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10.1245/ASO.2006.05.049
Annals of Surgical Oncology 13:1145-1155 (2006)
© 2006 Society of Surgical Oncology
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Original Article

Selective Blockade of Vascular Endothelial Growth Factor Receptor 2 With an Antibody Against Tumor-Derived Vascular Endothelial Growth Factor Controls the Growth of Human Pancreatic Adenocarcinoma Xenografts

Shane E. Holloway, MD1, Adam W. Beck, MD1, Latha Shivakumar, PhD1, Jessica Shih, BS1, Jason B. Fleming, MD1 and Rolf A. Brekken, PhD1,2

1 Department of Surgery, Division of Surgical Oncology, and Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390-8593, USA
2 Department of Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390-8593, USA

Correspondence: Address correspondence and reprint requests to: Rolf A. Brekken, PhD; E-mail: rolf.brekken{at}utsouthwestern.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, is critical for growth of human pancreatic adenocarcinoma. Preclinical studies demonstrate that blockade of VEGF activity can control the growth of pancreatic tumors in mice. In this study, we evaluated the efficacy of 2C3, an antibody that inhibits VEGF receptor 2 activation by human VEGF, to inhibit the growth of human pancreatic adenocarcinoma in mice.

Methods: Human pancreatic cancer cell lines (MiaPaca-2, Panc-1, and Capan-1) were used to establish xenografts in nu/nu mice. The expression of VEGF and its receptors was determined in each cell line. Proliferation of tumor cells in vitro and tumor growth in vivo in the presence of 2C3 or a control antibody was evaluated. The effect of 2C3 on tumor weight, total vessel density, number of pericyte-associated vessels, and tumor perfusion was determined, and the level of 2C3 in the serum of animals was measured by enzyme-linked immunosorbent assay.

Results: 2C3 did not affect the proliferation of cells in culture. 2C3 was present and active in the serum of tumor-bearing animals treated with 2C3, and these animals showed a decrease in tumor burden compared with control-treated mice. Therapy with 2C3 resulted in reduced vascular function, measured by a decrease in vessel density and in the percentage of vessels associated with pericytes. Furthermore, tumors derived from Capan-1 cells demonstrated decreased perfusion after treatment with 2C3.

Conclusions: Blockade of VEGF receptor 2 activation by tumor-derived VEGF decreases tumor vessel function and growth of some human pancreatic adenocarcinoma cell lines in mice.

Key Words: Pancreatic cancer • Angiogenesis • VEGF • Therapy • Monoclonal antibody


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer of the exocrine pancreas is characterized by extensive local invasion and metastases to the liver; this aggressive biology translates into a 5-year survival rate of 1% to 4% for all patients with a diagnosis of pancreatic adenocarcinoma.1 The current best therapy including surgery, radiation, and chemotherapy has done little to alter the cancer-related deaths of these patients,2 thus emphasizing the need for more effective therapy.

The progressive growth and metastasis of pancreatic cancer and other solid tumors is dependent on angiogenesis, the development of new vasculature from preexisting blood vessels and/or circulating endothelial stem cells.3 Vascular endothelial growth factor (VEGF) is a potent multifunctional cytokine that is a primary stimulant of angiogenesis in tumors.4,5 Blocking VEGF activity is an attractive strategy for antiangiogenic therapy of pancreatic tumors because human pancreatic adenocarcinoma cells secrete high levels of VEGF in vitro and in vivo.6 Additionally, VEGF and its receptors (VEGFR1 and VEGFR2) are expressed at higher levels in pancreatic adenocarcinoma tumors versus normal pancreatic tissue, as determined by Northern blot and immunohistochemical analysis.7 Furthermore, high levels of VEGF expression within the primary pancreatic tumor correlate with a decreased time to recurrence after curative resection, liver metastasis, and cancer-related death.8 Preclinical animal models of pancreatic cancer that evaluated different strategies to inhibit VEGF activity (DC101, a rat monoclonal antibody (mAb) specific for murine VEGFR2, and A.4.6.1, a mouse mAb specific for human VEGF) demonstrated reductions in tumor growth.9,10 However, recent phase II clinical trials with the humanized version of A.4.6.1, bevacizumab (Avastin; Genentech, South San Francisco, CA), in combination with gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN), a nucleoside analogue with known activity against pancreatic adenocarcinoma, have generated only modest therapeutic responses.11,12 The disparity between the results of preclinical animal studies and early clinical trials is likely due to multiple tumor and patient factors but suggests that different anti-VEGF strategies should be investigated.

