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Clinical Cancer Research Vol. 10, 1835-1842, March 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Inhibition of Orthotopic Human Bladder Tumor Growth by Lentiviral Gene Transfer of Endostatin

Eiji Kikuchi1, Silvia Menendez1, Makoto Ohori1, Carlos Cordon-Cardo1,2, Noriyuki Kasahara3 and Bernard H. Bochner1

Departments of 1 Urology and 2 Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York, and 3 Department of Medicine, University of California Los Angeles, Los Angeles, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Inhibitors of endothelial cell proliferation, such as endostatin, result in suppression of tumor-associated angiogenesis and can achieve growth-inhibitory effects depending on the type of tumor treated. The purpose of this study was to investigate whether local overexpression of endostatin could serve to diminish tumor growth of bladder cancer in vivo.

Experimental Design: We examined the capability of lentiviral-mediated gene transfer in vitro and therapeutic effects of lentivirus-based vectors expressing endostatin on tumor growth using an orthotopic human bladder tumor model.

Results: We found that self-inactivating lentivirus vectors containing green fluorescent protein, alone or in combination with endostatin, were capable of efficient and stable gene transfer to a variety of human bladder tumor cell lines. The production and secretion of endostatin from lentivirus-transduced KU-7 human bladder cancer cells was confirmed by Western blot and competitive enzyme immunoassay. Intravesical instillation of untransduced, green fluorescent protein control lentivirus-transduced, and endostatin-transduced KU-7 cells was performed in murine models to establish orthotopic tumors. Sustained long-term expression of endostatin was achieved in lentivirus-transduced orthotopic bladder tumors, and it was associated with decreased vascularization and inhibition of tumor growth. Lentivirus vector-mediated overexpression of endostatin did not affect the intrinsic production of basic fibroblast growth factor and vascular endothelial growth factor.

Conclusions: These findings suggest that lentivirus-mediated gene transfer might represent an effective strategy for expression of angioinhibitory peptides to achieve inhibition of human bladder cancer proliferation and tumor progression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The dependency of tumor growth on the ability to induce a neovascular response is supported by significant experimental and clinical data (1 , 2) . Angiogenesis is regulated by complex signaling pathways acting on endothelial cells that stimulate cell proliferation, migration, and subsequent tube formation (3) . The level of angiogenic activity within a tissue is determined by the balance between stimulatory and inhibitory molecular regulators of endothelial cell activation. As tumors progress, they must acquire the ability to switch from a physiologically inhibitory environment to one that promotes new blood vessel development.

The critical role of angiogenesis in bladder cancer progression is supported by laboratory and clinical correlative reports (4, 5, 6) . Microvessel density (MVD) within a bladder tumor is strongly associated with disease recurrence and overall survival in patients with invasive transitional cell carcinoma of the bladder (5) . Additionally, tumor angiogenesis has been established as a predictor of disease progression of bladder tumors (6) . These studies have led to the further characterization of angiogenic factors responsible for vascular density within bladder tumors. In addition to the overexpression of angiogenic inducers such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), reduction or loss in the expression of angiogenic inhibitors might play an important role in tumor progression. The loss of expression of the endogenous endothelial inhibitor thrombospondin-1 (THBS-1) has been found to play a central role in acquisition of the angiogenic phenotype in bladder cancer cell lines (7) . This suggests that blockade of tumor vascularization by endogenous inhibitors of angiogenesis might impact on tumor growth and might be promising as an approach to cancer treatment.

Endostatin is an endogenous angiogenic inhibitor, isolated from hemangioendothelioma cells as a COOH-terminal segment of collagen XVIII (8) . Elucidation of the detailed mechanisms by which endostatin affects tumor dormancy and angiogenesis is now under intense investigation. Endostatin can directly affect endothelial cell functions by inhibiting their proliferation and migration. Endostatin induces endothelial apoptosis via a mechanism that results in reduced Bcl-2 and Bcl-XL expression (9) and causes G1 cell cycle arrest in endothelial cells (10) . Endostatin has also been reported to interfere with fibroblast growth factor-2-induced signal transduction (11) and the activation of endothelial NO synthase (12) , integrins (13) , and glypicans (14) at the cell surface. In addition to its effects on endothelial cells, endostatin can also achieve direct inhibitory effects on the tumor cells themselves. Kim et al. (15) demonstrated that endostatin inhibits tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase-2.

