MB49 Cells Research Articles

Novel Immunotherapy for Metastatic Bladder Cancer Using Vaccine of Human Interleukin-2 Surface-modified MB 49 Cells

To develop a novel protein-anchor technology to immobilize human interleukin-2 on tumor cells to induce antitumor immunity. Interleukin-2 surface-modified MB49 cells were prepared as a vaccine. Subcutaneous and pulmonary metastatic mouse models of MB49 bladder cancer were used to evaluate the antitumor efficiency of the vaccine. Immunohistochemistry, flow cytometric, and cytotoxic T-lymphocyte assay were performed to assess the proportion and cytotoxicity of the T lymphocytes. The IL-2 surface-modified MB49 cell vaccine inhibited tumor growth and extended the survival of the mice, and the vaccine-cured mice effectively resisted the second MB49 but not the RM-1 prostate cancer cell challenge. Furthermore, more cytotoxicity on the MB49 cells and more CD4-positive, CD8-positive T cells appeared in the vaccine-treated group. The results of our study have demonstrated that the human interleukin-2 surface-modified MB49 bladder cancer cell vaccine induced specific antitumor immunity and was efficient against metastatic bladder cancer.

Preparation and application of GM-CSF-surface-modified MB49 cell vaccine

Objective Streptavidin (SA)-tagged cytokine was anchored to MB49 cells in vitro and biotinylated mucosal surface of bladder wall in vivo. The anchoring rate was calculated and the effects of anchoring and the relation between anchoring effect and time were observed. Methods Streptavidin ( SA) tagged green fluorescent protein (SA-GFP) was anchored to MB49 cells in vitro and observed under an fluorescence microscope. Female C57BL/6 mice with superficial bladder cancer were intravesically instilled with NHS-PE04-Biotin, PBS, SA-GM-CSF as SA-GM-CSF group, and PBS, SA-GM-CSF as PBS group.All the mice were killed to obtain their snap-frozen bladders for cryosectioning, and then immunohistochemistry analysis with biotin-conjugated HRP and DAB display liquid kit. Results SA-GFP was anchored to the surface of MB49 cells under the fluorescence microscopy, and the anchoring rate was 98.78%.SA-GM-CSF was detected on the biotinylated mucosal surface of bladder wall up to 7 days after its intravesical immobilization in SA-GM-CSF group, but no SA-GM-CSF was found in PBS group. Conclusion SA-GFP can be anchored to the surface of MB49 cells, and the anchoring rate was 98. 78%. Key words: Bladder carcinoma; Vaccination; GM-CSF; MB49; Surface modication

879 BLADDER EXPRESSION OF CYTOTOXIC EFFECTOR MOLECULES IN RESPONSE TO INTRAVESICAL MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUÉRIN (BCG)

