VACCINIA VIRUS MEDIATED p53 GENE THERAPY FOR BLADDER CANCER IN AN ORTHOTOPIC MURINE MODEL

0022-5347/05/1732-0604/0 THE JOURNAL OF UROLOGY® Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 173, 604 – 609, February 2005 Printed in U.S.A.

DOI: 10.1097/01.ju.0000143196.37008.2c

VACCINIA VIRUS MEDIATED p53 GENE THERAPY FOR BLADDER CANCER IN AN ORTHOTOPIC MURINE MODEL ISTVAN FODOR,* TATYANA TIMIRYASOVA, BELA DENES, JEFF YOSHIDA, HERBERT RUCKLE AND MICHAEL LILLY From the Center for Molecular Biology and Gene Therapy (IF, TT, BD, ML) and Departments of Biochemistry (IF), Urology (JY, HR) and Medicine (ML), Loma Linda University School of Medicine, Loma Linda, California

ABSTRACT

Purpose: We determined if vaccinia virus (VV) mediated delivery of human tumor suppressor p53 is safe and effective for bladder tumor therapy in an orthotopic murine model. Materials and Methods: We used recombinant VV (rVV) vectors to express transgenes in murine bladder cancer MB-49 cells in culture and those growing orthotopically in syngeneic mice. Cultured MB-49 cells were infected with rVV expressing reporter genes (rVV-L15) or p53 (rVV-TK-53) to measure virus infection and apoptosis induction. Orthotopic MB-49 tumors in C57/Bl6 mice were treated with intravesical instillation of rVV, and the tumor incidence, survival and transgene expression were determined. Results: Productive virus infection in vitro was observed in MB-49 cells, although at somewhat lower efficiency than in African Green Monkey kidney CV-1 cells (American Type Culture Collection, Manassas, Virginia). Expression of transgenes in vitro correlated with the virus dose. Cells infected with rVV underwent apoptosis with rVV-TK-53 inducing far greater cell death than rVV-L15. The rVV-L15 virus had no effect on tumor incidence but it increased mean survival compared with control. Instillation of rVV-TK-53 decreased the tumor incidence and 33% of mice survived treatment. At necropsy all nonsurviving mice had bladder tumor, whereas 2 survivors in the rVV-TK-53 treated group were tumor-free. Immunohistochemistry of tumors detected expression of the human p53 gene product in tumor cells. Conclusions: To our knowledge we report for the first time that recombinant vaccinia virus expressing human p53 can induce the death of MB-49 tumor cells in vivo, not only through the lytic effect of the virus, but also through expression of the death inducing p53 transgene. Further studies are needed to shed light on the mechanisms of rVV-TK-53 mediated tumor apoptosis and the antitumor immune response. KEY WORDS: bladder; bladder neoplasms; vaccinia; gene therapy; genes, p53

Mutations of the p53 tumor suppressor gene have been found in as many as 40% of cases of advanced transitional cell carcinoma and they are associated with poor prognosis and resistance to therapies.1, 2 Thus, the restoration of wildtype p53 to enhance cancer cell apoptosis and sensitize tumors to chemotherapy or radiation would provide a promising approach to bladder cancer therapy. Previously the biosafety of the intravesical delivery of p53 has been evaluated in mice3, 4 and patients5, 6 using adenovirus vectors. Although the applied vector was safe, transgene expression was minimal possibly due to down-regulation of adenovirus receptor CAR in many bladder cancer cells.7 Recombinant vaccinia virus (rVV) is an attractive vector for delivering foreign genes into cancer cells due to its wide host range, rapid infection and efficient expression of inserted transgenes. Intravesical instillation of rVV has been used as an immunogenic adjuvant rather than for the delivery and expression of pro-apoptotic genes.8, 9 Recent data have shown that such virus delivery may be clinically safe.10 In our previous studies we have observed that rVV medi-

ated delivery of the wild-type tumor suppressor p53 in murine glioma models has antitumor effects.11 In this report we present the results of rVV mediated delivery of the p53 transgene to bladder tumor cells in vitro and orthotopically established bladder tumors in vivo. MATERIALS AND METHODS