A variety of mAbs specific for VEGF have been generated,13 including a unique mAb (2C3) that blocks human VEGF from binding to VEGFR2 but not VEGFR1.14 2C3 is effective at controlling the growth of multiple human tumor xenografts in vivo by blocking the activity of tumor-derived VEGF.14–16 To evaluate the potential of 2C3 as a therapy for pancreatic cancer, we tested the effect of 2C3 on the in vivo growth and angiogenic activity in subcutaneous tumors derived from three well-characterized human pancreatic adenocarcinoma cell lines (Panc-1, MiaPaca-2, and Capan-1). These cell lines possess the principal oncogene and tumor-suppressor gene pro-file identified in >90% of pancreatic adenocarcinoma tumors.17,18 Tumors derived from these cell lines possess alterations in redundant signaling pathways that result in increased expression of VEGF. Alteration in VEGF expression and possibly in VEGF signaling might be responsible in part for the seemingly unremarkable effect of anti-VEGF therapy in patients with pancreatic adenocarcinoma.11,12 We demonstrate that blocking tumor-derived VEGF with 2C3 slows the growth and reduces the functional vasculature of human pancreatic tumor xenografts grown in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
MiaPaca-2 and Panc-1 human pancreatic adenocarcinoma cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Gemini Bio-Products, Woodland, CA). Capan-1 pancreatic adenocarcinoma cells (American Type Culture Collection) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum by using a standard tissue culture technique.

Reverse Transcriptase-Polymerase Chain Reaction
Expression of human or mouse VEGF A, VEG-FR1, VEGFR2, and neuropilin 1 (Npn-1) and -2 in cell lines and tumors was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) after total RNA was prepared by using TRIzol (Invitrogen) and the Qiagen RNeasy mini-kit method (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. Deoxyribonuclease treatment was performed by using DNA Free (Ambion, Inc., Austin, TX). The quality of the RNA was evaluated by gel electrophoresis before RT-PCR. Total RNA was used for RT-PCR by using a Titan one-step RT-PCR kit (Roche, Indianapolis, IN) according to the manufacturer’s directions. The sequences of the primer sets used are listed in Table 1Go. Products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining.


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  		TABLE 1. Primer sequences used for reverse transcriptase-polymerase chain reaction analysis
 
Enzyme-Linked Immunosorbent Assay
The amount of VEGF secreted into culture media was measured by enzyme-linked immunosorbent assay (ELISA). Cells were grown to 80% confluence, washed with Hanks’ balanced salt solution (Invitrogen), and incubated in serum-free media. After 24 hours, conditioned media were collected, and 1% bovine serum albumin (Calbiochem, La Jolla, CA) was added to stabilize the VEGF. VEGF was measured by using a Quantikine human VEGF immunoassay (R&D Systems, Minneapolis, MN) according to the manufacturer’s directions.

To measure the presence and activity of 2C3 within the systemic circulation of treated animals, blood was harvested from 2C3- and control-treated animals at necropsy. Serum was isolated and stored at –20°C. The sera were diluted serially and analyzed for binding to immobilized VEGF by standard indirect ELISA methods.

Cell Proliferation
Panc-1, Capan-1, and MiaPaca-2 cells were plated at a density of 1 x 103 cells per well in triplicate in a 96-well plate in appropriate media with either 2C3 or a control isotype-matched antibody (C44) at concentrations of 50, 5, or .5 µg/mL. Cell number was determined by the use of a 3-[4,5-dimethylthiazol-2-yl]-2-5 diphenyl tetrazolium bromide assay (Sigma, St. Louis, MO) performed according to the manufacturer’s instructions at 24, 48, and 72 hours after plating. The mean absorbance at 490 nm of 2C3- and control-treated cells at each time point was compared.

In Vivo Tumor Growth and Treatment With 2C3
All animal experiments were approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee. Tumor xenografts were established in female nu/nu mice (Charles River Laboratories, Wilmington, MA) by injecting 1 x 107 Panc-1, Mia Paca-2, or Capan-1 cells subcutaneously into the flank of each mouse. All mice were irradiated with 350 cGy from a cesium 137 source 24 hours before tumor cell injection. One day after tumor cell injection, therapy was initiated with intraperitoneal injections of 100 µg of 2C3 or C44. The mice were treated three times (Monday, Wednesday, and Friday) per week until the termination of the experiment. The tumor size and general status of the mice were recorded at the time of treatment. Vernier scale calipers were used to obtain perpendicular tumor diameters, and tumor volume was determined according to the following equation:


Formula

where D = largest diameter, and d = diameter perpendicular to D.

