PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of neoplasiaGuide for AuthorsAbout this journalExplore this journalNeoplasia (New York, N.Y.)
 
Neoplasia. 2005 October; 7(10): 912–920.
PMCID: PMC1550288

The Vascular-Targeting Fusion Toxin VEGF121/rGel Inhibits the Growth of Orthotopic Human Bladder Carcinoma Tumors1

Abstract

Vascular endothelial growth factor (VEGF) and its receptors (FLT-1 and KDR) are overexpressed by human bladder cancer cells and tumor endothelial cells, respectively. Strategies that target VEGF receptors hold promise as antiangiogenic therapeutic approaches to bladder cancer. A fusion protein of VEGF121 and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity experiments of VEGF121/rGel on the highly metastatic 253J B-V human bladder cancer cell line demonstrated that the VEGF121/rGel does not specifically target these cells, whereas Western blot analysis showed no detectable expression of KDR. Treatment with VEGF121/rGel against orthotopically implanted 253J B-V xenografts in nude mice resulted in a significant suppression of bladder tumor growth (~60% inhibition; P < .05) compared to controls. Immunohistochemistry studies of orthotopic 253J B-V tumors demonstrated that KDR is highly overexpressed in tumor vasculature. Immunofluorescence staining with antibodies to CD-31 (blood vessel endothelium) and rGel demonstrated a dramatic colocalization of the construct on tumor neovasculature. Treated tumors also displayed an increase in terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining compared to controls. Thus, VEGF121/rGel inhibits the growth of human bladder cancer by cytotoxic effects directed against the tumor vascular supply and has significant potential as a novel antiangiogenic therapeutic against human bladder cancer.

Keywords: Fusion toxin, VEGF, gelonin, vascular targeting, bladder cancer
Abbreviations: VEGF, VEGF-A, vascular endothelial growth factor A; Flt-1, FLT-1, VEGFR-1, vascular endothelial growth factor receptor 1; Flk-1, KDR, VEGFR-2, vascular endothelial growth factor receptor 2; rGel, gelonin; TCC, transitional cell carcinoma

Introduction

Transitional cell carcinoma (TCC) of the bladder is the fifth most common solid malignancy in the United States [1]. Over 63,000 new cases and over 13,000 deaths of this cancer are estimated for the year 2005 [2]. TCC is classified histopathologically into three types: superficial (papillary tumors), confined to the bladder wall (pT1 and pTa tumors), and invasive (stages T2–T4) [3]. Approximately 30% of patients with papillary tumors will progress to invasive TCC, where radical cystectomy is the standard therapy [1]. Unfortunately, this disease recurs in up to 50% of these patients despite surgery and is potentially lethal. Half of patients with muscle-invasive bladder cancer will develop metastatic disease [4].

The critical role of angiogenesis in tumor growth and metastasis has prompted major efforts to develop antiangiogenic therapies. Neovascularization is a normal, robust process that is controlled by numerous growth factors, including basic fibroblast growth factor, interleukin-8, hypoxia-inducible factors (1 and 2), and vascular endothelial growth factor A (VEGF-A) [5,6]. In addition, neovascularization appears to be an essential step to enable neoplastic growth and metastatic spread [7,8]. Animal models [9–13] as well as clinical studies [14–18] have identified elevated levels of these cytokines and growth factors during tumor progression and metastatic spread. In addition, receptors for many of these cytokines are overexpressed on the luminal surface of tumor vasculature and provide an opportunity for therapeutic targeting without the need to invade the tumor parenchyma.

VEGF-A is a predominant factor mediating angiogenesis and has been well-characterized for its pivotal involvement in tumor development and metastases [19––22]. The angiogenic actions of VEGF-A are primarily mediated through two closely related receptor tyrosine kinase domain receptors designated VEGFR-1 (Flt-1/FLT-1) and VEGFR-2 (Flk-1/KDR). These receptors are overexpressed in the endothelium of tumor vasculature [23–25] and are almost undetectable in the vascular endothelium of adjacent normal tissues. Therefore, they appear to be excellent targets for the development of therapeutic agents that inhibit tumor growth and metastatic spread through inhibition of tumor neovascularization. Recent studies have identified these receptors on breast [26], pancreatic [27,28], and leukemic human tumor cells [29,30].

We have previously described a novel fusion construct composed of VEGF121 and recombinant toxin gelonin (rGel), which is highly cytotoxic at nanomolar concentrations to both log phase and confluent endothelial cells that overexpress the KDR receptor and is not specifically cytotoxic to cells that overexpress the FLT-1 receptor [31]. rGel is a single-chain n-glycosidase that is similar in its action to ricin A chain [32], and immunotoxins and fusion constructs containing rGel have been shown to specifically kill tumor cells in vitro and in vivo [31–35]. In currently ongoing clinical studies, rGel does not appear to generate vascular leak syndrome, which limits the use of other toxins in its class [36]. The purpose of the current study is to evaluate the biologic activity of the vascular-targeting agent VEGF121/rGel on the growth of a highly metastatic human bladder carcinoma 253J B-V in an orthotopic model in nude mice. Our results show that VEGF121/rGel significantly inhibits tumor growth and suggests a potential clinical application of this vascular-targeting agent in the treatment of invasive bladder carcinoma.

Materials and Methods

Cell Lines

Porcine aortic endothelial (PAE) cells transfected with the human KDR receptor (PAE/KDR) were a generous gift from Dr. J. Waltenberger and developed as described [37]. The highly tumorigenic and highly metastatic 253J B-V human TCC cell line [38] was maintained as a monolayer culture in MEM medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, sodium pyruvate, nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and vitamins (CMEM). The 253J B-V cells were harvested by a 1-minute treatment with 0.025% trypsin-0.01% EDTA solution. The culture flask was tapped to detach the cells. The cells were washed in CMEM and resuspended in Hanks balanced salt solution (HBSS) in preparation for implantation into the mice.