Several studies have shown that recombinant endostatin inhibits tumor growth by preventing neovascularization in certain cancer models (16 , 17) . This form of angiostatic therapy requires long-term administration of the inhibitor to ensure suppression of tumor cells in vivo. Because long-term systemic delivery of recombinant molecules is expensive and time-consuming for patients, delivery of the molecule through a gene therapy approach might provide persistent expression as well as achieve high local levels of the antiangiogenic protein within the tumor microenvironment (18 , 19) .

The antiangiogenic and antitumor effects of endostatin have not yet been tested in a preclinical bladder tumor model, and furthermore, the potential utility of lentiviral vectors for efficient and stable gene transfer to bladder cancer cells has not been evaluated. Here, we have assessed the relative transduction efficiency of lentivirus vectors on a variety of bladder cancer cell lines for the purpose of achieving long-term expression of endostatin, and we report the results of an in vivo study using this lentivirus-mediated antiangiogenic gene therapy approach to treat orthotopic human bladder tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
Five human bladder cancer cell lines, including KU-7 (kindly provided by Dr. M. Murai; Keio University, Tokyo, Japan), J82, HT1376, T24, and UMUC-3 (American Type Culture Collection, Manassas, VA), were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 100 units of penicillin/streptomycin. In addition, 293T human kidney cells (American Type Culture Collection) were maintained in Eagle’s MEM in Eagle’s balanced salt solution with nonessential amino acids supplemented with 10% fetal bovine serum. These cell lines were maintained at 37°C in 5% CO2 humidified atmosphere.

Generation of Lentiviral Vector.
The BamHI/SalI fragment of MND-X-IRES-GFP (Ref. 20 ; kindly provided by D. B. Kohn; Children’s Hospital, Los Angeles, CA) was cloned into a HIV-1-based self-inactivating lentivirus vector, pRRLsinhCMVGFP (21) , replacing the existing green fluorescent protein (GFP) sequence. The resultant vector plasmid, pSin-GFP, was used as a control. The murine endostatin coding sequence was amplified by PCR using the plasmid pBacPAK8-endostatin (Ref. 8 ; kindly provided by J. Folkman; Harvard Medical School, Boston, MA) and fused with the sequence spanning the signal peptide of THBS-1 (kindly provided by D. Roberts; NIH, Bethesda, MD) by overlap-extension PCR. Subsequently, the product was cloned into pSin-GFP. The resulting vector plasmid pSin-Endo was used to generate the lentivirus Sin-Endo (Fig. 1)Citation . Lentivirus vectors were produced using a transient cotransfection system, as described previously (22) . Briefly, 1 x 107 293T cells were transfected with 7.5 µg of the envelope plasmid pMD.G encoding the G glycoprotein of vesicular stomatitis virus (VSV), 15 µg of the packaging plasmid pCMV{Delta}R8.91 (23) , and 15 µg of either pSin-GFP or pSin-Endo. The harvests of conditioned media containing recombinant lentivirus were collected 48–72 h later and concentrated by ultracentrifugation.



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  		Fig. 1. Schematic representation of the endostatin expression constructs. To produce pSin-Endo, endostatin was fused with human thrombospondin-1 signal peptide (SPhTHBS-1) and inserted into self-inactivating lentivirus vector plasmid pSin-GFP, in which most of the U3 region from Rous sarcoma virus (RSV) was deleted. Recombinant lentivirus vectors were produced by cotransfection of the packaging plasmid pCMV{Delta}R8.91, envelope plasmid pMD.G, and pSin-Endo. LTR, long terminal repeat; {Psi}, packaging signal; SD, splice donor; SA, splice acceptor; RRE, Rev response element; MCS, multiple cloning site; IRES, internal ribosome entry site; {Delta}U3, self-inactivating deletion in U3 region; poly(A), polyadenylation signal.

