Vascular endothelial cell growth factor (VEGF) regulates angiogenesis and metastasis of bladder cancer (transitional cell carcinoma, TCC) through binding to VEGF receptor-2 (VEGFR-2). In this study, we evaluated whether the anti-VEGFR monoclonal antibody (Mab) DC101 in combination with paclitaxel inhibited tumorigenesis, angiogenesis, and metastasis of human TCC growing within the bladder of athymic nude mice. In vivo therapy with Mab DC101 and paclitaxel induced significant regression of bladder tumors compared with either agent alone. Median bladder weights were reduced from 601 mg in untreated controls, 422 mg in mice treated with paclitaxel alone (P < 0.005),361 mg in mice treated with DC101 alone (P <0.005), and 113 mg in mice that received combination therapy(P < 0.0005). Only one of nine mice developed spontaneous lymph node metastasis after combined treatment, compared with seven of seven untreated controls (P < 0.0005), six of eight after DC101 (P < 0.01), and five of eight mice after paclitaxel (P < 0.05). Combined treatment with both paclitaxel and DC101 inhibited tumor-induced neovascularity compared with all other groups (P < 0.005), without altering the expression of VEGF or flk1. Mab DC101 and paclitaxel combined enhanced apoptosis in the tumor and endothelial cells compared with other treatment (P < 0.005). These studies indicate that Mab DC101, which blocks VEGFR-2 function, has significant efficacy against human TCC, especially when combined with the chemotherapeutic agent paclitaxel. The antitumor effect was mediated by inhibition of angiogenesis and induction of both tumor cell and endothelial cell apoptosis.
Tumor growth, invasion, and subsequent metastasis depend upon the establishment of an adequate blood supply through the process of angiogenesis (1). This process is regulated by the balance between stimulatory and inhibitory factors released by both tumor and host cells (2, 3, 4). A number of angiogenesis factors are expressed by the highly vascular TCC3 of the bladder (3), including bFGF and acidic FGF (5, 6),IL-8 (7), midkine (8), hepatocyte growth factor (9, 10), thymidine phosphorylase (11, 12), transforming growth factor-α (13, 14), and VEGF (15, 16, 17, 18). The level of VEGF expression by human TCC directly correlates with tumor progression (15, 16, 17, 18) so that the VEGF signaling pathway is a suitable therapeutic target for TCC. VEGF regulates angiogenesis after binding to its type I (flt) or II (flk1/KDR) receptor, both of which are relatively endothelial cell-specific transmembrane tyrosine kinases. VEGF-mediated signaling pathways mediate endothelial proliferation, migration, morphogenesis,and differentiation (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).
Previous studies have found that down-regulation of VEGF expression or VEGFR signaling pathways by neutralizing antibodies to VEGF, antisense VEGF constructs, dominant-negative VEGFRs, or tyrosine kinase inhibitors of VEGFR inhibited the growth and metastasis of human malignancies (31, 32, 33, 34, 35, 36, 37, 38). Systemic therapy with Mab DC101, an inhibitor of the murine VEGFR-2, inhibited the growth of human epidermoid, renal, pancreatic, and glioblastoma xenografts growing within the subcutis of athymic nude mice (39).
In the present study, we evaluated the therapeutic effect of the Mab DC101 in combination with paclitaxel against established metastatic TCC growing orthotopically within the bladder of athymic nude mice. Because TCC overexpresses VEGF and tumor endothelial cells overexpressed VEGFR (15, 16, 17, 18), we hypothesized that blockade of this receptor in combination with systemic chemotherapy provides a novel approach to the antiangiogenic treatment of human TCC which, to this point, has not been adequately explored.
Materials and Methods
Cell Lines and Culture.
The highly metastatic human bladder carcinoma cell line 253J B-V was grown as a monolayer in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum, vitamins, sodium pyruvate, l-glutamine, nonessential amino acids, and penicillin-streptomycin (40).
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 8–12 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 NIH.
Orthotopic Implantation of Tumor Cells.
Cultured 253J-BV cells (60–70% confluent) were prepared for injection as described previously (41). Mice were anesthetized with methoxyflurane. A lower midline incision was then made, and viable tumor cells in HBSS (1 × 106/0.05 ml) were orthotopically implanted into the bladder wall on day 0. Formation of a bulla indicated a satisfactory injection. The bladder was returned to the abdominal cavity, and the abdominal wall was closed with a single layer of metal clips.