Animals

Male athymic BALB/c nude mice were obtained from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). The mice were maintained in a laminar air flow cabinet under specific pathogen-free conditions and used at 8 to 12 weeks of age. All facilities were approved by the American Association for Accreditation of Laboratory Animal Care in accordance with the current regulations and standards of the United States Department of Agriculture, the Department of Health and Human Services, and the National Institutes of Health.

Expression and Purification of VEGF121/rGel

The construction, expression, and purification of VEGF121/rGel have been previously described [31]. The fusion toxin was stored in sterile phosphate-buffered saline (PBS) at -20°C.

In Vitro Cytotoxicity Assay

The cytotoxicity assay of VEGF121/rGel and rGel against log-phase PAE/KDR cells was performed as described previously [31], whereas that of VEGF121/rGel and rGel against log-phase 253J B-V cells was performed as follows. Log-phase cells (5 x 103) were plated in 96-well flat-bottom tissue culture plates and allowed to attach overnight. On the following day, the cells were replenished with medium supplemented with 2% serum and treated with various concentrations of VEGF121/rGel or rGel in triplicate. After incubation for 72 hours, the cells were treated with 50 µl of 1% MTT solution and incubated for 2 hours at 37°C. The MTT solution was removed and 100 µl of 10% DMSO was added to each well. Absorbance was read at 570 nm on a PowerwaveX 340 Microplate Scanning Spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT) using KC4 software.

Coculture of Endothelial Cells and 253J B-V Tumor Cells

253J B-V cells in log phase were plated into six-well plates (4 x 103 cells/well) and allowed to attach overnight. Sterile plastic inserts (0.4 µm) containing log-phase PAE/KDR cells (2 x 103 cells/well) were placed above the bladder cancer cells to permit a free passage of macromolecules between the two chambers. Bladder cancer cells without inserts were grown as controls. Cells were treated with various concentrations of VEGF121/rGel (4–500 nM) in triplicate. After incubation for 72 hours, the remaining adherent cells were stained with crystal violet (0.5% in 20% methanol) and solubilized with Sorenson's buffer (0.1 M sodium citrate, pH 4.2, in 50% ethanol). Absorbance was measured at 630 nm.

Western Blot Analysis

PAE/KDR and 253J B-V cells were cultured in 10-cm dishes with their respective media. Cells were treated with an IC50 dose of VEGF121/rGel for 24 hours. Control cells were treated with saline or VEGF (R&D Systems, Minneapolis, MN). Whole cell lysates of the treated and untreated PAE/KDR and 253J B-V cells were obtained by lysing cells in cell lysis buffer (50 mM Tris, pH 8.0, 0.1 mM EDTA, 1 mM DTT, 12.5 mM MgCl2, 0.1 M KCl, and 20% glycerol) supplemented with protease inhibitors [leupeptin (0.5%), aprotinin (0.5%), and PMSF (0.1%)]. Protein samples were separated by SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin, and the samples were analyzed for unphosphorylated (preactivated) KDR with mouse anti-Flk-1 monoclonal antibody (Transduction Laboratories, Lexington, KY) and for p-KDR using an antiphosphotyrosine antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate secondary antibodies were used at 1:5000 dilution for 1 hour and detected using an ECL detection kit (Amersham Biosciences, Buckinghamshire, England, UK).

Isolation of RNA and RT-PCR Analysis

Isolation of RNA and RT-PCR analysis was performed as previously described [39]. Briefly, total RNA was extracted using the RNeasy mini-kit (Qiagen, Valencia, CA), and its integrity was verified by electrophoresis on a denaturing formaldehyde agarose gel. RT-PCR analysis was performed by first subjecting the isolated RNA to first-strand cDNA synthesis, as described by the manufacturer of the Superscript First Strand synthesis system (Invitrogen, Carlsbad, CA). RT-PCR was performed using a Minicycler PCR machine (MJ Research, Inc., San Francisco, CA).

Orthotopic Implantation of Tumor Cells

A maximum tolerated dose of 40 mg/kg for VEGF121/rGel delivered intravenously under the conditions described below was established. For treatment purposes, 50% of the MTD was used. Each mouse was anesthetized with sodium pentobarbital (25 mg/kg, i.p.) and placed in the supine position. A lower midline incision was performed, and the bladder was exposed. The 253J B-V cells (3.5 x 105 cells in 50 µl of HBSS) were implanted into the wall of the bladder in the area of the bladder dome using 30-gauge needles on disposable 1-ml syringes. A successful implantation was indicated by a bleb in the bladder wall serosa. The abdominal wound was closed with metal wound clips on the first layer (Autoclips; Clay Adams, Parsippany, NJ). Thirty mice were randomized to three treatment groups. Treatment began on the third day after tumor injection into the bladder. The animals were treated with the following protocol: group 1—200 µl of saline every other day for 10 days (five treatments); group 2—29 µg of recombinant rGel in 200 µl of saline every other day for 10 days (five treatments); and group 3—80 µg of VEGF121/rGel in 200 µl of saline every other day for 10 days (five treatments). All treatment injections were delivered intravenously. Twenty-one days after tumor injection, the animals were sacrificed, and the bladders were harvested, weighed, and processed. For colocalization studies, a single dose of VEGF121/rGel (80 µg) or rGel (29 µg) was administered, and animals were sacrificed 4 hours later.

Histologic and Immunohistochemical Analysis

For histologic studies, 5-µm-thick sections were prepared and stained with hematoxylin and eosin. For immunohistochemical analysis, frozen tissue sections (8 µm thick) were fixed with cold acetone. Tissue sections (5 µm thick) of formalin-fixed paraffin-embedded specimens were deparaffinized in xylene, rehydrated in graded alcohols, and transferred to PBS. The slides were rinsed twice with PBS, and antigen retrieval was performed with pepsin for 12 minutes; endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 12 minutes. The samples were washed three times with PBS and incubated for 20 minutes at room temperature with a protein-blocking solution of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum. Excess blocking solution was drained, and the samples were incubated for 18 hours at 4°C with a 1:100 dilution of rat monoclonal anti-CD-31 antibody (PharMingen, San Diego, CA) or a 1:100 dilution of mouse anti-Flk-1 monoclonal antibody. The samples were then rinsed four times with PBS and incubated for 1 hour at room temperature with the secondary antibody. The slides were rinsed with PBS and incubated for 5 minutes with diaminobenzidine (Research Genetics, Huntsville, AL). The sections were then washed three times with PBS, counterstained with Gill's hematoxylin (Biogenex Laboratories, San Ramon, CA), and washed three times with PBS. The slides were mounted with Universal Mount mounting medium (Research Genetics).