Cell lines. We used the MB-49 mouse bladder cell line and CV-1 African Green Monkey kidney cells maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, New York) and antibiotic-antimycotic solution (Cellgro, Mediatech, Herndon, Virginia). Viruses. The Lister vaccine strain of VV was used as a parental virus. The construction of rVV-TK-53 containing a human p53 gene and rVV-L15 expressing firefly luciferase (luc) and lacZ reporters has been described previously.12 All recombinant virus constructs were replication competent. In situ lacZ assay. MB-49 and CV-1 cells were seeded and mock infected or infected with control rVV-L15 at multiplicity of infection (MOI) ⫽ 0.5 or 5. Infected cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde, and incubated with 1 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl-bDgalactoside), 5 mM potassium ferricyanide, 5 mM potassium ferricyanide and 2 mM MgCl2 in phosphate buffered saline (PBS) for 2 hours at 37C. ␤-Galactosidase (lacZ) activity was examined by phase contrast microscopy. Luciferase assay. MB-49 cells were infected with rVV-L15.

Submitted for publication April 5, 2004. Study received institutional animal care and use committee, Loma Linda University approval. Supported by funds from the Department of Defense (National Medical Test Bed) and Department of Urology, Loma Linda University. * Correspondence: Center for Molecular Biology and Gene Therapy, Loma Linda University School of Medicine, Loma Linda, California 92354 (telephone: 909-558-8777; FAX: 909-558-0177; e-mail [email protected]). 604

VACCINIA VIRUS MEDIATED p53 GENE THERAPY FOR BLADDER CANCER

Four, 8, 12 and 24 hours after infection luciferase activity was measured using a ML300 microplate luminometer (Dynatech Lab, Cambridge, Massachusetts).12 Results are expressed in relative light units (RLU) normalized to 1 ⫻ 106 cells. Immunocytochemistry. MB-49 cells seeded on chamber slides were mock infected or infected with viruses at MOI ⫽ 5.12 The cells were then washed, fixed with 4% paraformaldehyde and blocked with 3% bovine serum albumin and 0.05% Tween 20. The cells were immunolabeled with antibody against human p53 (clone DO-7, Vector Laboratories, Burlingame, California). Slides were visualized after incubation with Cy3 conjugated goat antimouse IgG (Jackson ImmunoResearch, West Grove, Pennsylvania) and counterstaining with DAPI (4,6-diamidino-2-phenylindole) by fluorescence microscopy. Detection of cell death in situ. Apoptosis related DNA strand breaks in MB-49 cells were detected by TUNEL assay (Roche Molecular Biochemicals, Mannheim, Germany) using fluorescence microscopy.12 Animals. C57/Bl6 female mice (Jackson Laboratories, Bar Harbor, Maine) at 4 to 6 weeks old were maintained in the animal care facility for at least 2 weeks prior to experiments. Biosafety level 2 precautions were implemented during animal housing and handling, as described in Guidelines for Research involving Recombinant DNA Molecules (National Institutes for Health Guidelines, 1994). Rapid CO2 sacrifice was humanely performed in compliance with the National Institutes for Health Guide for the Care and Use of Laboratory Animals. This study was approved by the institutional animal care and use committee of Loma Linda University. Induction and treatment of orthotopic murine bladder cancers. Mice were anesthetized by intraperitoneal injection of a combination of ketamine (80 mg/kg) and xylazine (5.2 mg/kg), and then catheterized via the urethra. A wound was produced in the bladder wall by introducing a cautery wire through the catheter into the bladder and applying a single 1-second pulse at 1 W. After removing the cautery wire 3 ⫻ 104 viable MB-49 cells in 0.1 ml PBS were instilled intravesically. The take rate appeared to be 100% based on physical examination and necropsy of experimental animals. Tumors were allowed to grow for 4 days, after which the animals were divided randomly into 3 groups with 14 or 15 per group. On day 4 the mice were treated intravesically with a single dose (1 ⫻ 106 pfu in 0.1 ml PBS) of rVV-L15, rVV-TK-53 or PBS. Virus inoculation required anesthesia and urethral catheterization. On day 7, 5 mice per experimental group were sac-

