 24 hours. HIF-1{alpha} is a nuclear protein; therefore, nuclear extracts were collected from both normoxic and hypoxic cells at these time points (Active Motif, Carlsbad, CA). The protein content of nuclear extracts was determined by the Bradford method (Bio-Rad). HIF-1{alpha} ELISA (BD Biosciences, Palo Alto, CA) was performed in triplicate on 30 µg of nuclear protein from each time point.

Construction of Hypoxia-Responsive Enhancer
Complementary 41–base pair oligonucleotides were constructed that encoded the HIF-1 recognition sequence from the promoter of the human VEGF gene (Invitrogen, Carlsbad, CA). These monomeric hypoxia-responsive enhancers (HREs) were designed with XhoI- and SalI-compatible ends for multimerization and cloning. The complementary sequences were as follows: 5'-TCGAGCCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTG-3' and 5'-TCGACAAGAGGACCTGTTGGAGCCCACGTATGCACTGTGC-3'.24 Paired oligomers were annealed by heating a 1:1 mixture to 94°C for 3 minutes in sodium Tris-ethylenediaminetetraacetic acid buffer (pH 8; Fisher Scientific, Pittsburgh, PA) and allowing the mixture to cool to room temperature. The 5' ends of the double-stranded HRE product were phosphorylated with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) for subsequent multimerization. Ten copies of the HRE fragment were tandemly ligated with Quick Ligase (New England Biolabs). The resulting multimerized hypoxia responsive enhancer (10 x HRE) was confirmed with sequence analysis and restriction digestion.

Enhancer Function
We cloned the 10 x HRE enhancer into the XhoI site of the pGL3 promoter vector (Promega, Madison, WI) upstream of the minimal SV40 promoter; this formed the luciferase reporter plasmid 10 x HRE/pGL3. AsPC1 cells were plated at 1.5 x 106 cells per well in six-well flat-bottom plates (Costar, Corning Inc.). Cells were transfected with 1.6 µg of the 10 x HRE/pGL3 plasmid by using Lipofectamine 2000 (Invitrogen). The native pGL3 plasmid was used as a control in these experiments. Cells were incubated in either 21% oxygen or 1% oxygen for 18 hours. Cell lysates were collected, and a luciferase assay (Luciferase Assay Kit; Promega) was performed by using a single injection luminometer (Berthold Technologies MicroLumat Plus, Oak Ridge, TN). Protein concentrations of cell lysates were determined by Bradford’s method (Bio-Rad) to normalize luciferase activity between transfected groups.

Construction of Vector Expressing Hypoxia-Responsive sFlk-1
A plasmid containing the sFlk-1 gene was obtained (Invivogen, San Diego, CA). The sFlk-1 gene was isolated by restriction digestion with NcoI and NheI (New England Biolabs). The NheI site was blunted through exonuclease digestion with Klenow (New England Biolabs). 10 x HRE/pGL3 was digested with NcoI and XbaI to remove the luciferase gene, and the XbaI site was blunted (Klenow). The sFlk-1 gene was ligated between the former NcoI and XbaI sites of 10 x HRE/pGL3 (Quick Ligase), thus forming the 10 x HRE/sFlk-1 plasmid. The sequence was confirmed by polymerase chain reaction (PCR) and restriction digestions.

Construction of HSV Amplicon Expressing 10 x HRE/sFlk-1
The pHSVminOriSmc vector, containing the HSV packaging sequences and lacking the genes required for viral replication, was used. The10 x HRE/sFlk-1 sequence with its poly-A tail was PCR-amplified from the 10 x HRE/sFlk-1 plasmid by using forward (5'-GATAAGGATCCGAGCTCTTACGCGTGCTAGC-3') and reverse (5'-TGACTGGGTTGAAGGCTCTCAAGGGCATCG-3') primers. These primers maintained a BamHI restriction site downstream of the poly-A tail and introduced a BamHI site upstream of 10 x HRE. The amplified PCR product was digested with BamHI (New England Biolabs) and ligated into pHSVminOriSmc at the BamHI site (Quick Ligase) to create HSV10 x HRE/sFlk-1. The sequence was confirmed by PCR and restriction digestion. A control amplicon (HSV-lacZ) expressing the ß-galactosidase (lacZ) reporter gene was constructed in the same fashion. These amplicon vectors were packaged and purified.25 Viral pellets were then resuspended in 100 µL of phosphate-buffered saline (PBS) and titered.26 Samples were stored at –80°C until use.