 
Lentiviral Transduction of Bladder Cancer Cells.
For the lentiviral transduction, KU-7 cells were plated in 6-well plates at a density of 1 x 105 cells/well and allowed to attach overnight. The medium was replaced with 1 ml of fresh complete medium, 100 µl of lentiviral supernatant, and 8 µg/ml hexadimethrine bromide (Sigma Chemical Co., St. Louis, MO) to assist the uptake of viral particles. Both pSin-GFP-transduced cells (KU-7GFP) and pSin-Endo-transduced cells (KU-7Endo) were collected 120 h after infection, and the number of GFP-positive cells by fluorescence-activated cell-sorting analysis was used as a parameter to estimate the genetically modified cell fraction, which was >80%.

To assess the relative infectivity of lentivirus vectors on the several types of human bladder cancer cells, 1 x 105 KU-7, J82, HT1376, T24, and UMUC-3 cells were infected with serial dilution of the Sin-Endo vector preparations, and GFP-positive cells were counted 120 h later by fluorescence-activated cell sorting. The respective titers were calculated according to the following formula: transduction units/ml = (cell number at the time of infection) x (percentage of GFP-positive cells) x (dilution factor).

Endostatin Expression on Transduced Cells.
To confirm the production and secretion of endostatin, parental KU-7, KU-7GFP, or KU-7Endo cells were plated in 6-well tissue culture plates at a density of 1 x 105 cells/dish and incubated for 48 h. Cell supernatants were collected and passed through a 0.45-µm filter. Twenty µg of each cell lysate were subjected to electrophoresis on a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline containing 5% nonfat milk powder for 1 h and then incubated overnight with an anti-GFP rabbit polyclonal antibody (Clontech, Palo Alto, CA), an anti-RAN goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-bFGF rabbit polyclonal antibody (Santa Cruz Biotechnology), an anti-VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology), or an antimouse endostatin rabbit polyclonal antibody (Cytimmune Sciences, College Park, MD). RAN protein content was used as a control for evaluating the equivalency of total protein loading for each sample. The membrane was then incubated for 1 h with species-specific appropriate secondary antibodies (Amersham-Phamacia, Little Chalfont, United Kingdom), and reactivity was detected by enhanced chemiluminescence system (Amersham-Phamacia). Endostatin concentrations in the supernatants were also determined by competitive enzyme immunoassay [EIA (Cytimmune)] according to the instructions of the manufacturer.

In Vitro Proliferation Assay.
The effects of endostatin on the in vitro growth of KU-7Endo cells were tested using spectrophotometric measurement. Briefly, parental KU-7, KU-7GFP, or KU-7Endo cells were seeded in each well of 96-well plates at 1 x 103 cells/well. The cell number was then assessed at 24-h periods over 5 consecutive days. At each time point, cells were harvested, washed, and then stained with 0.1% crystal violet. The absorbance value of each well was determined at 590 nm by a microplate reader (Bio-Rad Laboratories, Hercules, CA).

Effect of Endostatin Overexpression on an Orthotopic Growth of Bladder Tumor.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center. For tumor implantation, 8-week-old female nude (nu/nu) mice were anesthetized with an i.p. injection consisting of ketamine (100 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (20 mg/kg; Lloyd Laboratories, Shenandoah, IA). A 22-gauge catheter was inserted into the bladder transurethrally, and the urethra was tightly ligated with 3-0 silk suture. Subsequently, 1 x 107 KU-7 (n = 18), KU-7GFP (n = 13), or KU-7Endo (n = 18) cells in 100 µl of PBS were instilled into the bladder. Cells remained in the bladder for 3 h. On day 28, the mice were sacrificed. Immediately before sacrifice, urine was collected. After cystectomy, bladders were weighed, placed in OCT compound (Sakura, Tokyo, Japan), and immediately frozen in liquid nitrogen. Frozen sections (5 µm) were cut on a cryostat and stained with H&E. A total of 10 serial sections per bladder were obtained at 500-µm intervals, and the tumors were digitally determined by the medical image analysis program (Image Pro Plus Version 4.1; Media Cybernetics, Silver Spring, MD). The area of 10 sections was combined to determine an estimate of total tumor area. Fresh urine samples were centrifuged at 1500 rpm for 5 min at 4°C and frozen at -80°C. Concentrations of endostatin in urine were measured by EIA (Cytimmune).