You have accessJournal of UrologyBladder Cancer: Basic Research1 Apr 2011879 BLADDER EXPRESSION OF CYTOTOXIC EFFECTOR MOLECULES IN RESPONSE TO INTRAVESICAL MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUÉRIN (BCG) Matthew Knudson, Jonathan Henning, Michael O'Donnell, and Yi Luo Matthew KnudsonMatthew Knudson Iowa City, IA More articles by this author , Jonathan HenningJonathan Henning Iowa City, IA More articles by this author , Michael O'DonnellMichael O'Donnell Iowa City, IA More articles by this author , and Yi LuoYi Luo Iowa City, IA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.703AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder expression of cytokines and chemokines in response to intravesical (i.b.) BCG has been well documented. However, studies on BCG induction of bladder expression of cytotoxic effector molecules are underdeveloped. We investigated a panel of cytotoxic effector molecules, including IFN-γ, TNF-α, perforin, granzyme B, Fas ligand (FasL), and tumor necrosis factor related apoptosis-inducing ligand (TRAIL), in the BCG-treated bladders to determine whether expression of these molecules was correlated with the effect of BCG in treating bladder cancer. METHODS C57BL/6 female mice were treated i.b. with Pasteur strain BCG (0.1 OD/dose) twice weekly for a total of 7 treatments. Mice treated with PBS served as a control. Mice were sacrificed for bladder RNA extraction after treatments 2, 4, 6 and 7. RT-PCR was used to analyze mRNA levels of IFN-γ TNF-α, perforin, granzyme B, FasL and TRAIL. IP-10 (a Th1 chemokine) and TGF-β (an inhibitory cytokine) were included. GAPDH was used as an internal control. ImageJ software was used to quantify PCR products on agarose gels. In a parallel experiment C57BL/6 female mice were implanted i.b. with 5 X 105 syngeneic bladder cancer MB49 cells that expressed luciferase (MB49-Luc) at day 0 and treated i.b. with BCG (0.1 OD/dose) twice weekly starting at day 1 for a total of 6 treatments. Control mice were treated with PBS. Bioluminescence was measured once weekly with Xenogen IVIS. At day 23 mice were euthanized and bladder weights measured. Statistics was performed by one-way analysis of variance (ANOVA) of natural log transformed bladder weights followed by a post hoc Tukey's test. RESULTS I.b. BCG treatment led to an increase in bladder mRNAs of IFN-γ,TNF-α, IP-10 and perforin and a decrease in bladder mRNAs of TGF-β. There were no changes in bladder mRNAs of granzyme B, FasL and TRAIL. In MB49-Luc orthotopic tumor model, i.b. BCG treatment reduced bladder weight by 53% (p = 0.0503 vs. PBS-treated group). In addition, i.b. BCG treatment also led to a delay in tumor growth by bioluminescence compared to PBS-treated group. CONCLUSIONS Intravesical administration of BCG induced bladder expression of cytotoxic effector molecules IFN-γ, TNF-α and perforin, which was correlated with effective BCG treatment of bladder cancer. Increased expression of IP-10 and decreased expression of TGF-β, together with increased IFN-γ and TNF-α, support the role of BCG in the induction of Th1 immune responses in the bladders. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e352 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew Knudson Iowa City, IA More articles by this author Jonathan Henning Iowa City, IA More articles by this author Michael O'Donnell Iowa City, IA More articles by this author Yi Luo Iowa City, IA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A novel immunotherapy for superficial bladder cancer by the immobilization of streptavidin-tagged bioactive IL-2 on the biotinylated mucosal surface of the bladder wall

Intravesical administration of Bacillus Calmette-Guerin (BCG) after transurethral resection is by far the most effective local therapy for superficial bladder cancer, the fifth most common cancer in the world. However, approximately one-third of patients fail to respond and most patients eventually relapse. In addition, there are pronounced side effects of BCG therapy, such as BCG sepsis and a high frequency of BCG-induced cystitis. This study established a novel immunotherapy through immobilization of streptavidin-tagged human IL-2 (SA-hIL-2) on the biotinylated mucosal surface of bladder wall. A mouse orthotopic model of MB49 bladder cancer was established by perfusing MB49 cells into mouse bladders. The SA-hIL-2 fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall. Treatment began on day 1 after MB49 implantation, once every 3 days for 6 times. Immunohistochemical assay was performed to assess the persistence of SA-hIL-2 immobilized on the biotinylated mucosal surface of the bladder wall. The mice were monitored for tumor growth and survival. On day 60 after MB49 implantation, the SA-hIL-2-cured mice, which were found to have no hematuria or palpable tumors, were challenged with wild-type MB49 cells implanted into the pretreated bladder and monitored for survival. SA-hIL-2 could be immobilized efficiently and durably on the bladder mucosal surface as long as 7 days. On day 60 after MB49 implantation, 9 out of 20 SA-hIL-2-treated mice survived, but all mice in PBS control group died. More importantly, 5 out of 9 tumor-free mice in the SA-hIL-2 group were protected against a second intravesical wild-type MB49 tumor challenge. SA-hIL-2 fusion protein could significantly inhibit tumor growth and extend the survival time in the orthotopic model of MB49 bladder cancer.