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rificed. The bladders were removed and fixed in 10% buffered formalin for immunohistochemical analyses. The remaining mice (9 or 10 per group) were kept for survival studies. For pair comparisons of tumor incidence or survival we used the test for the equality of 2 proportions. Survival was not significantly different when the Z value was between ⫺1.96 and 1.96. An additional experiment used similarly treated cohorts (8 to 10 mice per group). The animals were sacrificed on day 14. Bladder tumors were then removed and weighed to estimate tumors bulk. Immunohistochemistry. Excised bladders were fixed in 10% buffered formalin and embedded in paraffin. Sections (5 ␮) were prepared and mounted on glass slides. Immunohistochemistry was performed using a MOM peroxidase kit, monoclonal anti-human p53 (clone DO-7, Vector Laboratories) and monoclonal anti␤-galactosidase (AB-1,Oncogene Research Products, Boston, Massachusetts). Finally, sections were counterstained with hematoxylin QS (Vector Laboratories). RESULTS

rVV infection of MB-49 bladder cancer cells. The susceptibility of cultured MB-49 cells to rVV infection was determined by several methods. Using control vector rVV-L15 we observed diffuse lacZ and luciferase activities in cultured cancer cells after virus infection (figs. 1 and 2). About 10% to 15% of MB-49 and 25% to 30% of CV-1 infected cells expressed the lacZ transgene at MOI ⫽ 0.5 (fig. 1, B and E), whereas at MOI ⫽ 5 the respective values were about 80% and 95% (fig. 1, C and F). Quantitative analysis of luciferase transgene expression correlated with the virus dose (fig. 2). Based on the data presented it is conceivable that at highest dose of infection (MOI ⫽ 10) all MB-49 cells were infected. Productive virus infection was confirmed using a plaque assay (fig. 3). Similar to the results shown in figures 1 and 2 transgene expression in MB-49 cells was somewhat lower than in CV-1 cells. The VV yield produced in MB-49 cells was 65% and 80% of that released from CV-1 cells at MOI ⫽ 1 and 5, respectively. Only rVV-TK-53 infected MB-49 cells expressed the p53 transgene in vitro in 40% to 50% cells (fig. 4, C), inducing cell death, as observed by TUNEL assay (fig 5). Most infected cells showed an apoptotic morphology containing condensed nuclei. Of rVV-L15 infected cells about 20% displayed apoptosis, whereas mock infected cells were completely unlabeled. Antitumor effect of VV transduced p53 in vivo. Mice bearing orthotopic MB-49 bladder tumors were infected with

FIG. 1. Expression of ␤-galactosidase by MB-49 and CV1 cells infected with rVV. MB-49 (A to C) and CV-1 (D to F) cells were mock infected (A and D) or infected with rVV-L15 at MOI ⫽ 0.5 (B and E) or 5 (C and F). At 24 hours after infection X-Gal staining was performed. Reduced from ⫻1,000.

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VACCINIA VIRUS MEDIATED p53 GENE THERAPY FOR BLADDER CANCER

FIG. 2. Luciferase expression in MB-49 bladder cancer cells infected at different MOIs by rVV-L15. At specific time points luciferase activity was measured. h, hours.

FIG. 3. rVV recovery after in vitro infection of MB-49 bladder cancer cells infected with rVV-L15. At 20 and 40 hours (h) after infection virus recovery was determined by plaque assay on CV-1 cells.