Experiments were terminated when the tumor volume exceeded 2000 mm3, or earlier if tumors showed signs of ulceration. At the time of necropsy, tumors were collected, weighed for final comparisons, and then divided. One portion of the tumor was snap-frozen in liquid nitrogen, and the other was fixed in methyl Carnoy’s fixative as described previously.19

Tumor Histology and Immunohistochemical Analysis
Methyl Carnoy’s fixed, paraffin-embedded tissues were sectioned by the molecular pathology core laboratory at the University of Texas Southwestern Medical Center. Antibodies for immunohistochemical analysis were as follows: rat anti-mouse endothelial cell antigen, MECA-3220 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), polyclonal rabbit anti-smooth muscle actin (SMA; Lab Vision Corporation, Freemont, CA), and polyclonal rabbit anti-vascular endothelial receptor 2 (T014).21 For vessel density analysis, 4- to 6-µm sections were softened at 60°C for 30 minutes followed by deparaffinization under standard conditions. Endogenous peroxide activity was blocked with 1% hydrogen peroxide in 50% methanol (v/v in Tris-buffered saline [TBS]) for 20 minutes. The tissue sections were blocked and permeabilized with 5% Aquablock (East Coast Biologics Inc., North Berwick, ME) and .02% BRIJ 35 (Calbiochem) in TBS. The sections were incubated with primary antibody for 1 hour, washed with TBS containing .2% Tween-20 (TBS-t), and incubated with the appropriate fluorescently labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA). Negative controls included primary antibodies of irrelevant specificity and/or secondary antibody only. Sections were examined on a Nikon E600 microscope, and images were captured with a Photometrics Coolsnap HQ camera by using Metamorph Software.

Total vessel density (TVD) was determined by counting the total number of MECA-32–positive blood vessels in 12 high-power fields from each slide (final magnification, x 400). Three slides from control and treated groups (a total of 36 fields per treatment group) were counted in a blinded fashion, and the TVD per group was calculated by dividing the total number of blood vessels counted by the total number of fields counted. The percentage of vessels associated with pericytes was determined by counting the number of MECA-32–positive blood vessels that were also positive for SMA by using the same quantification technique used to determine TVD.

Tumor Perfusion
Perfusion of the tumor and normal organs was evaluated as described previously.22 Briefly, 5 minutes before sacrifice, animals bearing Capan-1 tumors underwent a tail vein injection of 200 µL (10 mg/kg) of Hoechst 33342 (Sigma). Upon sacrifice, the animals were perfused with phosphate-buffered saline. The tumor and other tissues were collected and snap-frozen in liquid nitrogen. Five-micrometer frozen sections of tumor were air-dried for 30 minutes, fixed with acetone for 30 seconds, and rehydrated in TBS-t. Nonspecific staining was blocked by incubation in TBS containing 20% Aquablock for 2 hours. Sections were incubated with rabbit anti-VEGFR2 (10 µg/mL) for 1 hour, washed with TBS-t, and incubated with the appropriate fluorescein isothiocyanate–labeled secondary antibody. Slides were coverslipped in propidium iodide mounting media (Vector Laboratories, Burlingame, CA), and sections were evaluated microscopically.

Statistical Analysis
The data are expressed as the mean with the standard error of the mean and were tested for statistically significant differences by using a two-sample t-test. This analysis was performed with SigmaPlot version 8.0 (Systat Software, Inc., Point Richmond, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of VEGF and Its Receptors and the Effect of 2C3 In Vitro
All three human pancreatic adenocarcinoma cell lines produced detectable levels of VEGF at the messenger RNA (mRNA; Fig. 1A) and protein (Fig. 1BGo) level in vitro. Conditioned media from Capan-1 and Panc-1 cells had levels of VEGF protein near 2000 pg/mL, whereas MiaPaca-2 conditioned media had approximately 50% of that amount (Fig. 1BGo). The level of VEGFR1, VEGFR2, Npn-1, and Npn-2 expression by each cell line was also evaluated by RT-PCR (Fig. 1AGo). Each cell line made detectable levels of Npn-1 and Npn-2 (Fig. 1AGo), as reported previously.23 However, it was difficult to detect VEGFR1 or VEGFR2 in any of the cell lines. Figure 1AGo shows a low level of VEGFR1 in the Panc-1 cells, but VEG-FR1 could not be reproducibly detected even at 40 cycles of amplification in the MiaPaca-2 or Capan-1 cells. VEGFR2 was not found at the mRNA level in any of the cell lines (Fig. 1AGo). 2C3 had no effect on the proliferation (Fig. 2Go) or morphology (data not shown) of Panc-1, MiaPaca-2, or Capan-1 cells in vitro, even though each cell line produced VEGF and expressed cell-surface receptors for VEGF.