Assessment of Capillary Inhibition by Tube Formation Assay
AsPC1 cells were plated at 1 x 105 cells per well in six-well plates by using Opti-MEM (Memorial Sloan–Kettering Cancer Center, New York, NY) supplemented with 1% fetal calf serum and transduced with 5 x 104 transducing units (TU) of HSV10 x HRE/sFlk-1 or HSVlacZ. Cells were incubated at 21% oxygen or 1% oxygen for 3 days. After this incubation, conditioned medium from the AsPC1 cells was collected, supplemented with 10 ng/mL of VEGF (Sigma-Aldrich, St. Louis, MO), and used to plate HUVECs. Conditioned media without VEGF supplementation served as negative controls. After a 14-hour incubation, two independent observers counted capillary tubes.

Soluble Flk-1 Western Blot
The AsPC1 cells that had been used to condition media for the HUVEC assay were lysed with cell lysis buffer (Cell Signaling Technology). Protein concentrations of cell lysates were determined by Bradford’s method (Bio-Rad), and 50 µg of protein from each sample was loaded into sodium dodecyl sulfate polyacrylamide gels (Bio-Rad). After gel electrophoresis in Tris-glycine-sodium dodecyl sulfate (pH 8.4; Fisher Scientific), protein was transferred to polyvinylidene difluoride membranes for 1.5 hours in Trisglycine-sodium dodecyl sulfate containing 20% methanol at 250 mA and 4°C. Membranes were incubated for 16 hours in 4% milk containing either a 1/500 dilution of a mouse monoclonal anti-human Flk-1 antibody (Research Diagnostics Inc., Flanders, NJ) or a 1/1000 dilution of a control goat polyclonal anti–ß-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After primary antibody incubation, the membranes were blocked in PBS containing 4% milk. Flk-1 and ß-actin membranes were incubated in secondary antibodies (goat anti-mouse immunoglobulin G/horseradish peroxidase and donkey anti-goat immunoglobulin G/horseradish peroxidase, respectively [Santa Cruz Biotechnology]) diluted 1/1000 in PBS containing 4% milk. After membranes were washed in PBS, protein bands were visualized with a chemiluminescence kit (ECL Plus Western Blotting Detection System; Amersham Biosciences, Piscataway, NJ) and exposed on X-omat AR film (Kodak, Rochester, NY).

Establishment and Treatment of Flank Tumors
All animal procedures were performed with the approval of the Memorial Sloan-Kettering Institutional Animal Care and Use Committee. Six-week-old male athymic mice were obtained (Charles River Laboratories, Wilmington, MA). Mice were anesthetized with intraperitoneal injection of ketamine and xylazine (100 mg/kg of ketamine and 10 mg/kg of xylazine) for all procedures. Mice were housed three per cage and allowed food and water ad libitum.

Twelve athymic mice underwent bilateral subcutaneous flank injections with 1 x 106 AsPC1 cells. After tumor volumes reached approximately 30 mm3, mice were randomized to receive 1 x 106 TU of HSV10 x HRE/sFlk-1 (n = 8), 1 x 106 TU of HSVlacZ (n = 8), or 50 µL of PBS (n = 8) through direct intratumoral injection. The flank tumors of each animal received the same treatment. Tumor volumes were assessed over 5 weeks.

Statistical Analysis
Data are expressed as the mean ± SD. Comparisons between groups were performed with a two-tailed Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF Expression
AsPC1 cells incubated at 1% oxygen or 21% oxygen were examined for VEGF expression by ELISA. VEGF levels were 224 ± 27 pg/mL in hypoxic AsPC1 cell lysates and 30 ± 3 pg/mL in normoxic AsPC1 cell lysates. These levels represent a 7.5-fold increase in AsPC1 VEGF expression after 24 hours in hypoxia compared with normoxia (P < .001; Fig. 1AGo).