In Vivo Therapy of Human TCC Growing in the Bladders of Athymic Nude Mice.
Treatment commenced 21 days after tumor implantation. Tumors were harvested from one group of controls on day 21 when treatment began. The remaining mice were randomly separated into four groups(PBS-treated controls, DC101 only, paclitaxel only, and paclitaxel followed by DC101) and treated for 4 weeks according to the schedule shown in Fig. 1. Mice were closely monitored for any signs of progressive disease and killed if they became moribund.
The animals from each group were killed by cervical dislocation and necropsied on day 56. The bladder was removed and weighed, and the presence of metastases (lymph nodes and lung) was determined grossly and microscopically. The bladders were then either quickly frozen in liquid nitrogen for mRNA extraction, fixed in 10% buffered formalin,or placed in OCT compound (Miles Laboratories, Elkhart, IN). The lungs and lymph nodes were fixed in 10% buffered formalin or mechanically dissociated and put into tissue culture.
The remaining animals in each group (10 treated with paclitaxel and DC101 and 5 in each of the other treatment groups) were treated until they became moribund.
In Situ mRNA Hybridization Analysis.
Specific antisense oligonucleotide DNA probes were designed complementary to the mRNA transcripts based on published reports of the cDNA sequence: VEGF/vascular permeability factor (42);bFGF (41); IL-8 (43); and MMP-9 (44). The specificity of the oligonucleotide sequence was initially determined by a GenBank European Molecular Biology Library database and was also confirmed by Northern blot analysis (45). A poly d(T)20 oligonucleotide was used to verify the integrity and lack of degradation of mRNA in each sample. All DNA probes were synthesized with six biotin molecules and were reconstituted to 1 μg/μl in a stock solution containing 10 mm Tris (pH 7.6) and 1 mm EDTA.
In situ mRNA hybridization was performed as described previously, with minor modifications (46, 47). In situ hybridization was performed using the Microprobe Manual Staining System (Fisher Scientific, Pittsburgh, PA; Ref. 48). Tissue sections (4 μm) were dewaxed and rehydrated with Autodewaxer and Autoalcohol (Research Genetics), followed by enzymatic digestion with pepsin. Hybridization of the probe was performed for a total of 80 min at 45°C, incubated with alkaline phosphatase-labeled avidin for 30 min at 45°C, rinsed with alkaline phosphatase enhancer for 1 min, and then incubated with a chromogen substrate for 15 min at 45°C.
Quantification of Color Reaction.
Stained sections were examined using a Zeiss photomicroscope (Carl Zeiss, Thornwood, NY) equipped with a three-chip, charge-coupled device color camera (model DXC-969 MD; Sony Corp., Tokyo, Japan). The images were analyzed using Optimas image analysis software (version 4.10;Media Cybernetics, Silver Spring, MD). Images covering the range of staining intensities were captured electronically, a color bar(montage) was created, and a threshold value was set in the red, green,and blue mode of the color camera. All subsequent images were quantified based on this threshold. The integrated absorbance of each selected field was determined. Three different fields in each sample were quantified to derive an average value. The intensity of staining was determined by comparison with the integrated absorbance of poly d(T)20. The results were presented as the number of cells for each treatment group compared with the control, which was set to 100 (49).
For IHC 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 and rehydrated in graded alcohol. Antigen retrieval was carried out using pepsin for 12 min and then protein blocked with 5% normal horse serum and 1% normal goat serum. The samples were incubated for 18 h at 4°C with one of the following: a rat monoclonal anti-CD31 antibody (50), a 1:500 dilution of rabbit polyclonal anti-VEGF/vascular permeability factor antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a 1:500 dilution of rabbit polyclonal anti-bFGF antibody (Sigma Chemical Co., St. Louis, MO), a 1:50 dilution of a rabbit polyclonal anti-IL-8 antibody (Biosource International,Camarillo, CA), a 1:100 dilution of mouse monoclonal anti-MMP-9 antibody (Oncogene Research Products, Cambridge, MA), a 1:100 dilution of mouse monoclonal anti-PCNA antibody (Dako, Copenhagen, Denmark), or a 1:100 dilution of mouse monoclonal anti-flk1 antibody (Transduction Laboratories, Lexington, KY). The samples were then rinsed four times with PBS and incubated for 60 min at room temperature with the appropriate dilution of the secondary antibody. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine. The sections were then counterstained with Gill’s hematoxylin and mounted.