The efficacy of varying concentrations of intravesical apaziquone on murine bladder cancer in an animal model.

e15101 Background: This preclinical study compared the efficacy of varying concentrations of apaziquone, an intravesical chemotherapeutic agent currently in clinical trials, and placebo in an animal model of human bladder cancer. The standard dose in all ongoing clinical studies is 4 mg/ 40 mL (0.1mg/mL). Methods: A total of 1x105 MB49 cells were instilled into the bladder of C57BL/6 mice after electrocautery to establish the tumor model. Mice bearing orthotopic tumors received 0.1 mL intravesical instillation of varying concentrations of apaziquone (0.05, n = 10; 0.1, n = 10; 0.4, n = 8, and 0.8 mg/mL, n= 8) or placebo (0.85% saline, n = 8) one week after tumor implantation. Instillations were continued once a week for 4 weeks. Tumor incidence, overall and median survival is reported. Results: At the end of the observation period (80 days), all animals in the control (0.85% saline) and 0.05 mg/mL groups had died. Median survival of mice receiving apaziquone in concentration 0.1 mg/mL (42.5 days); 0.4 mg/mL (40.5 days) and 0.8 mg/mL (49 days) was longer than that of mice treated with 0.05 mg/mL (30 days) and 0.85% saline (27 days). Statistical analyses of survival data was control vs 0.1 mg/mL (P < 0.05); control vs 0.4 mg/mL (P < 0.01), control vs 0.8 mg/mL (P < 0.01); for 0.05 mg/mL vs 0.1 mg/mL (P < 0.05); 0.05 mg/mL vs 0.4 mg/mL (P < 0.01) and 0.05 mg/mL vs 0.8 mg/mL (P < 0.01) respectively. The median survival of the 0.1, 0.4 and 0.8 mg/mL treated groups did not differ, but 33.3% of the 0.1 mg/mL cohort, and 14.3% in the 0.4 mg/mL cohort were long-term survivors (>80 days). All other animals (n = 37) had died of cancer by the end of the observation period. Three mice did not survive anesthesia. At necropsy all mice had advanced local disease with deep invasion of the muscularis propria, and usually with multiple lung metastases. Conclusions: Apaziquone at the 0.1 mg/mL concentration demonstrates excellent antitumor activity compared to placebo or 0.05 mg/mL solutions. In this model there was no significant difference with higher concentrations (0.4 and 0.8 mg/mL) supporting continued development with the current clinical formulation (0.1mg/mL). Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Spectrum Pharmaceuticals Spectrum Pharmaceuticals Spectrum Pharmaceuticals Spectrum Pharmaceuticals

Intravesical Toll‐like receptor 7 agonist R‐837: Optimization of its formulation in an orthotopic mouse model of bladder cancer

To study the immune response caused by the intravesical administration of the immunomodulator R-837 in various formulations and to estimate its therapeutic potential for bladder cancer. Female C57BL/6 mice were intravesically treated with different formulations of R-837, a Toll-like receptor 7 agonist used for treating genital warts and skin malignancy. The tested formulation mixtures contained different ratios of lactic acid, a thermosensitive poloxamer polymer (Lutrol F127) and 2-(hydroxypropyl)-beta-cyclodextrin (HPbetaCD). Induction of tumor necrosis factor alpha (TNFalpha) and keratinocyte-derived chemokine (KC) was analyzed by Luminex microbeads assay. The therapeutic potential of intravesical administration of R-837 was assessed in an orthotopic, syngeneic mouse model of bladder cancer using MB49 cells. Intravesical administration of R-837 in lactic acid alone induced systemic and bladder TNFalpha and KC in a dose-dependent manner. Formulations including poloxamer decreased systemic absorption of R-837 and significantly reduced systemic and local induction of KC. Addition of HPbetaCD in the poloxamer formulation particularly reversed levels of systemic and local levels of TNFalpha and KC. Histological examination showed that poloxamer-HPbetaCD formulation allowed infiltration of mononuclear cells into urothelium and lamina propria. In studies using orthotopic mouse bladder cancer, the tumor loads in R-837-treated mice were significantly lower than those in vehicle-treated or non-treated mice. The optimized poloxamer-HPbetaCD formulation of R-837 shows therapeutic potential for bladder cancer while avoiding adverse side-effects.