Figure 1
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  		FIG. 1. Human pancreatic adenocarcinoma cells (AsPC1) increased vascular endothelial growth factor (VEGF) expression and hypoxia-inducible factor (HIF)-1{alpha} in hypoxia (1% oxygen). (A) After 24 hours of incubation in hypoxia or normoxia (21% oxygen), AsPC1 cell lysates underwent VEGF enzyme-linked immunosorbent assay (R&D Systems Inc., Minneapolis, MN). VEGF levels increased 7.5-fold in hypoxia versus normoxia (P < .001). (B) AsPC1 cells were incubated in either hypoxia or normoxia, and nuclear extracts were collected at 1, 5, and 24 hours. HIF-1{alpha} enzyme-linked immunosorbent assay (BD Biosciences, Palo Alto, CA) was performed on 30 µg of nuclear protein from each sample. At 24 hours, there was a 7.3-fold increase in HIF-1{alpha} expression in 1% oxygen compared with 21% oxygen (P < .005).

 
HIF-1{alpha} Expression
AsPC1 cells incubated at 1% oxygen or 21% oxygen were examined for HIF-1{alpha} expression by ELISA. AsPC1 cells demonstrated a 1.9-fold increase in HIF-1{alpha} expression after 1 hour of hypoxic incubation compared with normoxic cells (P < .005), a 5.0-fold increase after 5 hours (P < .005), and a 7.3-fold increase after 24 hours (P < .005; Fig. 1BGo).

Luciferase Activity
10 x HRE enhancer function was assessed by luciferase reporter assay. After incubation in 1% oxygen or 21% oxygen, AsPC1 cell lysates underwent analysis for luciferase expression. In 1% oxygen, AsPC1 cells transfected with 10 x HRE/pGL3 yielded a 49-fold increase in luciferase activity compared with cells transfected with pGL3 alone (P < .01; Fig. 2Go). In 21% oxygen, 10 x HRE/pGL3 transfection resulted in a 3-fold increase in luciferase activity compared with pGL3 (P < .05; Fig. 2Go). These experiments demonstrate that the 10 x HRE enhancer significantly improved paired gene expression, with marked specificity for an oxygen-deprived environment.


Figure 2
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  		FIG. 2. Luciferase assay evaluated a multimerized hypoxia-responsive enhancer (10 x HRE) function. After cloning into the pGL3 luciferase reporter vector (Promega, Madison, WI), AsPC1 human pancreatic cancer cells were transfected with 1.6 µg of our 10 x HRE/pGL3 construct. Cells were subsequently incubated in either hypoxia (1% oxygen) or normoxia (21% oxygen) for 18 hours. The native pGL3 promoter plasmid was used as a control. There was a 3-fold increase in luciferase activity in normoxia (P < .05) and a 49-fold increase in hypoxia (P < .01).

 
HUVEC Assay: HSV10 x HRE/sFlk-1 Transduction
HUVEC assay evaluated inhibition of capillary formation after HSV10 x HRE/sFlk-1 transduction. In normoxia, conditioned media from cells transduced with HSVlacZ yielded 84 ± 19 capillary tubes per well, whereas media from cells transduced with HSV10 x HRE/sFlk-1 had 54 ± 14 tubes per well. This represented a 36% reduction in capillary formation under normoxic conditions (P < .05; Fig. 3AGo). Media conditioned by hypoxic AsPC1 cells transduced with HSVlacZ exhibited a mean of 112 ± 27 capillary tubes per well. Conditioned media obtained from hypoxic cells transduced with HSV10 x HRE/sFlk-1 resulted in a mean of 27 ± 8 tubes per well. This represented a 76% reduction in capillary formation under hypoxic conditions (P < .005; Figs. 3BGo and 4Go). These results demonstrate that transduction with HSV10 x HRE/sFlk-1 can inhibit capillary formation, particularly under low-oxygen conditions.