Quantification of Immunostaining Intensity.
The intensity of the immunostaining of VEGF, bFGF, IL-8, MMP-9, and flk1 was measured in three different areas of each sample by an image analyzer using the Optimas software program (Media Cybernetics). Three different areas in each sample were quantified to yield an average measurement of intensity of immunostaining. The results are presented relative to the value for the control group, which was set at 100 (49).
Quantification of Microvessel Density.
Microvessel density was determined by light microscopy after immunostaining of sections with anti-CD31 antibodies using the procedure of Weidner et al. (51). Microvessel density is expressed as the average of the five highest areas identified within a single ×200 field.
For the TUNEL assay, tissue sections (5 μm thick) of formalin-fixed,paraffin-embedded specimens were deparaffinized and treated with a 1:500 proteinase K solution (20 μg/ml) for 15 min, and endogenous peroxidase was blocked by using of 3% hydrogen peroxide for 12 min. The samples were incubated for 10 min at room temperature with terminal deoxynucleotidyl transferase buffer and then for 18 h at 4°C with terminal transferase and biotin-16-dUTP. After counterstaining with peroxidase-conjugated streptavidin, the slides were incubated with diaminobenzidine, counterstained with Gill’s hematoxylin, and then mounted.
Quantification of Cell Proliferation and Apoptosis.
Cell proliferation and apoptosis were determined by light microscopy after immunostaining of sections with anti-PCNA and TUNEL assay. The density of proliferative cells and apoptotic cells is expressed as the average of the five highest areas identified within a single ×200 field.
Immunofluorescence Double Staining of Apoptotic Endothelial Cells.
For immunohistochemical analysis, frozen tissue sections (8 μm thick)were fixed with cold acetone. The samples were washed three times with PBS and incubated for 20 min 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 h at 4°C with a 1:400 dilution of rat monoclonal anti-CD31 antibody (PharMingen). The samples were then rinsed four times with PBS and incubated for 60 min at room temperature with a 1:200 dilution of secondary goat anti-rat IgG conjugated to Texas Red (Jackson ImmunoResearch Laboratory, Inc.). The sections were then washed two times with PBS. TUNEL assay was performed using a commercial kit according to the manufacturer’s protocol (Promega Corp., Madison, WI). The tissue sections were fixed in 4% paraformaldehyde at room temperature for 15 min. The tissue sections were permeabilized by incubating with 0.5% Triton X-100 in PBS for 5 min at room temperature. The slides were rinsed two times with PBS for 5 min each time and then incubated with equilibration buffer for 10 min. The equilibration buffer was then removed, and reaction buffer containing equilibration buffer, nucleotide mix, and terminal deoxynucleotidyl transferase enzyme was then added to the tissue sections and incubated in a humid atmosphere at 37°C for 1 h in the dark. EDTA was added to the slides for 5 min to stop the reaction.
To identify all cell nuclei, the slides were incubated with a 1:2000 dilution of Hoechst (300 μg/ml) at room temperature for 10 min. The slides were examined under a microscope (Inverted System IX70; Olympus,Melville, NY), and images were captured using a digital camera. When this procedure is used, the endothelial cells of CD31-positive microvessels are indicated by localized red fluorescence. Fluorescein-12-dUTP is incorporated at the 3′-OH ends of fragmented DNA, resulting in localized green fluorescence within the nucleus of apoptotic cells. All cell nuclei are indicated by blue fluorescence. The proportion of apoptotic endothelial cells is expressed as the ratio of apoptotic endothelial cells to the total number of endothelial cells in 10 random 0.011-mm2 fields at ×400.
The statistical differences for the number of vessels, proliferative cells, and apoptotic cells and for staining intensity for VEGF, bFGF,IL-8, MMP-9, and flk1 within the bladder tumors were analyzed by the Mann-Whitney test. The incidences of tumor and metastasis were analyzed by the χ2 test. A value of P <0.05 was considered significant.
Inhibition of Growth and Metastasis of Established Human TCC.
Therapy with DC101 and paclitaxel together was significantly more effective at reducing bladder tumors than was single-agent treatment with either paclitaxel or DC101 (Table 1). Combination therapy also resulted in significantly lower spontaneous lymph node metastasis (only one mouse thus treated had metastasis) compared with treatment with either paclitaxel or DC101 alone (Table 1).