Immunohistochemical Analysis.
For immunostaining, sections were fixed in acetone for 10 min (CD31) or in 4% paraformaldehyde for 1 h (GFP and endostatin). After endogenous peroxidase activity was blocked by use of 0.1% hydrogen peroxide for 15 min, sections were incubated for 30 min in a blocking solution containing 10% appropriate normal serum. Sections were incubated with antibodies specific for mouse endostatin (R&D Systems, Minneapolis, MN), CD31 (Clontech), or GFP (Molecular Probes, Eugene, OR) for 2 h. Slides were incubated with biotinylated species-specific appropriate secondary antibodies for 30 min and exposed to avidin-biotin-peroxidase complexes (Vector Laboratories, Inc., Burlingame, CA). Sections were treated with 0.06% 3,3'-diaminobenzidine (Sigma Chemical Co.), used as a final chromogen, and counterstained with hematoxylin. MVD was determined by calculating the areas of tumor capillary vessels/high-power field in sections stained with an anti-CD31 antibody. MVD was expressed as the mean percentage of vessel areas/field from three highly vascularized areas.

Statistical Analysis.
Data were presented as mean ± SE. Comparisons between groups were made using the Mann-Whitney U test or the Kruskal-Wallis test, where appropriate. Ps of <0.01 were considered to be statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Endostatin in Bladder Cancer Cells after Lentivirus-Mediated Gene Transfer in Vitro.
To test the utility of lentiviral vectors for gene transfer to bladder cancer cells, the transduction efficiency of VSV-G-pseudotyped lentiviral vectors (Fig. 1)Citation was first examined on various human bladder cancer cell lines by fluorescence-activated cell-sorting analysis, as listed in Table 1Citation . UMUC-3 was the most sensitive cell line for efficient gene transfer of lentiviral vectors, exhibiting transduction titers exceeding 107 viral particles/ml. Transduction efficacy of all other bladder cancer cell lines tested was also very efficient, on the order of 106 viral particles/ml. Because the transduction titer of lentivirus vectors on KU-7 cells was intermediate between that of J82 or HT1376 and T24 or UMUC-3, we chose this as a representative cell line for subsequent studies involving lentivirus-mediated gene transfer of endostatin in vitro and in vivo.


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  		Table 1 Comparative transduction efficiency of lentiviral vector on different bladder cancer cell lines

 
Subsequently, KU-7 cells were transduced with lentiviral vectors encoding either the GFP marker gene only or the endostatin coding sequence linked to the GFP marker via an internal ribosome entry site. Typically, at a multiplicity of infection of 1, >80% of lentivirally transduced KU-7 cells consistently exhibited GFP expression by fluorescence-activated cell-sorting analysis, and high levels of GFP protein expression were confirmed in cell lysates of virally transduced KU-7 cells by Western blot analysis (Fig. 2A)Citation . The resultant cells were designated KU-7GFP and KU-7Endo cells, respectively, and production of secreted forms of murine endostatin after lentiviral gene transfer was quantitated by EIA (Fig. 2B)Citation .



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  		Fig. 2. A, expression of green fluorescent protein (GFP), endostatin, vascular endothelial growth factor, and basic fibroblast growth factor in cell lysates of lentiviral transduced KU-7 cells. KU-7 cells transduced with lentivirus vector Sin-GFP (KU-7GFP) or lentivirus vector Sin-Endo (KU-7Endo) and nontransduced KU-7 cells were analyzed. Western blot analysis was performed using antibodies specific for GFP, endostatin, vascular endothelial growth factor, basic fibroblast growth factor, and RAN. B, endostatin concentrations in the supernatant of endostatin-transduced KU-7 cells measured with enzyme immunoassay kit for murine endostatin. Bars, SE.