Figure 3
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  		FIG. 3. After herpes simplex virus multimerized hypoxia-responsive enhancer soluble fetal liver kinase-1 (HSV10 x HRE/sFlk-1) transduction of AsPC1 human pancreatic adenocarcinoma cells, the human umbilical vein endothelial cell (HUVEC) assay was used to evaluate capillary inhibition. Medium was collected from AsPC1 cells after transduction with HSV10 x HRE/sFlk-1 or HSVlacZ and incubation in normoxia (21% oxygen) or hypoxia (1% oxygen). HUVECs were plated in the presence of this conditioned medium. (A) Media obtained from normoxic AsPC1 cells transduced with HSV10 x HRE/sFlk-1 resulted in 36% reduced capillary formation versus media conditioned by HSVlacZ-transduced cells grown under identical conditions (P < .05). (B) Media obtained from hypoxic AsPC1 cells transduced with HSV10 x HRE/sFlk-1 resulted in 76% reduced capillary formation versus media isolated from hypoxic, HSVlacZ-transduced cells (P < .005).

 

Figure 4
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  		FIG. 4. Human umbilical vein endothelial cell (HUVEC) assay evaluated capillary inhibition. (A) HUVECs plated in media derived from hypoxic AsPC1 pancreatic adenocarcinoma cells transduced with the HSVlacZ control amplicon. (B) HUVECs plated in media derived from hypoxic AsPC1 cells transduced with HSV10 x HRE/sFlk-1. There was a significant reduction in capillary tube formation after transduction with HSV10 x HRE/sFlk-1 compared with HSVlacZ.

 
10x HRE/sFlk-1 Expression
AsPC1 cells transduced with HSVlacZ or HSV10 x HRE/sFlk-1 underwent Western blot analysis for sFlk-1 expression. Greater sFlk-1 protein levels were demonstrated in AsPC1 cells transduced with HSV10 x HRE/sFlk-1 incubated in hypoxia compared with normoxia (Fig. 5Go). Nontransduced AsPC1 cells did not express sFlk-1 in hypoxia or normoxia. These results show that HSV10 x HRE/sFlk-1 preferentially expresses sFlk-1 under hypoxic conditions.


Figure 5
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  		FIG. 5. Western blot assessment of soluble vascular endothelial growth factor (VEGF) receptor (sFlk-1) expression after herpes simplex virus (HSV) amplicon transduction. After transduction with HSV multimerized hypoxia-responsive enhancer (10 x HRE)/sFlk-1 or HSVlacZ, AsPC1 human pancreatic adenocarcinoma cells were incubated in either hypoxia (1% oxygen) or normoxia (21% oxygen). Cell lysates were collected and underwent Western blot analysis for sFlk-1 protein by using a mouse monoclonal anti-human Flk-1 antibody (Research Diagnostics Inc., Flanders, NJ). Equal protein loading from each sample was confirmed by Western blot for ß-actin. Hypoxic AsPC1 cells transduced with HSV10 x HRE/sFlk-1 exhibited greater sFlk-1 expression compared with similarly transduced normoxic cells. AsPC1 cells did not express sFlk-1 after transduction with HSVlacZ.

 
Inhibition of Tumor Growth
AsPC1 cells were injected into the flanks of athymic mice, thus creating an in vivo tumor model. At the initiation of treatment, the animals receiving PBS had a mean tumor volume of 28.4 ± 3.5 mm3, those receiving HSVlacZ had a mean volume of 28.9 ± 4.1 mm3, and those receiving HSV10 x HRE/sFlk-1 had a mean volume of 27.8 ± 4.3 mm3. These tumor sizes were not significantly different. During the course of this experiment, no significant difference in volume was detected between tumors treated with HSVlacZ or PBS at any of the measured time points. However, as early as 1 week after HSV10 x HRE/sFlk-1 delivery, there was a significant inhibition of tumor growth. At this time, HSV10 x HRE/sFlk-1 treatment inhibited tumor growth by 23% versus HSVlacZ-injected mice (P < .01) and by 30% versus PBS-injected control animals (P < .001; Fig. 6Go). This disparity in tumor growth profiles persisted throughout the entire 5-week observation period. At the conclusion of the study, tumors treated with HSV10 x HRE/sFlk-1 occupied a mean volume of 93 ± 16 mm3, whereas tumors treated with HSVlacZ were 225 ± 28 mm3 and those treated with PBS were 232 ± 36 mm3. This represented a 59% reduction in tumor volume for mice treated with HSV10 x HRE/sFlk-1 versus HSVlacZ (P < .000001) and a 60% reduction versus PBS-treated animals (P < .00001; Fig. 6Go). These results demonstrate that intratumoral delivery of HSV10 x HRE/sFlk-1 can significantly retard tumor growth in vivo.