Immunofluorescence

Frozen tissue sections (8 µm thick) were fixed with cold acetone. The samples were washed three times with PBS and incubated for 20 minutes at room temperature with a protein-blocking solution containing PBS (pH 7.5), 5% normal horse serum, and 1% normal goat serum. Excess blocking solution was drained, and the samples were incubated for 18 hours at 4°C with rat anti-CD-31 monoclonal antibody (1:400), rabbit anti-rGel polyclonal antibody (1:400), and/or rabbit anti-Flk-1/KDR polyclonal antibody C20 (1:100). The samples were then rinsed four times with PBS and incubated for 1 hour at room temperature with goat antimouse FITC-conjugated secondary antibody (1:80), goat antirabbit TRITC-conjugated secondary antibody (1:80), or rabbit Cy5 secondary antibody (1:400). The sections were then washed twice with PBS. The slides were mounted and viewed.

Immunofluorescent Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin End Labeling (TUNEL) Analysis

Paraffin-embedded tissue sections were sectioned (6 µm) and deparaffinized in xylene, rehydrated in alcohol, and transferred to PBS. TUNEL was performed using a commercial kit (Promega Corp., Madison, WI) according to the manufacturer's instructions. Briefly, samples were fixed with 4% paraformaldehyde (methanol-free) for 10 minutes at room temperature, washed with PBS, and then permeabilized by incubating with 0.2% Triton X-100 in PBS (vol/vol) for 15 minutes. The samples were incubated with equilibration buffer (from the kit). A reaction buffer containing equilibration buffer (45 µl), nucleotide mix (5 µl), and terminal deoxynucleotidyl transferase (1 µl) was added to the sections and incubated in a humidified chamber for 1 hour at 37°C, protected from light. The reaction was terminated by immersing the samples in 2x SSC (30 mM NaCl, 3 mM sodium citrate, pH 7.2) for 15 minutes, followed by three washes to remove unincorporated fluorescein dUTP. Background fluorescence was determined by processing slides in the absence of terminal deoxynucleotidyl transferase (negative control). Nuclei were stained with propidiumiodide (1 µg/ml) for 10 minutes. Prolong (Molecular Probes, Eugene, OR) was used to mount coverslips. Immunofluorescence microscopy was performed using a Zeiss Plan-Neofluar lens on an epifluorescence microscope equipped with narrow bandpass excitation filters mounted in a filter wheel (Ludl Electronic Products, Hawthorne, NY) to individually select for green and red fluorescence. Images were captured using a cooled CCD camera (Photometrics, Tucson, AZ). DNA fragmentation was detected by localized green fluorescence within the nuclei of apoptotic cells.

Laser scanning cytometry (LSC)-mediated quantitative analysis was used to determine the number of TUNEL-positive cells in each tissue section, as previously described [40]. Negative control slides were used to set the gates on the scattergram, and each bladder tumor section was scanned by LSC to determine the percentage of apoptotic cells in 1 x 104 total cells per tumor.

Results

Cytotoxicity of VEGF121/rGel and rGel on 253J B-V Cells

Because VEGF121 binds only to KDR and FLT-1, we first examined RNA levels of these two receptors in 253J B-V cells. Total RNA was harvested from log-phase cells, analyzed for integrity, and subjected to RT-PCR with primers for KDR, FLT-1, and GAPDH (control). PAE/KDR and PAE/FLT-1 cells were used as positive controls. 253J B-V cells did not express detectable levels of KDR or FLT-1 RNA as determined by RT-PCR (Figure 1A). The direct cytotoxic effect of VEGF121/rGel and rGel on 253J B-V cells was initially evaluated in vitro and compared to their cytotoxic effect on PAE/KDR cells. As previously described [31], treatment of log-phase cells with VEGF121/rGel for 72 hours showed the greatest cytotoxic effect against PAE/KDR cells, with an IC50 of 1 nM (Figure 1B). In contrast, the IC50 of the untargeted rGel on these cells was approximately 100 nM, similar to the IC50 of VEGF121/rGel on 253J B-V cells. However, 253J B-V cells were approximately seven-fold more sensitive to the cytotoxic effects of the VEGF121/rGel construct compared to that of the untargeted rGel toxin (100 vs 700 nM, respectively). These values are similar to those for other tumor cells [31]. These values indicate some—but relatively poor—targeting of VEGF121/rGel to 253J B-V cells and that VEGF121/rGel is far more potent toward endothelial cells that overexpress the KDR receptor than to 253J B-V cells in vitro.

Figure 1
253J B-V cells are not targeted by VEGF121/rGel. (A) RT-PCR analysis demonstrating that 253J B-V cells do not express detectable levels of the VEGF121 receptors KDR and FLT-1. Endothelial cells transfected with KDR (PAE/KDR) and FLT-1 (PAE/FLT-1) are ...

Determination of KDR Expression on 253J B-V Cells by Western Blot Analysis

Previous studies in our laboratory show that the presence of the KDR receptor is critical to the development of VEGF121/rGel-mediated cytotoxicity. Therefore, we assessed the presence of KDR and p-KDR in PAE/KDR and 253J B-V cells. Cells were treated with VEGF121/rGel or VEGF165 for 24 hours and then processed as described. As shown in Figure 2, the KDR receptor was detected in PAE/KDR—but not 253J B-V—lysates, suggesting that 253J B-V cells do not express detectable amounts of KDR, in support of the cytotoxicity data obtained. PAE/KDR cells treated with VEGF121/rGel resulted in phosphorylation of the KDR receptor. However, p-KDR was not detected in 253J B-V lysates after treatment with either VEGF165 or with VEGF121/rGel.

