Murine Bladder Research Articles

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Tumor and Microenvironment Modification during Progression of Murine Orthotopic Bladder Cancer

The aim of this study was to monitor changes in the expression of immune-related genes in the bladder after tumor implantation. Mice were orthotopically implanted with MB49-PSA cells (C57BL/6 mice) on day 1 and terminated on days 7, 14, 21, and 28. Another mouse model (MBT-2/C3H mice) was examined at day 7. Gene expression analysis was performed using a TaqMan Low Density Mouse Immune Panel (Applied Biosystems, USA) on RNA extracted from the bladders. Selected genes were reconfirmed by real-time PCR analysis and RT-PCR on the mRNA from other animals. Immune suppressive (IL13, IL1β, PTGS2, NOS2, IL10, CTLA4, and CCL22) and immune stimulatory genes (CSF2, GZMB, IFNγ, CXCL10, TNFα, CD80, IL12a, and IL6) and AGTR2 were increased by day 7. By day 28, IL10, CCL2, CCL5, CXCL11, CTLA4, GZMB, IFNγ, CSF2, and IL6 were significantly increased. Therapeutic strategies involving TH1 induction and TH2 dampening may improve responses to immunotherapy.

Morphological plasticity promotes resistance to phagocyte killing of uropathogenic Escherichia coli

Uropathogenic Escherichia coli proceed through a complex intracellular developmental pathway that includes multiple morphological changes. During intracellular growth within Toll-like receptor 4-activated superficial bladder epithelial cells, a subpopulation of uropathogenic E. coli initiates SulA-mediated filamentation. In this study, we directly investigated the role of bacterial morphology in the survival of uropathogenic E. coli from killing by phagocytes. We initially determined that both polymorphonuclear neutrophils and macrophages are recruited to murine bladder epithelium at times coincident with extracellular bacillary and filamentous uropathogenic E. coli. We further determined that bacillary uropathogenic E. coli were preferentially destroyed when mixed uropathogenic E. coli populations were challenged with cultured murine macrophages in vitro. Consistent with studies using elliptical-shaped polymers, the initial point of contact between the phagocyte and filamentous uropathogenic E. coli influenced the efficacy of internalization. These findings demonstrate that filamentous morphology provides a selective advantage for uropathogenic E. coli evasion of killing by phagocytes and defines a mechanism for the essential role for SulA during bacterial cystitis. Thus, morphological plasticity can be viewed as a distinct class of mechanism used by bacterial pathogens to subvert host immunity.

The Physiological Significance of p27 KIP1 Expression in Detrusor Function

Bladder outlet obstruction results in smooth muscle cell hyperplasia, decreased bladder wall compliance, and lower and upper urinary tract pathology. The cyclin-dependent kinase inhibitor p27(KIP1) regulates bladder smooth muscle cell proliferation in response to bladder outlet obstruction but little is known about its physiological role in the bladder. We investigated the role of p27(KIP1) in the structure and function of the detrusor layer of the bladder wall. We used immunoblotting and reverse transcriptase-polymerase chain reaction to examine cell cycle regulation in response to increased mechanical tension in an in vitro model of tension induced smooth muscle cell proliferation and an in vivo model of bladder outlet obstruction. We compared unobstructed bladders of p27(+/+) and p27(-/-) mice (Jackson Laboratory, Bar Harbor, Maine) structurally by histological staining and functionally by in vivo cystometric measurements of bladder capacity, detrusor compliance and detrusor leak point pressure. Increased tension decreased p27(KIP1) at the protein level in human bladder smooth muscle cells and in intact murine bladder smooth muscle. p27(-/-) mice had bladder smooth muscle cell hyperplasia even in the absence of bladder outlet obstruction. While p27 loss had little effect on detrusor leak point pressure, p27(-/-) mice had significantly decreased bladder capacity and detrusor compliance. To our knowledge we provide the first report of the in vivo significance of p27(KIP1) in the regulation of detrusor function using a cystometric approach. We identified a role for p27(KIP1) in protecting against dysregulated smooth muscle cell proliferation, bladder capacity and detrusor compliance under normotensive conditions.