Abstract 5614: Interleukin-10 inhibits the induction of macrophage cytotoxicity against bladder cancer cells by Mycobacterium bovis bacillus Calmette-Guérin (BCG)

Abstract Intravesical BCG therapy has been used for the treatment of superficial bladder cancer for three decades. However, the mechanisms underlying BCG therapy currently remain elusive. We have focused on BCG induction of macrophage cytotoxicity against bladder cancer cells. Previously, we observed that macrophages are cytotoxic to bladder cancer cells upon stimulation with BCG in vitro. However, we found that macrophages from C57BL/6 mice are less potent than those from C3H/HeN mice for the killing of both bladder cancer MBT-2 cells and MB49 cells. This study was to determine whether macrophage-derived interleukin (IL)-10 is responsible for this discrepancy between these two commonly used murine bladder cancer models. Thioglycollate-elicited peritoneal exudate cells were harvested from mice 3 days after intraperitoneal injection of 2 ml 3% thioglycollate. Cells were incubated for 3 hours and non-adherent cells were removed after incubation. The remaining cells were 96% F4/80-positive cells as determined by flow cytometry. The prepared macrophages were then used for the analysis of BCG induction of cytotoxicity, cytokines, and nitric oxide (NO) in vitro. Studies demonstrated that BCG-stimulated C57BL/6 macrophages produced a substantially high level of IL-10 compared to BCG-stimulated C3H/HeN macrophages. This high IL-10 production correlated with reduced production of tumor necrosis factor (TNF)-α, IL-6 and NO in BCG-stimulated C57BL/6 macrophages. Neutralizing endogenous IL-10 during BCG stimulation increased C57BL/6 macrophage cytotoxicity against MB49 cells by 3.2-fold, along with increased production of TNF-α by 6.4-fold and NO by 3.6-fold, respectively. Macrophages from genetically modified C57BL/6 mice lacking IL-10 (IL-10−/−) also exhibited increased killing of MB49 cells and production of TNF-α and NO upon BCG stimulation. In addition, supplementation of exogenous recombinant IL-10 reduced BCG-induced C3H/HeN macrophage cytotoxicity against both MBT-2 cells and MB49 cells in a dose-dependent manner. Taken together, our results reveal the inhibitory role of IL-10 in BCG-induced macrophage cytotoxicity, suggesting that blockage of IL-10 may potentially enhance the effect of BCG in the treatment of bladder cancer patients, particularly for BCG non-responders who are often associated with high IL-10 production in response to BCG treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5614.

Effect of recombinant hIFN-alpha-2b-BCG on mouse bladder tumor MB49 cells in vitro

To investigate the antitumor effect of recombinant IFN-alpha-2b-BCG on mouse bladder cancer MB49 cells in vitro, and to explore its antitumor mechanisms. MB49 cells were co-cultured with recombinant BCG or wild BCG, and than were examined by light and transmission electron microscopy. The cell growth was assessed by MTT assay, and apoptosis rate and MHC-I of the MB49 cells was detected by flow cytometry using AO and Hoechst33258 fluorescence immunostaining. The hIFN-alpha-2b-BCG-treated tumor cells showed slow growth, detachment of some cells, and various degree of degeneration. Light microscopy revealed organelle disorganization, chromatin aggregation, nuclear pyknosis, and cytolysis in some cells. Cellular membrane bulged and some bubbles were seen under fluorescence microscope using AO staining. Hoechst33258 assay also depicted frequent apoptosis in the tumor cells. The MTT assay showed that rBCG more actively than the wild BCG inhibited the proliferation of MB49 cells. The apoptosis rate of the recombinant BCG group was 19.7% and 46.6% at the time point of 24 h and 48 h, respectively, significantly higher than 10.8% and 20.9%, respectively, in the wild BCG group. The results of flow cytometry indicated that both types of BCG enhanced the expression of MHC-I in the MB49 cells, but more effective in the recombinant BCG group. The recombinant hIFN-alpha-2b-BCG has more strong immuno-modulatory properties, anti-tumor effect on MB49 cells and induces apparent cytotoxicity in the bladder cancer cells in vitro.