Figure 2
KDR is not detectable on 253J B-V cells. Whole cell lysates were prepared and used in Western blot analysis as described in “Materials and Methods.” A 235-kDa protein corresponding to KDR was seen in PAE/KDR lysates. No KDR was detected ...

Evaluation of Flk-1/KDR Expression in 253J B-V Orthotopic Bladder Tumors In Vivo

This highly aggressive tumor model was assessed for its vascularity and the expression of Flk-1 on the vascular endothelium. As shown in Figure 3, numerous blood vessels were observed in 253J B-V tumors. In addition, all vessels demonstrated a colocalization of Flk-1 with CD-31, indicating that Flk-1 receptors are overexpressed only on vascular endothelial cells and are not detectable on tumor cells using this method.

Figure 3
Immunohistochemistry of 253J B-V bladder tumor tissue. Immunohistochemistry performed with anti-CD-31 and anti-Flk-1 antibodies demonstrates a colocalization of Flk-1/KDR with CD-31. Flk-1/KDR was not expressed by 253J B-V tumor cells, but was found only ...

Treatment of Orthotopic 253J B-V Tumors with VEGF121/rGel

The therapeutic and antiangiogenic effect of the fusion protein VEGF121/rGel was evaluated against human bladder cancer xenografts growing in athymic nude mice. The mice were treated every other day with a total of 20 mg/kg VEGF121/rGel, rGel, or saline. The mice were necropsied 21 days after tumor implantation, and bladder tumors were harvested, weighed, and processed. No difference was observed in tumor weight from mice treated with saline or rGel (Figure 4; P < .05). In contrast, tumors from mice treated with VEGF121/rGel weighed about 40% that of the control (P < .05).

Figure 4
Effects of VEGF121/rGel treatment on the in vivo growth of orthotopic 253J B-V cells. Tumor-bearing mice were treated intravenously with saline, rGel, or VEGF121/rGel. Mice were necropsied 21 days after tumor implantation, and bladder tumors were harvested. ...

To further understand the mechanisms leading to cytotoxic effects against the tumor in vivo, we performed coculture experiments of 253J B-V cells with PAE/KDR cells, simulating the in vivo setting of tumor cells and endothelial cells. We used VEGF121/rGel dose levels that are 80% to 100% cytotoxic to PAE/KDR cells but are mildly cytotoxic to 253J B-V cells. We observed no difference in VEGF121/rGel-mediated cytotoxicity between 253J B-V cells grown alone or in coculture (data not shown). The results suggest that VEGF121/rGel-mediated inhibition of the growth of human bladder cancer in vivo is not due to the effect per se of factors released by damaged endothelial cells. Rather, the results support our hypothesis that tumor cell death is a secondary effect due to specific damage to tumor vasculature.

Immunofluorescence and Localization of VEGF121/rGel in Tumors

Bladder tumors from treated mice were processed for histology and immunohistochemical analysis. Tissues for colocalization experiments were harvested 4 hours after VEGF121/rGel or rGel treatment. Immunofluorescence of tumor tissue sections with anti-CD-31 and anti-rGel antibodies showed a dramatic colocalization of VEGF121/rGel with CD-31 on the tumor neovasculature (Figure 5), indicating that VEGF121/rGel targets the tumor endothelium. In some instances, VEGF121/rGel did not colocalize with CD-31. VEGF121/rGel was not detected in other tissues (data not shown). Immunostaining with the anti-rGel antibody of tumors of animals treated with rGel was negative, indicating the specificity of VEGF121 as a targeting moiety for the tumor vasculature.

Figure 5
Immunofluorescence of tumor tissue sections of mice treated with rGel or VEGF121/rGel. Samples were processed as described in “Materials and Methods.” Panel A: CD-31 (green) is seen in tissue sections of mice treated with both VEGF121 ...

Immunofluorescence and Localization of Flk-1/KDR in Tumors

Bladder tumors from treated and control mice were harvested at the end of the study and processed for histology and immunohistochemical analysis. Immunofluorescence with the anti-CD-31 antibody of tumor tissue sections of mice treated with VEGF121/rGel revealed a vasculature that is less dispersed and more clustered compared to saline-treated mice, with far fewer vascular networks. Colocalization staining with anti-Flk-1/KDR antibody surprisingly showed an increase in the number of Flk-1/KDR receptors in tumors of mice treated with VEGF121/rGel versus rGel (Figure 6). The average increase in Flk-1/KDR levels was 34.4% after VEGF121/rGel treatment (P < .02). Almost all the Flk-1/KDR stainings colocalized with CD-31.

Figure 6
Effect of VEGF121/rGel treatment on Flk-1/KDR levels in orthotopic bladder tumors. CD-31 (red) is seen in tissue sections of mice treated with both VEGF121/rGel and rGel (control). VEGF121/rGel treatment results in a reduced network of tumor vessels. ...

TUNEL Analysis of Bladder Tumors

To study the in vivo effect on tumor cells as a result of VEGF121/rGel cytotoxicity on endothelial cells, bladder tumors from mice treated with VEGF121/rGel, rGel, and saline were stained for apoptotic effects. Both necrotic as well as non-necrotic regions were examined. As shown in Figure 7, tumors treated with either saline or rGel showed virtually no apoptotic regions. In comparison, treatment with VEGF121/rGel resulted in a slight increase in apoptosis. The results of LSC quantification are shown in Table 1. VEGF121/rGel-treated tumors showed a significantly higher TUNEL staining than rGel- or saline-treated tumor-bearing mice.