Figure 1
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  		FIG. 1. Pancreatic tumor cells produce vascular endothelial growth factor (VEGF) and VEGF receptors. Capan-1 (C), MiaPaca-2 (M), and Panc-1 (P) cells were grown to 80% confluence in vitro and analyzed by reverse transcriptase-polymerase chain reaction for the expression of VEGF and its receptors (A). Forty cycles were used for polymerase chain reaction analysis for each transcript except ß-actin, for which 25 cycles were used. The sequences of the primers used for reverse transcriptase-polymerase chain reaction analysis are listed in Table 1Go. RNA isolated from human microvascular endothelial cells (H) was used as a positive control. (B) Conditioned media were collected and analyzed for the level of VEGF protein by enzyme-linked immunosorbent assay. Studies were performed in duplicate, and results are displayed as the mean concentration in picograms per milliliter. The data shown are representative of at least three independent assays.

 

Figure 2
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  		FIG. 2. Inhibition of vascular endothelial growth factor receptor 2 activity does not influence tumor cell proliferation in vitro. Tumor cells (Panc-1, MiaPaca-2, and Capan-1) were grown in regular growth media in the presence of 2C3 at varying concentrations. Cell number was determined by 3-[4,5-dimethylthiazol-2-yl]-2-5 diphenyl tetrazolium bromide assay at 24, 48, and 72 hours after antibody addition. Results at 48 hours are shown and are expressed as a percentage of control for each cell type at 2C3 concentrations of .5, 5, and 50 µg/mL. The assay was performed twice, with similar results.

 
2C3 Controls the Growth of Pancreatic Tumor Xenografts
Palpable subcutaneous tumors were formed within 7 to 14 days of injection of the each tumor cell line into the flank of nu/nu mice. Administration of 2C3, but not the control antibody, reduced the growth of all three human pancreatic tumor xenografts. Sequential measurement of tumor volumes demonstrated a separation in mean tumor size at approximately 6, 10, and 12 weeks for the MiaPaca-2, Capan-1, and Panc-1 cells, respectively (data not shown).

After treatment with 2C3, animals with tumors derived from Panc-1, Capan-1, and MiaPaca-2 cells demonstrated an 80%, 40%, and 30% (Fig. 3AGo) reduction in tumor weight, respectively, when compared with control-treated tumors. Tumor weight at the time of sacrifice was different statistically (P < .05) in the MiaPaca-2 group and approached statistical significance in the Capan-1 group (P = .06). 2C3 prevented the growth of Panc-1 tumors in vivo, such that three of five animals had no evidence of tumor at the time of sacrifice, whereas all animals in the control group had well-established palpable tumors.


Figure 3
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  		FIG. 3. 2C3 controls the growth of pancreatic tumor xenografts. Flank tumors from each cell line were generated in nu/nu mice (n indicates the number of animals per group), and 1 day after tumor cell injection, the mice received systemic therapy with 2C3 or an isotype-matched control antibody (Control). The mice were treated with 100 µg of antibody given intraperitoneally three times a week for the duration of the experiment. Tumor volume (cubic millimeters) was measured weekly, and tumors were weighed at the conclusion of the study (A). When compared with controls, the final tumor weights were reduced in 2C3-treated animals, and this reduction was statistically significant in mice bearing MiaPaca-2 tumors (P < .05). (B) Tumors were harvested from control and 2C3-treated animals, fixed, paraffin-embedded, and stained with hematoxylin and eosin. Representative tumor sections from MiaPaca-2 and Capan-1 tumors from each treatment group are shown at a final magnification of x 100. Note that an edge of the tumor is shown on the left of each tumor section. The insets shown in the MiaPaca-2 sections are at a magnification of x 400 and demonstrate an increase in pyknotic nuclei in the tumors from 2C3-treated animals (bar = 50 µm).