HAMLET Treatment Delays Bladder Cancer Development

HAMLET is a protein-lipid complex that kills different types of cancer cells. Recently we observed a rapid reduction in human bladder cancer size after intravesical HAMLET treatment. In this study we evaluated the therapeutic effect of HAMLET in the mouse MB49 bladder carcinoma model. Bladder tumors were established by intravesical injection of MB49 cells into poly L-lysine treated bladders of C57BL/6 mice. Treatment groups received repeat intravesical HAMLET instillations and controls received alpha-lactalbumin or phosphate buffer. Effects of HAMLET on tumor size and putative apoptotic effects were analyzed in bladder tissue sections. Whole body imaging was used to study HAMLET distribution in tumor bearing mice compared to healthy bladder tissue. HAMLET caused a dose dependent decrease in MB49 cell viability in vitro. Five intravesical HAMLET instillations significantly decreased tumor size and delayed development in vivo compared to controls. TUNEL staining revealed selective apoptotic effects in tumor areas but not in adjacent healthy bladder tissue. On in vivo imaging Alexa-HAMLET was retained for more than 24 hours in the bladder of tumor bearing mice but not in tumor-free bladders or in tumor bearing mice that received Alexa-alpha-lactalbumin. Results show that HAMLET is active as a tumoricidal agent and suggest that topical HAMLET administration may delay bladder cancer development.

Interleukin-10 inhibitsMycobacterium bovisbacillus Calmette–Guérin (BCG)-induced macrophage cytotoxicity against bladder cancer cells

The mechanisms underlying bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer currently remain elusive. Previously, we demonstrated that macrophages were cytotoxic to bladder cancer cells upon BCG stimulation in vitro. However, macrophages from C57BL/6 mice were less potent than those from C3H/HeN mice for the killing of bladder cancer cells. This study was to determine whether interleukin (IL)-10 produced by macrophages in response to BCG is a causative factor for the reduced cytotoxicity in BCG-stimulated C57BL/6 macrophages. Thioglycollate-elicited peritoneal macrophages were prepared and analysed for the BCG induction of cytotoxicity, cytokines and nitric oxide (NO) in vitro. Compared to BCG-stimulated C3H/HeN macrophages, BCG-stimulated C57BL/6 macrophages exhibited reduced killing of bladder cancer MBT-2 cells and MB49 cells. Studies demonstrated further that BCG-stimulated C57BL/6 macrophages produced a high level of IL-10, which correlated with reduced production of tumour necrosis factor (TNF)-alpha, IL-6 and NO. Neutralizing endogenous IL-10 during BCG stimulation increased C57BL/6 macrophage cytotoxicity against MB49 cells by 3.2-fold, along with increased production of TNF-alpha by 6.4-fold and NO by 3.6-fold, respectively. Macrophages from C57BL/6 IL-10(-/-) mice also exhibited increased killing of MB49 cells and production of TNF-alpha and NO upon BCG stimulation. In addition, supplementation of exogenous recombinant IL-10 reduced BCG-induced C3H/HeN macrophage cytotoxicity against both MBT-2 cells and MB49 cells in a dose-dependent manner. These results reveal the inhibitory role of IL-10 in BCG-induced macrophage cytotoxicity, suggesting that blockage of IL-10 may potentially enhance the effect of BCG in the treatment of bladder cancer patients.