Figure 6
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  		FIG. 6. Treatment of AsPC1 human pancreatic adenocarcinoma flank tumors with herpes simplex virus multimerized hypoxia-responsive enhancer soluble fetal liver kinase-1 (HSV10 x HRE/sFlk-1) in athymic mice. AsPC1 flank tumor xenografts were treated with phosphate-buffered saline (PBS; n = 8), HSVlacZ (n = 8), and HSV10 x HRE/sFlk-1 (n = 8). There was no significant difference in tumor volumes between the PBS-treated and HSVlacZ-transduced groups. HSV10 x HRE/sFlk-1 transduction significantly reduced tumor volume versus both PBS-treated and HSVlacZ-transduced tumors as early as 7 days after infection. Five weeks after treatment, HSV10 x HRE/sFlk-1 yielded a 60% reduction in tumor volume versus tumors treated with PBS (P < .00001) and yielded a 59% reduction in volume versus tumors treated with HSVlacZ (P < .000001).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
VEGF plays a critical role in capillary tube formation and survival of newly formed blood vessels.12 Recent research has further elucidated the role of VEGF as a key regulator of cancer-related angiogenesis. VEGF has been shown to induce endothelial cell proliferation and tumor growth, potentially contributing to metastatic spread.3,4 Studies have demonstrated that blocking VEGF at either the protein or RNA level can decrease tumor vascularization and volume.27,28 Moreover, increased levels of VEGF have been correlated with increased invasiveness and a worse prognosis in various malignancies.29–32 Hypoxia is the primary inducer of VEGF expression in tumors.33 Therefore, targeting tumor hypoxia may enhance cancer treatments that aim to suppress VEGF.

There are two principal VEGF receptors: VEGF receptor-1 (fms-like tyrosine kinase 1 [Flt-1]) and VEGF receptor-2 (Flk-1).13,14 However, only Flk-1 is expressed primarily on endothelial cells.17 After VEGF binds to membrane-bound Flk-1, there is receptor dimerization and transduction of an intracellular signal. Through receptor tyrosine kinase activity, Flk-1 phosphorylates several intracellular endothelial proteins, such as phospholipase C-{gamma}, phosphatidylinositol-3 (PI-3) kinase, and Ras guanosine triphosphatase–activating protein.34 This pathway results in the angiogenic stimulation of endothelial cells.

Soluble forms of both Flk-1 and Flt-1 have been shown to inhibit endothelial cell activation by VEGF. Soluble Flk-1 delivered by an adenoviral vector has demonstrated decreased angiogenesis during wound healing in mice.35 In the treatment of cancer, soluble forms of Flt-1 have demonstrated efficacy in inhibiting tumor xenograft growth. Adenovirus expressing sFlt-1 administered to athymic mice inhibited capillary formation in flank tumors derived from a lung cancer cell line.36 Furthermore, an embryonic renal cell line expressing sFlt-1 inhibited HUVEC growth in vitro and follicular thyroid cancer xenografts in vivo. Immunohistochemical analysis confirmed decreased microvessel densities in treated tumors.37 Soluble VEGF receptors have therefore shown significant utility in blocking angiogenesis and inhibiting solid tumor growth.