 
Western blot analysis demonstrated that GFP was expressed in cell lysates of both lentiviral transduced KU-7 cell lines, and endostatin was expressed only by KU-7Endo cells (Fig. 2A)Citation . EIA revealed that the KU-7Endo cells secreted endostatin into the culture medium at a concentration of 636.9 ± 12.4 ng/ml/106 cells, whereas no endostatin was detected in the conditioned medium obtained from the parental KU-7 and KU-7GFP cells (Fig. 2B)Citation .

In Vitro Growth of Endostatin-Producing KU-7 cells.
To evaluate the effects of endostatin transduction and expression on the growth of KU-7 cells in vitro, the relative growth rates of KU-7Endo, KU-7GFP, and parental KU-7 cells were compared by spectrophotometric measurement of viable cells. As shown in Fig. 3Citation , there was no significant difference between the growth rates of KU-7Endo, KU-7GFP, and parental KU-7 cells, suggesting that neither the lentivirus transduction procedure nor the overexpression of either GFP or endostatin affected the intrinsic rate of cellular proliferation in these cells. Furthermore, because lentiviral vectors immediately achieved highly efficient levels of gene transfer into the bladder cancer cells, no selection was necessary to obtain relatively pure populations of transduced cells, and the potential for any selection bias is significantly reduced.



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  		Fig. 3. In vitro proliferation curve of transduced KU-7 cells. Growth rate of KU-7 (•), KU-7GFP ({blacksquare}), and KU-7Endo ({blacktriangleup}) was measured by quantitative crystal violet assay. Each value represents the mean ± SE derived from at least three individual experiments. In vitro growth characteristics were similar among the transduced cell lines.

 
In Vivo Growth of Endostatin-Overexpressing KU-7 Cells in an Orthotopic Bladder Tumor Model.
To investigate the effect of endostatin on the establishment of orthotopic bladder tumors, we inoculated KU-7, KU-7GFP, and KU-7Endo cells transurethrally into nude mice. At 28 days after instillation, bladders were removed and weighed. The mean bladder wet weight was significantly smaller in mice inoculated with KU-7Endo cells than in those inoculated with KU-7 or KU-7GFP cells (Table 2)Citation . Orthotopically implanted tumor masses were confirmed histologically in all bladder samples examined. The tumor incidence in mice inoculated with parental KU-7 cells and KU-7GFP cells was 94.4% and 92.3%, respectively. However, only 55.6% of mice inoculated with KU-7Endo cells had histological evidence of residual established tumors (Table 2)Citation . Analysis of total tumor area acquired from 10 sections was used to estimate the size of the implanted tumors (Fig. 4A)Citation . The mean total tumor area in mice inoculated with KU-7Endo cells was 1.86 ± 0.79, which was significantly smaller than those in mice inoculated with parental KU-7 (25.81 ± 6.64) or KU-7GFP (10.23 ± 4.23) cells (P < 0.01).


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  		Table 2 Effect of lentiviral transduced endostatin on orthotopic implanted KU-7 bladder tumor

 


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  		Fig. 4. A, inhibition of tumor growth by transduction of KU-7 cells with an endostatin-expressing lentiviral vector. KU-7 cells, control green fluorescent protein (GFP)-transduced KU-7 cells (KU-7GFP), or endostatin-transduced KU-7 cells (KU-7Endo) were orthotopically instilled into the bladder of nude mice. At 28 days after the inoculation of cells, bladders were removed and stained with H&E. Total area of tumor masses was measured in 10 sections prepared from each bladder. Each value represents the mean ± SE. The differences between the KU-7Endo group and control groups (KU-7 or KU-7GFP) were statistically significant (*, P < 0.01). B, endostatin concentrations in urine of mice implanted with endostatin-transduced KU-7 cells measured with enzyme immunoassay kit for murine endostatin. Bars, SE. Level of endostatin concentrations (means) in urine of mice with KU-7Endo established tumors was significantly higher than those in urine of mice with KU-7 or KU-7GFP established tumors. *, P < 0.01 versus KU-7 or KU-7GFP group.

 
We also examined the concentration of endostatin in the urine of KU-7, KU-7GFP, and KU-7Endo tumor-bearing animals (Fig. 4B)Citation . Mean urine endostatin levels were 107.5 ± 37.9 ng/ml from mice with established KU-7Endo tumors, which was significantly higher those from mice with established KU-7 (3.6 ± 2.2) or KU-7GFP (10.9 ± 1.1) tumors (P < 0.01).