Singlet oxygen-induced apoptosis of cancer cells using upconversion fluorescent nanoparticles as a carrier of photosensitizer

The photodynamic effect of upconversion nanoparticles loaded with a photosensitizer was studied on murine bladder cancer cells (MB49). Mesoporous silica was coated onto sodium yttrium fluoride upconversion nanocrystals to form a core-shell structure and then loaded with the photosensitizer zinc (II)-phthalocyanine into the porous silica. The nanoparticles displayed a uniform spherical shape with an average diameter of about 50 nm and showed good dispersibility in water. Intracellular uptake study in MB49 cells revealed a time- and concentration-dependent accumulation of these nanoparticles. Upon irradiation with 980-nm near-infrared light, their efficiency in activating the loaded zinc (II)-phthalocyanine to generate singlet oxygen molecules was confirmed in live cells. The cytotoxic effect of the released singlet oxygen from the nanoplatform was proven by cell viability assay, confocal microscopy, DNA agarose gel electrophoresis, cytochrome c–releasing assay, and prostate-specific antigen–enzyme-linked immunosorbent assay, all of which showed a strong photodynamic effect of the nanoparticles on MB49 cells. This suggests the efficacy of sodium yttrium fluoride upconversion nanoparticles as a carrier for photosensitizers and their use in photodynamic therapy of cancer and some other diseases. From the Clinical Editor In this study, the photodynamic effect of upconversion nanoparticles loaded with a photosensitizer was investigated on murine bladder cancer cells, with strongly positive results, which may pave its way to future clinical use in malignant tumors and potentially other diseases.

MB49 Murine Urothelial Carcinoma: Molecular and Phenotypic Comparison to Human Cell Lines as a Model of the Direct Tumor Response to Bacillus Calmette-Guerin

The mouse urothelial carcinoma cell line MB49 is widely used as an in vitro and in vivo model of urothelial carcinoma. Little comparative data exist on the molecular and phenotypic responses of this cell line relative to human cell lines. We compared the effect of bacillus Calmette-Guerin on the MB49 cell line relative to responses previously observed in the human urothelial carcinoma lines T24 (ATCC) and 253J. Molecular end points in MB49 cells after bacillus Calmette-Guerin exposure were signaling pathway activation (NF-kappaB, AP1 and C/EBP), gene expression (IL-6 and p21), HMGB1 release/responsiveness and gene expression profiling at 6 hours. Phenotypic response end points were direct cytotoxicity using dye exclusion, viability on MTT assay, apoptotic sensitivity and cell cycle compartmentalization. NF-kappaB, AP1, C/EBP, IL-6 and p21 reporter constructs were activated in MB49 cells in response to bacillus Calmette-Guerin. Gene expression profiles showed an inflammatory/immune clustering response. Bacillus Calmette-Guerin decreased cell viability and induced G1 cell cycle arrest. Treatment of MB49 cells with bacillus Calmette-Guerin induced caspase independent cell death while simultaneously decreasing sensitivity to pro-apoptotic agents. Cell death was associated with release of the necrotic cell death marker HMGB1. MB49 cells expressed HMGB1 receptors and activated intracellular NF-kappaB signaling pathways in response to bacillus Calmette-Guerin. MB49 cells show molecular and phenotypic responses to bacillus Calmette-Guerin that replicate those observed in human urothelial carcinoma lines. MB49 cells appear to be an excellent model in which to study bacillus Calmette-Guerin as an antitumor agent for urothelial carcinoma.

Novel Invasive Orthotopic Bladder Cancer Model With High Cathepsin B Activity Resembling Human Bladder Cancer

We developed and characterized an orthotopic invasive bladder tumor model. The MB49-I invasive bladder tumor cell line was obtained after 13 consecutive in vivo passages of primary tumor obtained by subcutaneous inoculation of MB49 bladder tumor cells in C57Bl/6J male mice. MB49-I tumor local invasiveness, tumor weight and spontaneous metastatic capacity were higher than in MB49 tumors. In MB49-I bladder tumors increased vimentin was observed, suggesting epithelial mesenchymal transition. In vitro the MB49-I cell line showed higher invasive properties associated with an increase in cathepsin B, metalloproteinase 9 and urokinase-type plasminogen activator proteolytic activities. Orthotopic bladder tumors induced by electrocautery of the bladder wall and subsequent instillation of MB49 and MB49-I bladder cancer cells generated superficial and invasive bladder tumors, respectively. The new murine bladder model described resembles human bladder disease, making it a useful tool for studying the molecular mechanisms of tumor progression and metastasis, and assaying antimetastatic and anti-invasive agents.