In this study, we used sFlk-1 to inhibit VEGF, because binding of VEGF to membrane-bound Flk-1 has been clearly established as the main endothelial cell stimulus during tumor angiogenesis.17 We targeted production of sFlk-1 to tumor hypoxia, the primary stimulus for VEGF expression. We demonstrated that AsPC1 human pancreatic adenocarcinoma cells upregulated HIF-1{alpha} and VEGF levels in hypoxia. Our multimerized HRE significantly increased paired gene expression in hypoxic cells and supported the development of a hypoxia-inducible VEGF inhibitor (10 x HRE/sFlk-1). An HSV amplicon vector expressing 10 x HRE/sFlk-1 was genetically engineered and demonstrated preferential sFlk-1 production at low oxygen concentrations. This amplicon further inhibited HUVEC capillary formation in vitro and AsPC1 flank tumor xenograft growth in vivo.

HSV amplicons are efficient gene-delivery vehicles. Packaged amplicon vectors have the same viral envelope, tegument, and capsid as replication-competent herpesviruses.38 They therefore are able to transduce a wide range of dividing and postmitotic cell types.19 Recently, the ability to produce HSV amplicons without helper viruses has nearly eliminated their contamination with cytotoxic replication-competent viral strains.38,39 We used helper virus-free HSV amplicons in our current experiments because they have demonstrated minimal cytopathic effects in vitro.20 Other advances in amplicon processing and purification have allowed for higher titers of amplicon stocks, approaching 108 TU/mL.19,38 Furthermore, the possibility of recombination and reversion to wild-type, replication-competent virus is extremely low, because amplicons carry <1% of the HSV genome.38 A potential limitation of amplicon-mediated gene transfer is the transient duration of gene expression. Decreasing levels of gene expression over time are related to loss of vector DNA during cell division, as well as DNA degradation within the cell.19 Nevertheless, the ability of HSV10 x HRE/sFlk-1 to inhibit tumor growth over 5 weeks is not unexpected, because HSV amplicons have maintained stable gene transduction for >2 months.40 Recently, integrated forms of the HSV vector platform have been described that exhibit profiles of longer duration.41 These newer forms of the HSV amplicon could provide more stable maintenance of hypoxia-regulated sFlk-1 expression and, thus, enhance the therapeutic benefit.

Although the oxygen tension can vary greatly between different areas of solid tumors, the median oxygen concentration of many pancreatic cancers is <1% oxygen; surrounding normal pancreatic tissue has concentrations that reach 12% oxygen.2 We therefore used 1% oxygen to mimic pancreatic tumor hypoxia in our in vitro experiments. Furthermore, because the atmospheric oxygen concentration of 21% is used in routine cell culture, this level was chosen as our in vitro normoxic standard. The actual oxygen concentration of normal tissues in vivo may be lower than this.42,43 Reports reveal, however, that there is little difference in multimerized HRE enhancer activity between 5% oxygen and 21% oxygen.1 Therefore, our 10 x HRE enhancer function at 21% oxygen may reflect its activity in physiologic normoxia.

Despite the reported lack of HRE inducibility above 5% oxygen, there remains a low baseline level of gene expression under normoxic conditions.1 This activity may have contributed to the less pronounced, but significant, reduction in capillary formation demonstrated in normoxic pancreatic cancer cells transduced with HSV10 x HRE/sFlk-1. However, the side effects of inhibiting high VEGF levels in normoxic tissues are limited in adults, because increased levels of VEGF are restricted to wound healing and the menstrual cycle.44,45 Further studies are necessary to assess the potential systemic effects of antiangiogenic gene therapy directed toward VEGF.

We have demonstrated that a novel HSV amplicon expressing sFlk-1 under hypoxic control can be used as definitive therapy for established pancreatic adenocarcinoma by reducing angiogenesis and tumor growth. In addition, hypoxia is a common solid-tumor condition, and HSV amplicons are able to transduce a wide array of cell types. This hypoxia-driven angiogenic inhibitor may therefore have broad therapeutic applicability.