 
Histological examination of tumors from control and 2C3-treated animals showed that prolonged therapy with 2C3 resulted in substantial changes in the number of viable cells in the interior of both MiaPaca-2 and Capan-1 tumors (Fig. 3BGo). Tumors from 2C3-treated mice showed an increase in the number of condensed nuclei and evidence of tissue stress that was not present in tumors from the control groups.

The level of anti-human VEGF activity (2C3) in the serum of 2C3- or control-treated mice was evaluated by indirect ELISA. Sera from 2C3-treated animals had an anti-human VEGF titer of >1:25,000 at the time of sacrifice (Fig. 4Go), whereas sera from control-treated animals did not react with human VEGF.


Figure 4
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  		FIG. 4. 2C3 is present and biologically active in the serum of treated animals. Serum from 2C3- and control-treated animals was collected at the time of sacrifice and analyzed for binding to vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay. The mean reactivity of serial dilutions of serum samples from three 2C3- and control-antibody treated animals is shown and demonstrates the presence of anti-human VEGF activity specifically in the 2C3-treated animals.

 
Treatment of mice bearing MiaPaca-2 tumors with 2C3 did not alter the expression of VEGF or its receptors at the mRNA level in vivo (Fig. 5Go). In contrast, 2C3 treatment of mice bearing Capan-1 tumors resulted in a slight but noticeable increase in the level of mouse Npn-2 compared with tumors from control-treated animals (Fig. 5Go). RT-PCR analysis of both human and mouse VEGF expression in MiaPaca-2 and Capan-1 tumors demonstrated that the expression level of mouse VEGF was higher than the expression level of human VEGF. Surprisingly, treatment with 2C3 did not alter the level of human or mouse VEGF in these animals. We were unable to demonstrate specifically a transcript corresponding to human VEGFR2 in MiaPaca-2 and Capan-1 tumors.


Figure 5
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  		FIG. 5. 2C3 treatment does not alter the expression of vascular endothelial growth factor (VEGF) or its receptors at the messenger RNA level. RNA was isolated from tumors harvested from 2C3- or control-treated animals bearing either Capan-1 or MiaPaca-2 tumors. The RNA was used for reverse transcriptase-polymerase chain reaction analysis of human (A) and mouse (B) VEGF, VEGFR1, VEGFR2, Npn-1, and Npn-2. The level of expression of ß-actin was used as a loading control. The same primers for ß-actin were used for both mouse and human ß-actin RNA analysis. The number of cycles used for polymerase chain reaction was 35 to 40 except for ß-actin, for which 25 cycles were used. Three representative tumors from each group were analyzed and are displayed.

 
2C3 Therapy Decreases Vascular Function in Pancreatic Tumor Xenografts
Immunohistochemical analysis of tumors from control and 2C3-treated animals demonstrated that 2C3 therapy reduced TVD and the percentage of vessels that were associated with SMA-positive pericytes in tumors derived from MiaPaca-2 and Capan-1 cells (Figs. 6Go and 7Go). There was also a decrease in the number of vessels positive for VEGFR2 and the intensity of VEGFR2 expression by endothelial cells in tumors from mice treated with 2C3 (Fig. 6Go). Inhibition of VEGFR2 activity with 2C3 also decreased the perfusion of the DNA-binding dye Hoechst 33342 (Fig. 8Go). The areas of Hoechst uptake in tumors from both 2C3 and control animals correspond to areas in the tumor that were fed by MECA-32–positive and VEGFR2-positive blood vessels (Fig. 8Go).


Figure 6
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  		FIG. 6. 2C3 treatment reduces tumor blood vessel density. (A) Total vessel density in the control and 2C3-treated MiaPaca-2 and Capan-1 tumors was determined by immunohistochemical analysis with the mouse endothelial specific antibody MECA-32. Representative MECA-32 reactivity in each tumor group is shown. The number of MECA-32–positive blood vessels per high-power field (hpf) is quantified in the box plots shown. Data within the box define the vessel counts within 1 SD, with the thick bar representing the mean and the thin bar the median number of vessels within each group. The whiskers define the distribution of vessel counts within 2 SD, with dots marking the outliers. (B) The expression of vascular endothelial growth factor receptor 2 (VEGFR2) was determined by immunohistochemistry. Representative sections of VEGFR2 expression in MiaPaca-2 tumors from animals treated with C44 (Control) or 2C3 are shown. VEGFR2 expression in Capan-1 tumors from animals treated with C44 (Control) or 2C3 was determined by immunofluorescence. MECA-32–positive blood vessels are shown in red, whereas VEGFR2 reactivity is shown in green. Yellow indicates colocalization of the two proteins (bar = 50 µm).