Research Highlights

Research Highlights

The therapeutic potential of recombinant BCG expressing the antigen S1PT in the intravesical treatment of bladder cancer

Purpose Bacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy. Materials and methods A tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-γ, TNF-α), and Th2 (IL-5, IL-6, IL-10, TGF-β) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival. Results Both BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-α in the BCG treated group, as well as increases in TNF-α and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups. Conclusions rBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG.

Modulation of protective T cell immunity by complement inhibitor expression on tumor cells.

Complement-inhibitory proteins expressed on cancer cells can provide protection from antitumor antibodies and may potentially modulate the induction of an immune response to tumor-associated antigens. In the current study, we investigated the consequences of complement inhibitor down-regulation on the effector and inductive phases of an immune response. Stable small interfering RNA-mediated down-regulation of the complement inhibitor Crry on MB49 murine bladder cancer cells increased their susceptibility to monoclonal antibody and complement in vitro. In a syngeneic model of metastatic cancer, the down-regulation of Crry on i.v.-injected MB49 cells was associated with a significant decrease in tumor burden and an increase in the survival of challenged mice. However, monoclonal antibody therapy had no additional benefit. There was an antitumor IgG response, but the response was not effected by Crry down-regulation on inoculated tumor cells. Down-regulation of Crry on MB49 cells resulted in an enhanced antitumor T-cell response in challenged mice (measured by lymphocyte IFN-gamma secretion), and CD8+ T cell depletion of mice prior to injection of MB49 cells completely abrogated the effect of Crry down-regulation on tumor burden and survival. Deficiency of C3 also abrogated the effect of Crry down-regulation on the survival of MB49-challenged mice, indicating a complement-dependent mechanism. These data indicate that complement inhibitors expressed on a tumor cell can suppress a T cell response and that enhancing complement activation on a tumor cell surface can promote protective T cell immunity.

Expression-targeted gene therapy for the treatment of transitional cell carcinoma

Targeted gene delivery for induced apoptosis of transitional cell carcinomas was carried out in vivo in mice via utilization of the murine cyclooxygenase type 2 (Cox-2) promoter (Tis10). MB49 cells, which constitutively overexpress Cox-2 like numerous other carcinomas, selectively expressed delivered genes that utilized this transcriptional control element. The products of the delivered genes were artificially inducible forms of caspases 3 and 9, which remained inactive until a chemical inducer of dimerization was later injected intraperitoneally. The genes were delivered intravesically as plasmids complexed with poly(ethylenimine). Significant improvements, in the form of reduced bladder mass, reduced tumor volume, anti-angiogenesis and inhibition of tumor growth were seen versus untreated or unactivated controls. In some instances, tumors were seen to go into complete remission. There were no apparent bystander effects associated with the treatments. This targeted gene therapy regimen could have wide applicability to numerous cancers due to constitutive overexpression of Cox-2.

Histopathological characterization of a syngeneic orthotopic murine bladder cancer model