   ACKNOWLEDGMENTS
 
The authors thank Guojie Ye, PhD, for his advice with cloning procedures and knowledge of HSV vectors. They also thank Sam Yoon, MD, for his help with the HUVEC assay and Wade Narrows for preparation of helper virus-free HSV amplicon stocks. Supported by grants RO1 CA 76416 and RO1 CA/DK80982 (Y.F.) from the National Institutes of Health, grant MBC-99366 (Y.F.) from the American Cancer Society, grant BC024118 from the US Army, and the Rochester Nathan Shock Center.

Received for publication March 20, 2004. Accepted for publication July 8, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Shibata T, Giaccia AJ, Brown JM. Development of a hypoxia-responsive vector for tumor-specific gene therapy. Gene Ther 2000;7:493–8.[CrossRef][Medline]
  2. Koong AC, Mehta VK, Le QT, et al. Pancreatic tumors show high levels of hypoxia. Int J Radiat Oncol Biol Phys 2000;48:919–22.[CrossRef][Medline]
  3. Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res 1996;56:4509–15.[Abstract/Free Full Text]
  4. Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res 1996;56:941–3.[Abstract/Free Full Text]
  5. Tannock I, Guttman P. Response of Chinese hamster ovary cells to anticancer drugs under aerobic and hypoxic conditions. Br J Cancer 1981;43:245–8.[Medline]
  6. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin 2002;52:23–47.[Abstract/Free Full Text]
  7. Bush RS, Jenkin RD, Allt WE, et al. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br J Cancer Suppl 1978;37:302–6.
  8. Duffy JP, Eibl G, Reber HA, Hines OJ. Influence of hypoxia and neoangiogenesis on the growth of pancreatic cancer. Mol Cancer 2003;2:12–22.[CrossRef][Medline]
  9. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995;92:5510–4.[Abstract/Free Full Text]
  10. Buchler P, Reber HA, Buchler M, et al. Hypoxia-inducible factor 1 regulates vascular endothelial growth factor expression in human pancreatic cancer. Pancreas 2003;26:56–64.[CrossRef][Medline]
  11. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–76.[CrossRef][Medline]
  12. Benjamin LE, Keshet E. Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal. Proc Natl Acad Sci U S A 1997;94:8761–6.[Abstract/Free Full Text]
  13. Shibuya M, Yamaguchi S, Yamane A, et al. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene 1990;5:519–24.[Medline]
  14. Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL, Shows TB. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene 1991;6:1677–83.[Medline]
  15. Millauer B, Wizigmann-Voos S, Schnurch H, et al. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 1993;72:835–46.[CrossRef][Medline]
  16. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998;273:13313–6.[Abstract/Free Full Text]
  17. Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 1994;367:576–9.[CrossRef][Medline]
  18. Ogawa T, Takayama K, Takakura N, Kitano S, Ueno H. Anti-tumor angiogenesis therapy using soluble receptors: enhanced inhibition of tumor growth when soluble fibroblast growth factor receptor-1 is used with soluble vascular endothelial growth factor receptor. Cancer Gene Ther 2002;9:633–40.[CrossRef][Medline]
  19. Sena-Esteves M, Saeki Y, Fraefel C, Breakefield XO. HSV-1 amplicon vectors—simplicity and versatility. Mol Ther 2000;2:9–15.[CrossRef][Medline]
  20. Fraefel C, Jacoby DR, Lage C, et al. Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors. Mol Med 1997;3:813–25.[Medline]
  21. Tung C, Federoff HJ, Brownlee M, et al. Rapid production of interleukin-2-secreting tumor cells by herpes simplex virus-mediated gene transfer: implications for autologous vaccine production. Hum Gene Ther 1996;7:2217–24.[Medline]
  22. Jarnagin WR, Delman K, Kooby D, et al. Neoadjuvant interleukin-12 immunogene therapy protects against cancer recurrence after liver resection in an animal model. Ann Surg 2000;231:762–71.[CrossRef][Medline]