Immunohistochemical Analysis of Orthotopic Bladder Tumors.
Immunohistochemical analysis using an anti-GFP antibody revealed that GFP expression was only present in the bladder tumors established by lentiviral transduced KU-7 cells (KU-7GFP and KU-7Endo cells; Fig. 5Citation ). The area stained with anti-GFP antibody was consistent with the actual tumor area visualized histologically. We also analyzed endostatin expression in bladder tissues from mice inoculated with KU-7, KU-7GFP, and KU-7Endo cells. Because endostatin is an endogenous inhibitor of angiogenesis, endostatin was found to be expressed in some of the blood vessels throughout the tissues of normal and tumor-bearing regions, including arteries and capillaries (data not shown). Bladders in mice inoculated with the KU-7 and KU-7GFP control cells demonstrated a similar pattern of endogenous endostatin staining in the blood vessels, whereas none of the tumor cells demonstrated any evidence of endostatin expression. In contrast, tumor cells of the bladder in mice inoculated with KU-7Endo cells were strongly positive for endostatin within the cytoplasm, suggesting that high amounts of endostatin were being produced by orthotopic KU-7Endo tumors. The extent of vascularization of KU-7, KU-7GFP, and KU-7Endo tumors was determined by immunohistochemical staining using the endothelial marker CD31. A significant reduction in the number of endothelial cells within the KU-7Endo tumors, compared with the control KU-7 or KU-7GFP tumors, was observed. The quantitative MVD count in KU-7Endo tumor was 2.3 ± 0.4%/high-power field, which was significantly smaller than those of untransduced KU-7 (6.2 ± 0.8%/high-power field) or KU-7GFP (5.2 ± 0.6%/high-power field) tumors (P < 0.01; Fig. 6Citation ).



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  		Fig. 5. Histological and immunohistochemical analysis of the growing orthotopic bladder tumors 28 days after instillation of 1 x 107 KU-7, KU-7GFP, or KU-7Endo cells. Histological staining by H&E demonstrated that bladder inoculated with KU-7Endo cells had much smaller tumor area than those inoculated with KU-7 or KU-7GFP cells (bars, 1 mm). Immunohistochemical detection of the green fluorescent protein (GFP) marker protein revealed that tumors formed by KU-7 cells showed no staining with GFP antibody, whereas extensive staining was observed in tumors formed by KU-7GFP and KU-7Endo cells (bars, 1 mm). Immunohistochemical staining of endostatin demonstrated that tumors formed by KU-7 and KU-7GFP cells had staining only at the perivascular zone, whereas strong staining was seen not only around tumor blood vessels but also on tumor cells themselves in tumors formed by KU-7Endo cells (bars, 25 µm). Immunohistochemical analysis with anti-CD31 monoclonal antibodies to highlight microvessels revealed that tumors formed by KU-7 and KU-7GFP cells showed more extensive vascularization than those formed by KU-7Endo cells (bars, 50 µm).

 


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  		Fig. 6. Tumor microvessel densities (means) in sections from tumors formed by KU-7, KU-7GFP, and KU-7Endo cells. Bars, SE. Frozen sections were immunostained with antibodies against CD31. The areas of tumor capillary vessels/high-power field were calculated, and the microvessel density was expressed as the mean percentage of vessel areas/field from three highly vascularized areas. *, P < 0.01 versus KU-7 or KU-7GFP group.