Figure 7
TUNEL analysis of orthotopic bladder tumors. Tumors treated with VEGF121/rGel show a much higher TUNEL staining compared to controls. Negative control denotes cells analyzed for TUNEL without the addition of terminal deoxynucleotidyl transferase. Control ...
Table 1
LSC-Mediated Analysis of TUNEL Staining of Orthotopically Placed 253J B-V Bladder Tumors of Mice Treated with rGel or VEGF121/rGel.

Discussion

Although bladder cancer responds to traditional chemotherapeutic approaches, resistance and relapse occur even in those patients who attain a complete response, and patients with metastatic disease generally face a poor prognosis. Bladder primary tumors and metastatic sites appear to be highly vascular and depend on numerous soluble factors and their receptors to mediate the growth and development of the intratumoral vascular tree. Targeting angiogenic mechanisms within these tumors potentially offers another therapeutic modality that may not be susceptible to resistance. However, this is a difficult endeavor because there are so many factors capable of controlling angiogenesis.

We have previously reported that VEGF121/rGel is cytotoxic to endothelial cells overexpressing the KDR receptor rather than the FLT-1 receptor [31], and that endothelial cells expressing the KDR receptor below a threshold number (approximately 2 x 103 receptors per cell) are several hundred-fold more resistant to VEGF121/rGel than are cells overexpressing KDR (1–3 x 105 receptors per cell) [31]. Although Flt-1 binds VEGF with a 50-fold higher affinity than KDR [41], most of the VEGF angiogenic properties (mitogenicity, chemotaxis, and induction of morphologic changes) are mediated by interactions with KDR [37]. The IC50 of VEGF121/rGel for PAE/KDR, an endothelial cell line that overexpresses KDR (mimicking proliferating endothelial cells during angiogenesis), is 1 nM. VEGF121/rGel cytotoxicity toward other tumor cell lines in vitro is much higher, and in vivo efficacy studies suggest that the activity of VEGF121/rGel at low doses is due to its cytotoxicity toward endothelial cells and not the tumor. To test the cytotoxicity of VEGF121/rGel toward the bladder cell line 253J B-V, we performed an in vitro cytotoxicity assay and found that the IC50 of VEGF121/rGel for this highly metastatic cell line was 100 nM, comparable to that of VEGF121/rGel with other tumor cell lines and much higher than the IC50 toward proliferating endothelial cells. The cytotoxicity of the free toxin to PAE/KDR cells is different compared to the cytotoxicity to 253J B-V cells. As observed previously, it is possible that the rate or route of entry of rGel is different based on the rate of cell division, which can vary from one cell type to another. Although the higher cytotoxicity of VEGF121/rGel than rGel toward 253J B-V cells suggests the presence of a VEGF121 receptor, the receptor levels are fairly low, as supported by RT-PCR, Western and immunohistochemistry data point to the importance of high receptor levels in VEGF121/rGel-mediated cytotoxicity and destruction of the tumor vasculature. Indeed, immunostaining of orthotopic 253J B-V tumors localized p-KDR to tumor-associated endothelial cells [42]. To date, there are only two reports of KDR expression on TCCs [43,44].

Our results show an average increase of 34.4% in Flk-1/KDR levels in VEGF121/rGel-treated mice compared to controls (P < .02), the majority of which appear to be on the vasculature. Note that VEGF121/rGel treatment results in a vasculature that is less dispersed and more clustered, subjecting tumor cells that are distant from the vasculature to hypoxic stress. The increase in total Flk-1/KDR levels is similar to that observed in the treatment of 253J B-V tumors with DC101, an anti-Flk-1 antibody [42]. Previous results suggest that induction of Flk-1 occurs preferentially within regions of relative hypoxia (i.e., in the tumor core) [42]. In addition, VEGF plays a role in the paracrine stimulation of angiogenesis [45,46] but may also act through an auto-regulatory pathway that is activated when VEGF signal transduction is interrupted [47]. Growth-regulatory pathways are often controlled by feedback loops that reduce the expression of pathway components when signal transduction is active and increase levels when signaling is inactive. In the orthotopic 253J-BV tumor model, upregulation of Flk-1/KDR could serve to rapidly reestablish the vascular network once therapy is terminated.

Treatment of the orthotopic 253J B-V model with VEGF121/rGel resulted in a 60% inhibition of tumor growth compared to rGel-treated mice. This compares favorably to therapy with a blocking antibody against murine VEGFR-2 in the same tumor model [42]. Although bladders in this experiment were harvested, weighed, and processed 21 days after tumor injection, it is possible that a higher rate on inhibition can be observed at different time points after tumor injection, depending on the expression of VEGFR-2 in the tumor vascular endothelium. Further studies are warranted.

Molecular engineering has enabled the synthesis of novel chimeric molecules that have therapeutic potential. Chimeric fusion constructs targeting the IL-2 receptor [48,49], EGF receptor [50], and other growth factor cytokine receptors have been described. Studies by Olson et al. [51] and Ramakrishnan et al. [52] showed that a chemical conjugate of VEGF and truncated diphtheria toxin (DT) displayed an impressive cytotoxic activity on cell lines expressing receptors for VEGF. Further studies with VEGF-DT fusion constructs demonstrated selective toxicity to Kaposi's sarcoma cells and dividing endothelial cells in vitro and in vivo [53].

The association of bladder cancer progression with proangiogenic agents has been suggested for at least 20 years, with the demonstration that urine from cancer patients induces neovascularization in a rat cornea model [54]. In addition, endothelial cells in bladder tumors overexpress the mRNA for Flt-1 and KDR [55]. Studies of non-small cell lung cancer and melanoma have since confirmed a link between neovascularization, microvessel density, and survival or prognosis; however, this remains somewhat controversial [56–58]. Previous studies using agents such as interferon have demonstrated an ability to inhibit tumor growth in this bladder tumor model primarily through an antiangiogenic mechanism [1,59]. Because the VEGF121/rGel fusion toxin is primarily cytotoxic to vascular endothelial cells rather than directly cytotoxic to bladder tumor cells, the tumor growth-inhibitory effects noted in this model are due solely to effects on tumor vasculature. The mechanism of action of VEGF121/rGel on endothelial cells in vitro appears to be necrotic (data not shown), and the proapoptotic effects observed on bladder tumor cells appear to result from hypoxic stress and pH changes secondary to the vascular disruption effects of the construct because this agent does not appear to directly cause apoptotic effects in intoxicated cells.