Ten mice in the paclitaxel and DC101 group and 5 animals from each of the other therapy groups underwent a prolonged course of therapy. Seven of 10 animals treated with both paclitaxel and DC101 were alive with both primary tumor and metastases at 132 days after initiation of therapy, whereas all control animals became moribund, and all were necropsied by day 53 (Fig. 2).
Inhibition of VEGF, bFGF, IL-8, MMP-9, and flk1 Expression and Microvessel Density after flk1/KDR Blockade and Paclitaxel.
The expressions of VEGF, bFGF, IL-8, and MMP-9 mRNA and protein were analyzed by in situ hybridization and IHC, respectively;flk1 was analyzed by IHC (Table 2). Compared with results for the control group, mRNA and protein expressions of VEGF, bFGF, IL-8, and MMP-9 were not significantly altered in any of the three experimental groups.
However, microvessel density was significantly less in tumors of mice treated with Mab DC101 alone or with both Mab DC101 and paclitaxel compared with controls. Paclitaxel alone had no significant effect on microvessel density (Fig. 3). The expression of flk1 was not affected by treatment.
Enhancement of Apoptosis and Inhibition of Proliferation by Treatment with DC101 and Paclitaxel.
We evaluated the effect of Mab DC101 and paclitaxel on cellular proliferation and apoptosis by PCNA and TUNEL assays, respectively(Table 3; Fig. 3). The number of PCNA-positive cancer cells counted per ×200 field was significantly lower after treatment with either paclitaxel alone or Mab DC101 alone,compared with controls (P < 0.005). The combination of Mab DC101 and paclitaxel significantly inhibited proliferation compared with the use of either agent alone (P < 0.005).
The number of apoptotic cancer cells counted per ×200 field was significantly increased from 4 ± 2 in PBS controls to 11 ±4 and 15 ± 5 after therapy with Mab DC101 and paclitaxel,respectively (P < 0.005). The combination of Mab DC101 and paclitaxel significantly enhanced apoptosis compared with each agent alone (26 ± 7; P < 0.005).
Induction of Endothelial Cell Apoptosis after Treatment with Mab DC101 and Paclitaxel.
We investigated whether the increased apoptosis seen after therapy with Mab DC101 and paclitaxel was attributable to tumor or endothelial cell apoptosis, using double staining immunofluorescence with anti-CD31 and TUNEL (Table 4; Fig. 3). Endothelial cells are detected by localized red fluorescence, whereas green fluorescence is detected within the nuclei of apoptotic cells. Double labeling of endothelial cells undergoing apoptosis results in localized yellow fluorescence. The number of apoptotic endothelial cells counted per ×400 field was calculated as the ratio of double-labeled endothelial cells to total endothelial cells. The number of apoptotic endothelial cells was significantly greater in mice treated with either paclitaxel alone or Mab DC101 alone than in control mice(P < 0.005). Combination therapy with both paclitaxel and Mab DC101 resulted in a significantly greater apoptotic index for endothelial cells compared with control mice or those treated with paclitaxel alone (P < 0.005), indicating that the higher numbers of apoptotic cells included both endothelial and tumor cells.
Although TCC of the bladder is a chemosensitive tumor, most deaths from bladder cancer are caused by metastases that resist conventional chemotherapy (52, 53, 54). Most patients with advanced TCC will have an initial response to chemotherapy, but chemoresistant disease rapidly ensues (53, 54, 55). Therefore, new chemotherapeutic strategies must be developed if we are to improve the outcome for patients with advanced bladder cancer.
Recently, it has been appreciated that biological agents such as IFN-α, epidermal growth factor, and Her-2/neu receptor antagonists (49, 56, 57) modulate host responses and enhance the efficacy of standard cytoreductive chemotherapy. The anti-EGFR Mab C225 (58, 59, 60, 61) has significant antitumor effect against TCC of the bladder when combined with paclitaxel, a chemotherapeutic agent that is active against advanced human TCC (62, 63, 64, 65). Paclitaxel inhibits cell replication by enhancing polymerization of tubulin monomers into stabilized microtubule bundles that cannot reorganize into the proper structures for mitosis (62, 63, 64, 65, 66, 67, 68). This results in cell cycle blockade in mitosis and subsequent activation of an apoptotic pathway (64, 65). When combined, Mab C225 and paclitaxel enhanced apoptosis within tumor cells and down-regulated tumor-induced neovascularization.