had died of cancer (fig. 6). Median survival of nonsurviving mice receiving rVV-L15 (36 days) or rVV-TK-53 (36 days) was significantly longer than that of mice treated with PBS (21 days). Statistical analyses showed that survival values for rVV-L15 vs PBS (Z ⱖ-2.361 to ⱕ-2.9277) and rVV-TK-53 vs PBS (Z ⱖ-1.9896 to ⱕ-2,5567) were significantly different. Although the median survival of the 2 virus treated groups did not differ, 33% of the mice in the rVV-TK-53 cohort were long-term survivors, while all rVV-L15 treated mice had died of cancer by day 54. All mice that died had primary bladder tumors, as demonstrated by necropsy. In the PBS and rVV-L15 cohorts a 100% tumor incidence was recorded. Necropsy also showed that 33% to 40% of mice in the control (PBS) and rVV-TK-53 cohorts also had clear evidences of metastasis (see table). The surprisingly high incidence of metastases (90%) in rVV-L15 treated mice should be validated with repeat experiment involving larger cohorts. On day 70 the surviving 3 mice in the rVV-TK-53 treated cohort were sacrificed for necropsy. One mouse had bladder tumor and metastasis, whereas the other 2 were tumor and metastasis-free. In a separate experiment treated cohorts of animals were sacrificed on day 14 after the intravesical instillation of MB-49 cells and bladder weight was measured. While bladder weight was not statistically different among the groups, animals treated with rVV-TK-53 had the smallest bladder weight, while animals treated with PBS had the largest bladder weight (fig. 7). These data may support the results of the survival experiment, as described. rVVs express their transgenes during in vivo infection. All tumor sections, whether from control or virus treated tumor, showed patches of necrosis within the tumor with modest leukocyte (mostly granulocyte) infiltration (fig. 8). Tumors from virus infected animals had 15% to 20% more leukocytes than PBS treated tumors based on quantitated counts of leukocyte nuclei on hematoxylin QS stained sections. Immunohistochemistry of bladder tumor sections showed patchy expression of LacZ protein in tumors treated with rVV-L-15 but not in normal bladder mucosa (fig. 8). Expression of the p53 transgene was also detected irregularly, mostly in areas of necrotic tumor only. Normal bladder epithelium and viable tumor cells expressed no human p53 protein. DISCUSSION

rVVs by a single intravesical instillation and followed for overall survival. By the end of the observation period (70 days) all animals in the control (PBS) and rVV-L15 groups

Bladder cancer presents unique issues with regard to virus mediated gene therapy. The so-called glycosaminoglycan layer of the bladder urothelium represents a significant bar-

FIG. 4. Expression of human p53 protein in MB-49 cells that were mock infected (A) or infected with rVV-L15 (B) or rVV-TK-53 (C) at MOI ⫽ 5. Immunocytochemistry was performed 40 hours after infection. Reduced from ⫻1,000.

FIG. 5. TUNEL assay of apoptotic MB-49 cells that were mock infected (A) or infected with rVV-L15 (B) or rVV-TK-53 (C) at MOI ⫽ 5. At 40 hours after infection apoptotic cells (green areas) were detected by TUNEL assay and fluorescence microscopy. Reduced from ⫻1,000.

VACCINIA VIRUS MEDIATED p53 GENE THERAPY FOR BLADDER CANCER

FIG. 6. Survival of tumor bearing mice implanted intravesically with MB-49 cells. On day 4 single treatment with rVV-L15, rVVTK-53 (rVV-p53) or PBS was performed. Cohorts of 9 or 10 mice each were monitored for survival until day 70.

Incidence of metastasis in tumor bearing mice No. Treatment

Overall Survived Bladder tumor Metastases: Lungs Kidneys Mesenteric lymph nodes Intestine There were no liver metastases.

PBS

L-15

p53

10 0 10 4

10 0 10 9 4 5 1 3

9 3 7 3 1 3

3 1

rier to any potential orthotopic gene therapy.13 These experiments demonstrated that vaccinia virus successfully infected cultured MB-49 cells and established bladder tumor cells in vivo, as determined by the expression of reporter and p53 gene products. Tumor bearing mice treated with rVVL15 and rVV-TK-53 showed a statistically significant increase in median survival compared with that in the mock infected cohort. Long-term survival in the rVV-TK-53 cohort was superior compared with the cohort that received rVVL15 treatment or the untreated control. In addition, bladder tumor weight was lowest in the rVV-TK-53 treatment group. These data suggest that the vaccinia virus and its expressed transgene can contribute to tumor growth inhibition. However, further experiments are required to understand the mechanisms involved in the therapeutic effect. The higher median survival seen with either VV suggests oncolytic and immunological effects that can occur within days of VV treatments. The long-term survival and lower tumor incidence of rVV-TK-53 treated animals may be partly explained by p53 mediated apoptosis and immune response. Immunohistochemical analysis of rVV-p53 treated tumors confirmed p53 expression in tumor cells. These data are in agreement with our previous finding of the greater antitumor effect of VV expressing p53 compared with control VV in a glioma model,11 at least partly due to p53 mediated apoptosis.12 Interestingly VV delivered p53 in vitro suppressed glioma cell growth and induced cell apoptosis without regard to the status of endogenous p53. However, immunological or inflammatory events caused by p53 mediated tumor cell apoptosis and the over expression of highly immunogenic p53 alone might also contribute to the stronger antitumor effect of rVV-TK-p53. Thus, strong humoral and cellular immune response against human p53 in