The development of targeted therapeutic approaches for bladder cancer generally includes agents directed against the tumor cells themselves [60]. Thiesen et al. [61] initially reported a promising approach using antibodies recognizing transitional bladder carcinoma cells linked to ricin A or B chains. More recently, Zang et al. [62] reported on the clinical efficacy of a BD1-RT immunotoxin administered after resection through the intravesicular route in patients with bladder cancer. The comparison to the control group treated with mitomycin C demonstrated no statistically significance differences between the groups in terms of recurrence rates; however, the side effect rates were reportedly lower for the immunotoxin group.

Intravesicular therapy is an attractive option for bladder cancer because relatively high doses of therapeutic agents can be delivered by establishing a direct contact with the bladder tumor, thereby providing minimal systemic side effects. However, agents targeting the tumor vasculature, such as VEGF121/rGel, operate in the luminal side of the blood vessels; therefore, intravesicular therapy does not appear to be an option. However, multimodality therapy using vascular-targeted agents delivered intravenously—in combination with chemotherapeutic or biologic agents targeting the tumor cells themselves or other aspects of the neovascularization process and delivered either systemically or intravesicularly—may have some benefits. Recent studies suggest that the use of antiangiogenic agents may potentiate the efficacy of chemotherapy. This area is currently under intensive investigation [63].

In conclusion, VEGF121/rGel is a promising cytotoxic agent that targets the neovasculature of bladder carcinoma and is an excellent candidate for clinical trials and potential combination therapy for the treatment of bladder cancer.

Footnotes

1This research was conducted, in part, by the Clayton Foundation for Research and supported by the SPORE grant “M. D. Anderson Cancer Center SPORE in Genitourinary Cancer” (P50 CA91846).

2Khalid A. Mohamedali and Daniel Kedar contributed equally to this work.

3Current address: Institute of Urology, Rabin Medical Center, Beilinson Campus, Petach-Tikvah, Israel.