 

Figure 7
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  		FIG. 7. 2C3 treatment reduces the number of tumor blood vessels associated with pericytes. MiaPaca-2 and Capan-1 tumors were harvested from control and 2C3-treated animals, snap-frozen, and sectioned for immunofluorescence analysis. Tumor sections were incubated with primary antibodies specific for a pan marker of endothelium (MECA-32; red) and pericytes ({alpha}-smooth muscle actin [SMA]; green). The number of SMA + MECA-32–positive blood vessels per high-power field (hpf) is quantified in the box plots shown. Data within the box define the vessel counts within 1 SD, with the thick bar representing the mean and the thin bar the median number of vessels within each group. The whiskers define the distribution of vessel counts within 2 SD, with dots marking outliers.

 

Figure 8
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  		FIG. 8. 2C3 treatment reduces vascular function in Capan-1 tumors. Vascular perfusion of Capan-1 tumors was evaluated by intravenous injection of the DNA-binding dye Hoechst 33342 20 minutes before necropsy. All cells present in the tumor were stained with propidium iodide (PI). Perfused regions of the tumor stained with Hoechst (blue); a decrease in Hoechst signal and vascular endothelial growth factor receptor 2 (VEGFR2; green) is evident in 2C3-treated tumors. When the Hoechst and VEGFR2 images were merged (Merge), the tumor regions with reduced Hoechst signal (reduced perfusion) corresponded to the observed reduction in VEGFR2-positive vessels.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings to emerge from this study were that (1) inhibition of VEGFR2 by an mAb specific for tumor-derived VEGF can control the growth of MiaPaca-2 human pancreatic tumor cells in mice and that (2) inhibition of tumor-derived VEGF alters the function of the vasculature in solid tumors. The dependence of solid tumors on angiogenesis for development, growth, and metastasis is well established.24 VEGF is a primary stimulant of angiogenesis3,4,25,26 and, as such, is the focus of many therapeutic strategies aimed at inhibiting angiogenesis in solid tumors.4,27 VEGF is expressed prominently in pancreatic ductal adenocarcinoma6 and has been targeted for inhibition in animal models9,10,28,29 of pancreatic cancer, as well as in clinical trials.12 These studies used antibodies or small-molecular-weight kinase inhibitors that block activation of both VEGFR1 and VEGFR2. Although the results are encouraging, they are less than optimal and suggest that improvement in anti-VEGF therapy for the treatment of pancreatic cancer is possible. The efficacy of anti-VEGF therapy depends on the inhibition of specific functions of VEGF, including VEGF-induced cell (endothelial and tumor) survival, VEGF-induced cell proliferation, VEGF-induced migration, and, potentially, VEGF-induced vascular permeability.

Bevacizumab (Avastin) is the only anti-VEGF mAb that has been tested clinically in the United States in patients with pancreatic cancer and is the agent against which similar agents should be compared. Avastin is the humanized version of mouse mAb A.4.6.1, which blocks human VEGF from binding to VEGFR1 and VEGFR2.30–32 It is clear that VEGFR2 is the dominant signaling VEGF receptor33 and that inhibition of VEGFR2 alone can be effective at controlling tumor growth in animal models.9,14,34,35 However, it is not clear whether VEGFR1 functions as an angiogenic or regulatory receptor in the tumor microenvironment. Mice lacking VEGFR1 die in utero,36 thus demonstrating that VEGFR1 is required for development. It is interesting to note that mice lacking VEGFR1 die as a result of an excess of endothelial cells and a disorganized vascular tree.37 Furthermore, mice lacking only the intracellular domain of VEGFR1 are viable and seem to have a normal vascular network.38 These studies suggest that in certain contexts, VEGFR1 negatively regulates the activity of VEGF by sequestering the ligand away from the dominant signaling receptor (presumably, VEGFR2). These results are in contrast to other studies that suggest that VEGFR1 can function as a signaling receptor that mediates VEGF-induced angiogenesis,39 recruitment of endothelial progenitors,40 and VEGF-induced macrophage chemotaxis.41