Human TCC is characterized by an overexpression of VEGF relative to normal urothelium (15, 16, 17, 18). The blockade of the VEGF/VEGFR signaling pathway by VEGF-neutralizing antibodies or by VEGF antisense or dominant-negative VEGF receptors has been shown repeatedly in all cases to significantly inhibit tumor growth and metastasis (31, 32, 33, 36, 37, 38, 39). Various antibodies directed to the extracellular domain of the VEGFR type II have been developed (35, 37, 38, 39). These agents block the binding of VEGF to its receptor, inhibit VEGF-induced signaling in endothelial cells, and inhibit the growth of human tumors in mice by an antiangiogenic mechanism (35, 37, 38, 39).
In the present study, we observed that treatment of human TCC growing within the bladder of athymic nude mice with Mab DC101, which targets the VEGFR-2 in combination with paclitaxel, enhanced apoptosis and inhibited tumorigenesis, angiogenesis, and metastasis. The survival of mice was also prolonged by Mab DC101 and paclitaxel.
The mechanisms responsible for the interaction between Mab DC101 and paclitaxel are unclear but likely involve an increase in apoptosis within endothelial cells coupled with a decrease in proliferation. The enhanced apoptosis represents the summation of effects on separate pathways regulating apoptosis (69, 70). Paclitaxel has a significant antitumor effect for TCC of the bladder, either as a single agent or in combination with other cytotoxic agents. Paclitaxel increases microtubule stability by preventing tubulin depolymerization,leading to tubulin bundling (71). These cytoskeleton changes result in cell cycle arrest and apoptosis within 20 h of paclitaxel exposure (72). VEGF signaling inhibits apoptotic cell death in endothelial cells by several mechanisms,including the induction of the antiapoptotic proteins Bcl-2 (73, 74) and MCL1 (75), phosphorylation of phosphatidylinositol 3-kinase (76, 77, 78), the phosphorylation of ERK1 and ERK2 (79), and inhibition of stress-activated protein kinase/c-Jun-NH2-kinase (80). The antiapoptotic effect of VEGF was reversed in capillaries by flk1/KDR fusion proteins (36, 81) and by mitogen-activated protein kinase/ERK inhibitors (79, 82).
The results of the present study are in accordance with these previous observations. We observed a significant increase in apoptotic death of both tumor and endothelial cells after treatment with Mab DC101 and paclitaxel. Apoptotic tumor cells were seen clustered around apoptotic endothelial cells, suggesting an interaction between paclitaxel and relative hypoxia within the tumor caused by the regression of the neovasculature. Alternatively, it has been shown that VEGF signaling inhibits apoptosis induced by chemotherapeutics, thereby possibly rendering these agents relatively ineffective (75). Mab DC101 may enhance the effects of paclitaxel by overcoming this VEGF-induced inhibition of apoptosis (83, 84). Simultaneously, cellular proliferation as measured by PCNA was maximally inhibited by therapy with Mab DC101 and paclitaxel. The antiproliferative effect of therapy was maximized by treatment with paclitaxel followed by Mab DC101.
In summary, our experiments demonstrate that paclitaxel enhances the antitumor effect of Mab DC101 against metastatic human TCC growing within the bladder of athymic nude mice and improved the survival of mice with metastatic human TCC growing within their bladders. The improved response to this strategy seems to result from enhancement in the induction of apoptosis by paclitaxel and Mab DC101. The results of this preclinical study justify the clinical application of this novel form of combination therapy as treatment of advanced bladder cancer.
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.
Supported by Cancer Center Core Grant CA 16672,NIH Grants CA 67952 (to R. R.) and CA 67914 (to C. P. N. D.), and a grant from Imclone.
The abbreviations used are: TCC, transitional cell carcinoma; VEGF, vascular endothelial cell growth factor; VEGFR,vascular endothelial cell growth factor receptor; bFGF, basic fibroblast growth factor; IL, interleukin; Mab, monoclonal antibody;MMP-9, matrix metalloproteinase type 9; IHC, immunohistochemical staining; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PCNA, proliferating cell nuclear antigen; ERK,extracellular signal regulated kinase.