 
VEGF and bFGF Expression in Endostatin-Overproducing KU-7 Cells.
Expression of VEGF and bFGF was evaluated in KU-7, KU-7GFP, and KU-7Endo cells by Western blot analysis (Fig. 2A)Citation . KU-7 cells demonstrated identifiable levels of both VEGF and bFGF expression. The levels of VEGF expression from KU-7GFP and KU-7Endo cells were unchanged compared with that of KU-7 cells. Although there may be a difference in the level of bFGF expressed by the lentivirally transduced cells compared with the untransduced tumor cells, this difference appears to be relatively small. This difference can be attributed to the cellular response to viral transduction because the KU-7GFP and KU-7Endo cells demonstrate similar levels of bFGF expression. We also analyzed VEGF expression in bladder tissues from mice inoculated with KU-7, KU-7GFP, and KU-7Endo cells. Bladders in mice inoculated with KU-7, KU-7GFP, and KU-7Endo demonstrated similar patterns of VEGF staining, suggesting that overproduction of endostatin in KU-7 cells does not affect the production of VEGF protein (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Data from the present study revealed that lentiviral vectors are capable of efficient gene transfer to all of the bladder cancer cell lines tested. Because the coding sequence of endostatin represents the COOH-terminal fragment of collagen XVIII, this sequence alone is not sufficient for efficient secretion in mammalian cells. Thus, the signal peptide of human THBS-1 was used to enable significant amounts of endostatin to be expressed and secreted into the culture medium of our bladder cancer cell lines, as confirmed by Western blot analysis and EIA in our present study. The endostatin-producing KU-7 cells and the control KU-7 cells demonstrated equal in vitro growth rates, suggesting that the lentiviral transduction of cells, as well as expression of endostatin, did not change the in vitro growth characteristics of KU-7 cells. Our in vivo studies, however, clearly demonstrated that the establishment and growth of endostatin-producing KU-7 tumors was significantly inhibited compared with the controls in the orthotopic bladder cancer model. Immunohistochemical analysis confirmed the expression of a high level of endostatin within the KU-7Endo tumors, with an associated reduction in vascular density. Taken together, these results suggest that the observed tumor growth inhibition results from the antiangiogenic activity of endostatin. To our knowledge, this is the first report to describe the therapeutic effects of lentivirus-based vectors expressing endostatin on growth of bladder cancer in vivo.

Potentially, there may be effects on lentivirally transduced tumor cells that may result in differences in their tumorogenic behavior. Whereas no statistically significant difference was observed, it is suggested by the results of the mean total tumor area in our lentiviral controls (KU-7GFP) compared with parental KU-7 cells. It is for this reason that the lentivirally transduced KU-7GFP controls were included to correct for the effects of viral transduction. Alternatively, although we used nude animals in these experiments, some residual natural killer cell and B-cell activity may be active. GFP has been reported to have a cytotoxic effect that might work through an immunological mechanism (24 , 25) . Inclusion of the GFP-producing KU-7GFP controls allowed us to better obtain an accurate assessment of the overall inhibitory effect of endostatin overexpression.

The crucial role of angiogenesis in bladder cancer progression is supported by the investigation of endogenous angiogenic inhibitors. Decreasing levels of THBS-1, the most investigated endogenous inhibitor of angiogenesis, have been associated with increased MVD, development of p53 mutation, increased rate of recurrence, and reduced survival from bladder cancer (26) . Campbell et al. (7) demonstrated reduced levels of THBS-1 in bladder cancer despite equal levels of angiogenic inducers when compared with normal urothelium. Endostatin, another endogenous inhibitor of angiogenesis, has prompted no specific research into its potential use in bladder cancer, although endostatin has seen success in numerous animal models of other cancers. Data from this report have highlighted the effectiveness of endostatin in reducing the vascularity and growth rate of human transitional cell carcinoma in an orthotopic bladder tumor model.

It is well-known that the expression of VEGF and bFGF is up-regulated in bladder cancer (27 , 28) . Bernardini et al. (28) showed that the serum levels of VEGF were significantly associated with bladder tumor stage and grade. Interestingly, urinary VEGF was also associated with tumor recurrence rates, suggesting that quantification of urinary VEGF might provide a valuable noninvasive marker for the early detection of bladder tumor (29) . In the present studies, we found that VEGF and bFGF were both expressed from KU-7 human bladder cancer cells, however, levels of these proangiogenic factors appeared unchanged by the overexpression of endostatin. Dixelius et al. (11) have reported that only in the presence of bFGF could endostatin disturb cell-matrix and cell-cell adhesion. They have concluded that, in in vivo situations, the antiangiogenic effect of endostatin would become evident only in areas of high growth factor stimulation (11) . Kim et al. (30) have demonstrated that endostatin directly interacts with KDR/Flk-1 and blocks the binding of VEGF to endothelial cells. We speculated that, in our orthotopic bladder tumor model, a high amount of endostatin produced by lentiviral transduced KU-7 cells might block VEGF and/or bFGF signal. In addition, it can inhibit the local invasiveness of KU-7 cells, as well as the migration of endothelial cells, without affecting the production of these angiogenic proteins.