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FIG. 7. Bladder tumor weight. Bars represent mean ⫾ SE of 8 to 10 preparations.

mice inoculated with VV-p53 has been demonstrated previously.14 It had also been observed that cell injury and death of tumor cells could release potent adjuvants that stimulate the cytotoxic T lymphocyte response.15 Infiltrating leukocytes, mostly granulocytes, were detected in bladder tumor tissues of all treatment cohorts. Somewhat increased numbers of leukocytes observed in virus treated mice are likely to have been recruited by efficient virus mediated apoptosis of infected tumor cells, leading to the release of inflammatory stimuli and antigens. In the C6 glioma model we observed a significant increase in granulocytes in the spleen and blood of VV treated mice compared with that in healthy or tumor bearing cohorts.16 We can speculate that VV infection induces activation of the nuclear factor-␬B (NF␬B) pathway, a key regulator of inflammatory and immune response,17 apparently through Toll-like receptor signaling.18 On the other hand, NF-␬B activation is a powerful positive regulator of granulocyte survival.19 Thus, in response to VV infection the granulocyte increase may also be mediated by virus induced NF-␬B signaling. Interestingly immunohistochemical analysis showed transgene expression only in tumors, whereas bladder mucosa remained virus-free. These data differ from a report claiming that rVV could infect urothelium.8 This may have been due to the low pathogenicity of our vaccine strain of VV compared with the laboratory strain used by others. Recently we imaged live mice bearing MB-49 bladder tumors after systemic delivery of a recombinant virus expressing luciferase and green fluorescent protein. Visualization of these optical reporters confirmed preferential virus replication in tumors but not in normal tissues (data not shown). Other investigators have also reported a remarkable degree of tumor selectivity for rVV infection in other tumor models.20 A single instillation of therapeutic VV was performed in this study. This treatment increased survival but did not cure all mice. The possible reasons could be a patchy, limited extent of viral spread within the tumors, antiviral immunological responses, which can occur within 6 days of viral exposure,21 and the induction of resistance genes, known to occur in many types of mammalian cells. However, repeat instillation of the rVV-TK-53 virus is likely to increase further the efficacy of VV mediated therapy, as we reported earlier in glioma model12 and others observed with adenovirus vectors in bladder cancer models.6, 22 CONCLUSIONS

Our study describes a new approach to bladder cancer gene therapy using rVV mediated local delivery of p53 in a syngeneic, orthotopic murine tumor model. Survival of tumor bear-

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VACCINIA VIRUS MEDIATED p53 GENE THERAPY FOR BLADDER CANCER

FIG. 8. Expression of rVV encoded transgenes in MB-49 tumors treated with intravesical virus. Animals were treated with PBS (A and B), rVV-L15 (C to E) or rVV-TK-53 (F to K). Tissue sections were then examined by immunohistochemical analysis for expression of p53 (A, B and F to K) or lacZ (C to E). Patchy lacZ expression was shown in tumor cells (white arrowhead) but not normal urothelium (blue arrowhead) (C to E). Areas of viable tumor as well as necrotic areas were infiltrated with leukocytes, that is mostly granulocytes (red arrowheads), in rVV-TK-53 treated tumors (H and K). p53 expression was seen in tumor cells in necrotic areas (white arrowheads) (G, H, J and K) but not in normal urothelium (blue arrowheads) or in viable tumor (F and I). Reduced from ⫻100 (A, C, D, F, G, I and J) and ⫻1,000 (B, E, H and K).

ing mice treated with rVV-TK-53 was 33%, whereas all mice in the rVV-L15 and PBS treated cohorts died of bladder cancer. Thus, apparently rVV can induce antitumor effect in the orthotopic MB-49 tumor model, not only through viral lytic effect and immunological means, but also through expression of a death inducing transgene. However, further studies in larger treatment cohorts are required to confirm the observed effects and shed light on the mechanisms of VV mediated tumor apoptosis and immune response. Dr. Eric Lattime provided the MB-49 mouse bladder cell line. Daila S. Gridley, Loma Linda University provided advice and statistical analysis. REFERENCES

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