References

1. Izawa JI, Sweeney P, Perrotte P, Kedar D, Dong Z, Slaton JW, Karashima T, Inoue K, Benedict WF, Dinney CP. Inhibition of tumorigenicity and metastasis of human bladder cancer growing in athymic mice by interferon-beta gene therapy results partially from various antiangiogenic effects including endothelial cell apoptosis. Clin Cancer Res. 2002;8:1258–1270. [PubMed]
2. Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ. Cancer statistics, 2005. CA Cancer J Clin. 2005;55:10–30. [PubMed]
3. Fauconnet S, Lascombe I, Chabannes E, Adessi GL, Desvergne B, Wahli W, Bittard H. Differential regulation of vascular endothelial growth factor expression by peroxisome proliferator-activated receptors in bladder cancer cells. J Biol Chem. 2002;277:23534–23543. [PubMed]
4. Kausch I, Bohle A. Molecular aspects of bladder cancer III. Prognostic markers of bladder cancer. Eur Urol. 2002;41:15–29. [PubMed]
5. Jung YD, Ahmad SA, Liu W, Reinmuth N, Parikh A, Stoeltzing O, Fan F, Ellis LM. The role of the microenvironment and intercellular cross-talk in tumor angiogenesis. Semin Cancer Biol. 2002;12:105–112. [PubMed]
6. Torisu H, Ono M, Kiryu H, Furue M, Ohmoto Y, Nakayama J, Nishioka Y, Sone S, Kuwano M. Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: possible involvement of TNFalpha and IL-1alpha. Int J Cancer. 2000;85:182–188. [PubMed]
7. Aranda FI, Laforga JB. Microvessel quantitation in breast ductal invasive carcinoma. Correlation with proliferative activity, hormonal receptors and lymph node metastases. Pathol Res Pract. 1996;192:124–129. [PubMed]
8. Fox SB, Leek RD, Bliss J, Mansi JL, Gusterson B, Gatter KC, Harris AL. Association of tumor angiogenesis with bone marrow micrometastases in breast cancer patients. J Natl Cancer Inst. 1997;89:1044–1049. [PubMed]
9. Furumatsu T, Nishida K, Kawai A, Namba M, Inoue H, Ninomiya Y. Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth. Int J Cancer. 2002;97:313–322. [PubMed]
10. Schimer M, Hoffmann J, Menrad A, Schneider MR. Anti-angiogenic chemotherapeutic agents: characterization in comparison to their tumor growth inhibition in human renal cell carcinoma models. Clin Cancer Res. 1998;4:1331–1336. [PubMed]
11. Tsuzuki Y, Mouta CC, Bockhorn M, Xu L, Jain RK, Fukumura D. Pancreas microenvironment promotes VEGF expression and tumor growth: novel window models for pancreatic tumor angiogenesis and microcirculation. Lab Invest. 2001;81:1439–1451. [PubMed]
12. Vajkoczy P, Thurnher A, Hirth KP, Schilling L, Schmiedek P, Ullrich A, Menger MD. Measuring VEGF-Flk-1 activity and consequences of VEGF-Flk-1 targeting in vivo using intravital microscopy: clinical applications. Oncologist. 2000;5(Suppl 1):16–19. [PubMed]
13. Westphal JR, Van't Hullenaar R, Peek R, Willems RW, Crickard K, Crickard U, Askaa J, Clemmensen I, Ruiter DJ, De Waal RM. Angiogenic balance in human melanoma: expression of VEGF, bFGF, IL-8, PDGF and angiostatin in relation to vascular density of xenografts in vivo. Int J Cancer. 2000;86:768–776. [PubMed]
14. Bikfalvi A, Bicknell R. Recent advances in angiogenesis, anti-angiogenesis and vascular targeting. Trends Pharmacol Sci. 2002;23:576–582. [PubMed]
15. Desai SB, Libutti SK. Tumor angiogenesis and endothelial cell modulatory factors. J Immunother. 1999;22:186–211. [PubMed]
16. Ferrara N, Gerber HP. The role of vascular endothelial growth factor in angiogenesis. Acta Haematol. 2001;106:148–156. [PubMed]
17. Gasparini G, Barbareschi M, Boracchi P, Verderio P, Caffo O, Meli S, Palma PD, Marubini E, Bevilacqua P. Tumor angiogenesis predicts clinical outcome of node-positive breast cancer patients treated with adjuvant hormone therapy or chemotherapy. Cancer J Sci Am. 1995;1:131. [PubMed]
18. Gasparini G, Harris AL. Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. J Clin Oncol. 1995;13:765–782. [PubMed]
19. Kumamoto H, Ohki K, Ooya K. Association between vascular endothelial growth factor (VEGF) expression and tumor angiogenesis in ameloblastomas. J Oral Pathol Med. 2002;31:28–34. [PubMed]
20. Kurebayashi J, Otsuki T, Kunisue H, Mikami Y, Tanaka K, Yamamoto S, Sonoo H. Expression of vascular endothelial growth factor (VEGF) family members in breast cancer. Jpn J Cancer Res. 1999;90:977–981. [PubMed]
21. Obermair A, Kucera E, Mayerhofer K, Speiser P, Seifert M, Czerwenka K, Kaider A, Leodolter S, Kainz C, Zeillinger R. Vascular endothelial growth factor (VEGF) in human breast cancer: correlation with disease-free survival. Int J Cancer. 1997;74:455–458. [PubMed]
22. Oehler MK, Caffier H. Diagnostic value of serum VEGF in women with ovarian tumors. Anticancer Res. 1999;19:2519–2522. [PubMed]
23. Fine BA, Valente PT, Feinstein GI, Dey T. VEGF, flt-1, and KDR/flk-1 as prognostic indicators in endometrial carcinoma. Gynecol Oncol. 2000;76:33–39. [PubMed]
24. Senger DR, Van de WL, Brown LF, Nagy JA, Yeo KT, Yeo TK, Berse B, Jackman RW, Dvorak AM, Dvorak HF. Vascular permeability factor (VPF, VEGF) in tumor biology. Cancer Metastasis Rev. 1993;12:303–324. [PubMed]
25. Shibuya M. Role of VEGF-flt receptor system in normal and tumor angiogenesis. Adv Cancer Res. 1995;67:281–316. [PubMed]
26. Price DJ, Miralem T, Jiang S, Steinberg R, Avraham H. Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells. Cell Growth Differ. 2001;12:129–135. [PubMed]
27. Itakura J, Ishiwata T, Shen B, Kornmann M, Korc M. Concomitant over-expression of vascular endothelial growth factor and its receptors in pancreatic cancer. Int J Cancer. 2000;85:27–34. [PubMed]
28. von Marschall Z, Cramer T, Hocker M, Burde R, Plath T, Schirner M, Heidenreich R, Breier G, Riecken EO, Wiedenmann B, et al. De novo expression of vascular endothelial growth factor in human pancreatic cancer: evidence for an autocrine mitogenic loop. Gastroenterology. 2000;119:1358–1372. [PubMed]
29. Ferrajoli A, Manshouri T, Estrov Z, Keating MJ, O'Brien S, Lerner S, Beran M, Kantarjian HM, Freireich EJ, Albitar M. High levels of vascular endothelial growth factor receptor-2 correlate with shortened survival in chronic lymphocytic leukemia. Clin Cancer Res. 2001;7:795–799. [PubMed]
30. Padro T, Bieker R, Ruiz S, Steins M, Retzlaff S, Burger H, Buchner T, Kessler T, Herrera F, Kienast J, et al. Overexpression of vascular endothelial growth factor (VEGF) and its cellular receptor KDR (VEGFR-2) in the bone marrow of patients with acute myeloid leukemia. Leukemia. 2002;16:1302–1310. [PubMed]