Whereas s.c. tumor models in rodents are frequently used because of the greater ease with which tumor growth kinetics can be monitored by caliper measurements, preclinical experiments of tumor growth should be examined in the organ of origin because heterotopic tumor models do not accurately reflect the interactions between the microenvironment of the organ and the tumor (31 , 32) . Furthermore, mechanisms regulating angiogenesis are considered to be tissue specific (33) , and the angiogenic phenotype is dependent on the differential expression of cytokines and growth factors within the microenvironment of the organ itself (34) . The concept of organ-specific angiogenesis is particularly important for the interpretation of preclinical studies evaluating angiogenesis and antiangiogenic therapies for human bladder cancers. Thus, orthotopic human bladder cancer models using nude mice, a more relevant system compared with other models, have been developed to mimic the microenvironment of human bladder cancer. Our current work is the first example of the tumor growth-inhibitory effects of local overexpression of endostatin using an in vivo, orthotopic bladder cancer model. This work represents novel data that support the potential use of endostatin as a therapeutic agent in the treatment of bladder cancer and provides data that intravesically administered lentiviral gene therapy might prove to be a viable strategy to deliver the endostatin gene. Additional intravesical studies will be required to fully evaluate the clinical utility of lentiviral vectors for the treatment of bladder cancer.

Lentiviral vectors are attractive tools for human cancer gene therapy (35, 36, 37) . In addition to their ability to achieve stable integration into the chromosomes and their relatively large cloning capacity, lentiviruses offer the advantage that they can transduce nondividing cells. This feature is a great advantage for gene transfer in cancer cells because nondividing cancer cells are usually concentrated in the hypoxic core of tumors (38) and represent a chemoresistant population (39) . In this study, we used VSV-G-pseudotyped lentivirus vectors, which have a potential advantage in achieving efficient gene delivery to cancer cells, even if they lack specific proteins that normally act as receptors for virus entry, because the VSV-G envelope is thought to bind to cell surface phospholipids (40) . In contrast, whereas adenoviruses are also useful vectors for gene transfer in a variety of cell types, a significant obstacle to their use for cancer gene therapy in vivo is the loss of coxsackievirus-adenovirus receptor expression in many cancer cells, particularly in more advanced malignancies. Previous studies have shown that T24, J82, and HT1376 bladder cancer cells that have little expression of coxsackievirus-adenovirus receptor, the primary receptor for adenovirus infection, were resistant to adenovirus-mediated gene delivery without any modification (41, 42, 43) . Our present results demonstrated that even these bladder cancer cell lines might be transduced by the VSV-G-pseudotyped lentivirus with high efficiency.

Whereas lentiviruses are attractive and useful vectors, biosafety issues should be considered when considering the use of HIV-based vectors for human gene therapy. The self-inactivating HIV-1 vector used in the study relies on the introduction of a 400-nucleotide deletion in the U3 region of the 3'-long terminal repeat (21) . This deletion abolishes the long terminal repeat promoter activity, resulting in reduced likelihood of generating replication-competent lentiviruses by reduction of overlap homology and enhancement of the level of transgene expression by prevention of interference between the long terminal repeat and internal promoters.


   ACKNOWLEDGMENTS
 
We thank Maria E. Dudas and Minglan Lu (Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY) for expert technical assistance.


   FOOTNOTES
 
Grant support: Supported by American Cancer Society Grant RPG-99-355-01-MGO.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Bernard H. Bochner, Department of Urology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021. Phone: (646) 422-4387; Fax: (212) 988-0759; E-mail: bochnerb{at}mskcc.org

Received 8/26/03; revised 11/ 7/03; accepted 11/21/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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