  23. Wade-Martins R, Smith ER, Tyminski E, Chiocca EA, Saeki Y. An infectious transfer and expression system for genomic DNA loci in human and mouse cells. Nat Biotechnol 2001;19:1067–70.[CrossRef][Medline]
  24. Post DE, Van Meir EG. Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells. Gene Ther 2001;8:1801–7.[CrossRef][Medline]
  25. Bowers WJ, Howard DF, Brooks AI, Halterman MW, Federoff HJ. Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers. Gene Ther 2001;8:111–20.[CrossRef][Medline]
  26. Bowers WJ, Howard DF, Federoff HJ. Discordance between expression and genome transfer titering of HSV amplicon vectors: recommendation for standardized enumeration. Mol Ther 2000;1:294–9.[CrossRef][Medline]
  27. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993;362:841–4.[CrossRef][Medline]
  28. Saleh M, Stacker SA, Wilks AF. Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res 1996;56: 393–401.[Abstract/Free Full Text]
  29. Imoto H, Osaki T, Taga S, Ohgami A, Ichiyoshi Y, Yasumoto K. Vascular endothelial growth factor expression in non-small-cell lung cancer: prognostic significance in squamous cell carcinoma. J Thorac Cardiovasc Surg 1998;115:1007–14.[Abstract/Free Full Text]
  30. Ishigami SI, Arii S, Furutani M, et al. Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer. Br J Cancer 1998;78:1379–84.[Medline]
  31. Gasparini G, Toi M, Gion M, et al. Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma. J Natl Cancer Inst 1997;89:139–47.[Abstract/Free Full Text]
  32. Toi M, Hoshina S, Takayanagi T, Tominaga T. Association of vascular endothelial growth factor expression with tumor angiogenesis and with early relapse in primary breast cancer. Jpn J Cancer Res 1994;85:1045–9.[CrossRef][Medline]
  33. Minchenko A, Bauer T, Salceda S, Caro J. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest 1994;71:374–9.[Medline]
  34. Guo D, Jia Q, Song HY, Warren RS, Donner DB. Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation. J Biol Chem 1995;270:6729–33.[Abstract/Free Full Text]
  35. Jacobi J, Tam BY, Sundram U, et al. Discordant effects of a soluble VEGF receptor on wound healing and angiogenesis. Gene Ther 2004;11:302–9.[CrossRef][Medline]
  36. Takayama K, Ueno H, Nakanishi Y, et al. Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ. Cancer Res 2000;60:2169–77.[Abstract/Free Full Text]
  37. Ye C, Feng C, Wang S, et al. sFlt-1 gene therapy of follicular thyroid carcinoma. Endocrinology 2004;145:817–22.[Abstract/Free Full Text]
  38. Saeki Y, Ichikawa T, Saeki A, et al. Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors. Hum Gene Ther 1998;9:2787–94.[Medline]
  39. Stavropoulos TA, Strathdee CA. An enhanced packaging system for helper-dependent herpes simplex virus vectors. J Virol 1998;72:7137–43.[Abstract/Free Full Text]
  40. Wang Y, Yu L, Geller AI. Diverse stabilities of expression in the rat brain from different cellular promoters in a helper virus-free herpes simplex virus type 1 vector system. Hum Gene Ther 1999;10:1763–71.[CrossRef][Medline]
  41. Costantini LC, Jacoby DR, Wang S, Fraefel C, Breakefield XO, Isacson O. Gene transfer to the nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors. Hum Gene Ther 1999;10:2481–94.[CrossRef][Medline]
  42. Hockel M, Schlenger K, Knoop C, Vaupel P. Oxygenation of carcinomas of the uterine cervix: evaluation by computerized O2 tension measurements. Cancer Res 1991;51: 6098–102.[Abstract/Free Full Text]
  43. Dachs GU, Greco O, Tozer GM. Targeting cancer with gene therapy using hypoxia as a stimulus. Methods Mol Med 2004;90:371–88.[Medline]
  44. Licht P, Russu V, Lehmeyer S, Wissentheit T, Siebzehnrubl E, Wildt L. Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function. Fertil Steril 2003;80: 1228–33.[Medline]
  45. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol 2001;280:C1358–66.[Abstract/Free Full Text]




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