We developed and characterized by histopathology and immunohistochemistry a syngeneic murine bladder tumor model derived from the MB49 tumor cell line. Bladder tumor implantation was achieved by intravesical instillation of 5 x 105 MB49 tumor cells in C57BL/6 mice. A chemical lesion of the bladder was performed in order to promote intravesical tumor implantation. The bladder wall lesion was accomplished by transurethral instillation of silver nitrate (AgNO3). After 15 days, the animals were sacrificed, examined macroscopically for intravesical tumor and bladder weight. Histology and immunohistochemistry were performed using cytokeratin 7 (CK7), carcinoembrionic antigen (Dako-CEA), p53 and c-erbB2 oncoprotein (Her2/neu). Twenty-nine out of 30 animals (96.7%) developed intravesical tumors in a 15-day period. Macroscopically, the mean bladder weight was 0.196g (0.069-0.538g), 10 to 15 times the normal bladder weight. The immunohistochemical analysis showed significant membrane expression of CEA and CK7: a similar finding for human urothelial cancer. We also characterized absence of expression of p53 and anti-Her2/neu in the murine model. High tumor take rates were achieved by using the chemical induction of the bladder tumor. Although electric cauterization is widely described in the literature for syngeneic orthotopic animal models, the technique described in this study represents an alternative for intravesical bladder tumor implantation. Moreover, the histopathology and immunohistochemical analysis of the murine bladder tumor model derived from the MB49 cell line showed a resemblance to human infiltrating urothelial carcinoma, allowing clinical inference from experimental immunotherapy testing.

Cathepsin B is involved in the apoptosis intrinsic pathway induced by Bacillus Calmette-Guérin in transitional cancer cell lines

Bacillus Calmette-Guérin (BCG) is the most effective treatment for superficial and in situ transitional bladder cancer. Although the complete mechanisms for its effect are not fully understood yet, both immunological and direct effects on tumor cells have been proposed. It has been proposed that apoptotic tumor cells could be better inducers of immunity than necrotic ones. Thus, apoptosis of bladder cancer cells could contribute to a global response to BCG. Lysosomal hydrolase cathepsin B (CB) is involved in the apoptotic process and has a key role in breast cancer cell programmed death through the activation of a pro-apoptotic protein BID. Truncated BID participates in the mitochondrial apoptotic pathway that involves the activation of pro-caspase 9. The possibility that CB can be involved in apoptosis of TCC line has not been explored yet. Therefore, we analyzed the participation of CB in BCG-induced apoptosis of human and murine TCC lines. Apoptosis was evaluated by a morphologic assay and CB activity by a substrate-specific colorimetric method. Expression of CB, BID and pro-caspase 9 was determined by Western blotting. BCG induced apoptosis of murine (MBT2, MB49) and human (T24) TCC lines. An increase in both CB activity and protein was also observed. The apoptosis of T24 and MB49 cell lines was mediated by activation of pro-caspase 9 and BID, both proteins are involved in mitochondrial apoptosis. Apoptosis and activation of pro-caspase 9 and BID were inhibited by CA-074Me (CA), a cell permeable CB inhibitor. Thus, CB is involved in BCG-induced apoptosis of TCC lines, using at least in part the mitochondrial pathway.

New potential therapy for orthotopic bladder carcinoma by combining HVJ envelope with doxorubicin

To establish a new therapeutic method to treat bladder carcinoma, we investigated the therapeutic potential of doxorubicin hydrochloride (DXR) combined with hemagglutinating virus of Japan-envelope vector (HVJ-E) in an orthotropic mouse bladder cancer model. DXR and/or HVJ-E were instilled into the bladder after implantation of MB49 cells. Antitumor effects of combination therapy were evaluated by histological analysis of the bladder on day 14 after tumor implantation. The survival rate of MB49-disseminated mice was examined for 60 days after single or double administration of DXR alone or DXR/HVJ-E. The surviving mice were re-challenged with intravesical injection of MB49 cells, and the bladder was observed after 3 weeks. Combined intravesical instillation of HVJ-E and DXR resulted in a significantly higher rate of tumor-free mice (11/21) compared with mice treated using DXR alone (3/19, P<0.05). Median survival was >60 days for intravesical instillation of HVJ-E and DXR, compared with the 29 days for DXR instillation alone (P<0.05). After combination therapy, surviving mice formed no tumors in the bladder following intravesical re-instillation of MB49. HVJ-E increased antitumor effects in combination with chemotherapeutic agent (DXR). Antitumor immunity appeared to be enhanced using HVJ-E.