31. Veenendaal LM, Jin H, Ran S, Cheung L, Navone N, Marks JW, Waltenberger J, Thorpe P, Rosenblum MG. In vitro and in vivo studies of a VEGF121/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors. Proc Natl Acad Sci USA. 2002;99:7866–7871. [PubMed]
32. Stirpe F, Olsnes S, Pihl A. Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A. J Biol Chem. 1980;255:6947–6953. [PubMed]
33. Rosenblum MG, Murray JL, Cheung L, Rifkin R, Salmon S, Bartholomew R. A specific and potent immunotoxin composed of antibody ZME-018 and the plant toxin gelonin. Mol Biother. 1991;3:6–13. [PubMed]
34. Rosenblum MG, Zuckerman JE, Marks JW, Rotbein J, Allen WR. A gelonin-containing immunotoxin directed against human breast carcinoma. Mol Biother. 1992;4:122–129. [PubMed]
35. Rosenblum MG, Cheung L, Kim SK, Mujoo K, Donato NJ, Murray JL. Cellular resistance to the antimelanoma immunotoxin ZME-gelonin and strategies to target resistant cells. Cancer Immunol Immunother. 1996;42:115–121. [PubMed]
36. Talpaz M, Kantarjian HM, Freireich EJ, Lopez V, Zhang W, Cortes-Franco J, Scheinberg DA, Rosenblum MG. Phase I clinical trial of the anti-CD-33 immunotoxin Hum195/rGel. American Association for Cancer Research; Abstract R5362, 94th Annual Meeting; 2003. p. 1066.
37. Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem. 1994;269:26988–26995. [PubMed]
38. Dinney CP, Fishbeck R, Singh RK, Eve B, Pathak S, Brown N, Xie B, Fan D, Bucana CD, Fidler IJ. Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice. J Urol. 1995;154:1532–1538. [PubMed]
39. Ran S, Mohamedali KA, Luster TA, Thorpe PE, Rosenblum MG. The vascular-ablative agent VEGF(121)/rGel inhibits pulmonary metastases of MDA-MB-231 breast tumors. Neoplasia. 2005;7:486–496. [PMC free article] [PubMed]
40. Davis DW, Weidner DA, Holian A, McConkey DJ. Nitric oxide-dependent activation of p53 suppresses bleomycin-induced apoptosis in the lung. J Exp Med. 2000;192:857–869. [PMC free article] [PubMed]
41. de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science. 1992;255:989–991. [PubMed]
42. Davis DW, Inoue K, Dinney CP, Hicklin DJ, Abbruzzese JL, McConkey DJ. Regional effects of an antivascular endothelial growth factor receptor monoclonal antibody on receptor phosphorylation and apoptosis in human 253J B-V bladder cancer xenografts. Cancer Res. 2004;64:4601–4610. [PubMed]
43. Gakiopoulou-Givalou H, Nakopoulou L, Panayotopoulou EG, Zervas A, Mavrommatis J, Giannopoulos A. Non-endothelial KDR/flk-1 expression is associated with increased survival of patients with urothelial bladder carcinomas. Histopathology. 2003;43:272–279. [PubMed]
44. Song S, Zeng L, Wu J. Cloning and monoclonal antibody preparation of VEGF receptor KDR extracellular V–VII domain and KDR expression in carcinomas of different origins. Zhonghua Zhong Liu Za Zhi. 1999;21:96–98. [PubMed]
45. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev. 1997;18:4–25. [PubMed]
46. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 1999;13:9–22. [PubMed]
47. Jackson MW, Roberts JS, Heckford SE, Ricciardelli C, Stahl J, Choong C, Horsfall DJ, Tilley WD. A potential autocrine role for vascular endothelial growth factor in prostate cancer. Cancer Res. 2002;62:854–859. [PubMed]
48. Duvic M, Cather J, Maize J, Frankel AE. DAB389IL2 diphtheria fusion toxin produces clinical responses in tumor stage cutaneous T cell lymphoma. Am J Hematol. 1998;58:87–90. [PubMed]
49. Frankel A, Tagge E, Chandler J, Burbage C, Hancock G, Vesely J, Willingham M. IL2-ricin fusion toxin is selectively cytotoxic in vitro to IL2 receptor-bearing tumor cells. Bioconjug Chem. 1995;6:666–672. [PubMed]
50. Osborne CK, Coronado-Heinsohn E. Targeting the epidermal growth factor receptor in breast cancer cell lines with a recombinant ligand fusion toxin (DAB389EGF) Cancer J Sci Am. 1996;2:175. [PubMed]
51. Olson TA, Mohanraj D, Roy S, Ramakrishnan S. Targeting the tumor vasculature: inhibition of tumor growth by a vascular endothelial growth factor-toxin conjugate. Int J Cancer. 1997;73:865–870. [PubMed]
52. Ramakrishnan S, Olson TA, Bautch VL, Mohanraj D. Vascular endothelial growth factor-toxin conjugate specifically inhibits KDR/flk-1-positive endothelial cell proliferation in vitro and angiogenesis in vivo. Cancer Res. 1996;56:1324–1330. [PubMed]
53. Arora N, Masood R, Zheng T, Cai J, Smith DL, Gill PS. Vascular endothelial growth factor chimeric toxin is highly active against endothelial cells. Cancer Res. 1999;59:183–188. [PubMed]
54. Chodak GW, Hospelhorn V, Judge SM, Mayforth R, Koeppen H, Sasse J. Increased levels of fibroblast growth factor-like activity in urine from patients with bladder or kidney cancer. Cancer Res. 1988;48:2083–2088. [PubMed]
55. Brown LF, Berse B, Jackman RW, Tognazzi K, Manseau EJ, Dvorak HF, Senger DR. Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas. Am J Pathol. 1993;143:1255–1262. [PubMed]
56. Decaussin M, Sartelet H, Robert C, Moro D, Claraz C, Brambilla C, Brambilla E. Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival. J Pathol. 1999;188:369–377. [PubMed]
57. Koukourakis MI, Giatromanolaki A, Thorpe PE, Brekken RA, Sivridis E, Kakolyris S, Georgoulias V, Gatter KC, Harris AL. Vascular endothelial growth factor/KDR activated microvessel density versus CD31 standard microvessel density in non-small cell lung cancer. Cancer Res. 2000;60:3088–3095. [PubMed]
58. Straume O, Akslen LA. Expression of vascular endothelial growth factor, its receptors (FLT-1, KDR) and TSP-1 related to microvessel density and patient outcome in vertical growth phase melanomas. Am J Pathol. 2001;159:223–235. [PubMed]
59. Singh RK, Gutman M, Bucana CD, Sanchez R, Llansa N, Fidler IJ. Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas. Proc Natl Acad Sci USA. 1995;92:4562–4566. [PubMed]
60. Syrigos KN, Deonarian DP, Epenetos AA. Use of monoclonal antibodies for the diagnosis and treatment of bladder cancer. Hybridoma. 1999;18:219–224. [PubMed]
61. Thiesen HJ, Juhl H, Arndt R. Selective killing of human bladder cancer cells by combined treatment with A and B chain ricin antibody conjugates. Cancer Res. 1987;47:419–423. [PubMed]
62. Zang Z, Xu H, Yu L, Yang D, Xie S, Shi Y, Li Z, Li J, Wang J, Li M, et al. Intravesical immunotoxin as adjuvant therapy to prevent the recurrence of bladder cancer. Chin Med J (Engl) 2000;113:1002–1006. [PubMed]
63. Tiguert R, Lessard A, So A, Fradet Y. Prognostic markers in muscle invasive bladder cancer. World J Urol. 2002;20:190–195. [PubMed]

Articles from Neoplasia (New York, N.Y.) are provided here courtesy of Neoplasia Press