2C3 is a mouse mAb that binds to VEGF but selectively inhibits binding and activation of VEG-FR2, but not VEGFR1. Because of this unique property of 2C3, it is an attractive tool to dissect out the contribution of VEGFR1 to the development and progression of pancreatic adenocarcinoma. Although A.4.6.1 has a higher affinity42 (KD {approx} 4 x 10–10 M) for VEGF than 2C3,13 previous animal experiments that compared the antitumor effect of the mAbs in the same tumor model showed little difference between the two antibodies.14 These results suggest that inhibition of VEGFR1 is not required for effective control of tumor growth, at least in the models that have been tested thus far.14–16 It is possible that VEGF bound by 2C3 is capable of binding to VEGFR1, which could stimulate a signal cascade that negatively affects the proangiogenic pathway initiated by VEGF binding to VEGFR2 or other putative receptors.43,44 This could be why an mAb of lower affinity seems to be as effective as A.4.6.1 in the treatment of various human tumors in mice.

Surprisingly, treatment of tumor-bearing mice with 2C3 did not affect the level of mRNA expression of host or tumor-derived VEGF or its primary receptors expressed on tumor cells or host cells in the tumor (Fig. 5Go). It is possible that there are posttranslational changes that are induced by 2C3 treatment. We did, however, detect a noticeable increase in the expression of murine Npn-2 in mice bearing Capan-1 tumors treated with 2C3. This might be due to a change in the bioavailability of VEGF, or it could represent a compensatory mechanism by the host due to inhibition of VEGF-VEGFR2 interaction by 2C3.

Mice bearing human pancreatic xenografts that are treated with 2C3 show a reduced tumor burden when compared with control-treated animals; however, tumors continued to grow in the face of therapy. We postulate two potential mechanisms for this. First, 2C3 inhibits tumor-derived (human) VEGF but has no effect on host-derived (mouse) VEGF, of which there are substantial levels in each tumor type tested (Fig. 5Go). Second, there are multiple other angiogenic pathways (e.g., fibroblast growth factor 2) that could be contributing to the development and maintenance of the vascular system in solid tumors.45 Our results and those of others that have specifically blocked VEGFR2 activity in human tumor xenografts systems strongly suggest that tumor-derived VEGF is a critical factor for the survival and maintenance of vascular function in solid tumors. These studies also imply that tumor xenograft models underestimate the efficacy of anti-VEGF strategies that are specific for human VEGF and that inhibition of both tumor- and host-derived VEGF is likely to be more effective.

The quality and character of the vascular system in tumors from mice treated with 2C3 was altered significantly when compared with control-treated animals. Mice treated with 2C3 had tumors that showed a decrease in overall vessel density, VEG-FR2-expressing vessels, and the number of pericyte-associated vessels compared with tumors from control-treated animals. These results are consistent with previous reports in which 2C3 was used in an animal model of human breast carcinoma.15 We also observed a decrease in the vascular perfusion of Capan-1 tumors in mice treated with 2C3, which is consistent with a decrease in tumor microvessel density and previous reports linking alterations of microvessel density with alterations of vascular perfusion.46,47

In summary, we report that selective inhibition of VEGFR2 with an antibody specific for tumor-derived VEGF is effective at controlling the growth of pancreatic tumor xenografts in mice. Consistent with this is the fact that inhibition of tumor-derived VEGF activation of VEGFR2 results in altered vascular function in treated tumors and highlights the function of VEGFR2 and tumor-derived VEGF in the development and maintenance of the vascular system in solid tumors.


   ACKNOWLEDGMENTS
 
Supported by grants from the National Pancreas Foundation (R.A.B.), the James Ewing Foundation (J.B.F.), the American Cancer Society (J.B.F.), the EPe Marie Cain Research Scholarship in Angiogenesis Research (R.A.B.), and a Sponsored Research Agreement from Peregrine Pharmaceuticals Inc. (R.A.B.). The authors thank Peregrine Pharmaceuticals Inc. for providing purified 2C3 for these studies and Mishel Davis and Tamara Hart for technical support. The hybridoma MECA-32, developed by Dr. Eugene C. Butcher, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, Iowa).


   FOOTNOTES
 
Presented at the 57th Annual Meeting of the Society of Surgical Oncology, New York, New York, March 2004.

Dr. Brekken is a paid consultant for, receives research support through a sponsored research agreement with, and has an equity interest in Peregrine Pharmaceuticals Inc., a company that has licensed the exclusive rights to 2C3 from the University of Texas.

Received for publication June 16, 2005. Accepted for publication December 12, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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