Enterococcal Biofilm Formation and Virulence in an Optimized Murine Model of Foreign Body-Associated Urinary Tract Infections

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial UTIs and pose significant clinical challenges. Enterococcal species are among the predominant causative agents of CAUTIs. However, very little is known about the pathophysiology of Enterococcus-mediated UTIs. We optimized a murine model of foreign body-associated UTI in order to mimic conditions of indwelling catheters in patients. In this model, the presence of a foreign body elicits major histological changes and induces the expression of several proinflammatory cytokines in the bladder. In addition, in contrast to naïve mice, infection of catheter-implanted mice with Enterococcus faecalis induced the specific expression of interleukin 1β (IL-1β) and macrophage inflammatory protein 1α (MIP-1α) in the bladder. These responses resulted in a favorable niche for the development of persistent E. faecalis infections in the murine bladders and kidneys. Furthermore, biofilm formation on the catheter implant in vivo correlated with persistent infections. However, the enterococcal autolytic factors GelE and Atn (also known as AtlA), which are important in biofilm formation in vitro, are dispensable in vivo. In contrast, the housekeeping sortase A (SrtA) is critical for biofilm formation and virulence in CAUTIs. Overall, this murine model represents a significant advance in the understanding of CAUTIs and underscores the importance of urinary catheterization during E. faecalis uropathogenesis. This model is also a valuable tool for the identification of virulence determinants that can serve as potential antimicrobial targets for the treatment of enterococcal infections.

A combination of the metabolic enzyme inhibitor APO866 and the immune adjuvant L-1-methyl tryptophan induces additive antitumor activity

Many types of malignant cells have a higher nicotinamide adenine dinucleotide (NAD) turnover rate than normal cells, as well as the ability to escape immune responses. Indoleamine 2,3-dioxygenase (IDO) is reported to be a negative immune regulator. Overexpression of IDO in dendritic cells is observed in tumor-draining lymph nodes. IDO-expressing dendritic cells suppress T-cell activation and promote immune tolerance. The nicotinamide phosphoribosyl transferase (NAMPT) inhibitor APO866 (also called FK866 or WK175) selectively inhibits tumor growth through intracellular NAD depletion. The IDO-specific inhibitor L-1-methyl-tryptophan (L-1MT) activates immune responses and reduces tumor volume in murine tumor models. We combined L-1MT and APO866 treatments and tested their antitumor effects in the murine gastric and bladder tumor models. In immune-competent mice, a combination of APO866 and L-1MT had a better therapeutic effect than did either L-1MT or APO866 alone. The intracellular level of NAD was suppressed by APO866 but not L-1MT. However, an additive inhibitory effect on tumor growth was not observed in tumor-bearing immune-deficient mice. The new strategy of combining a metabolic inhibitor and an immune adjuvant induced a potent therapeutic effect.

435 LOXL2 as a new marker of basal-like phenotype in breast cancer

The aim of the present study was to investigate the biological and molecular basis of intraepithelial spreading (IES) of carcinomas in situ (CIS) of the urinary bladder, which were considered to be precursors of nodular invasive carcinomas.The propensity for IES of human transitional carcinoma cells was examined by inoculation into murine renal pelvis and urinary bladder, and the biological character of the cells with a high propensity for IES was explored in vitro.Three of 6 cell lines exhibited a high propensity for IES. When cultured on laminin, these IES cells scattered, whereas 3 non-IES cells and 2 immortalized transitional epithelial cells did not. IES cells showed strong adhesiveness, haptotaxis and enhanced migration on laminin compared with both non-IES and immortalized transitional epithelial cells. In IES cells, expression of the integrin beta4 subunit was markedly reduced and the integrin alpha6 beta1 complex was predominant compared with the integrin alpha6 beta4 complex. Transfection of IES cells with integrin beta4 subunit cDNA inhibited their ability to migrate on laminin and their propensity for IES. In addition, expression of the integrin beta4 subunit was reduced in surgically resected specimens of CIS of the urinary bladder.The results indicate that a reduction in integrin beta4 plays a role in IES of CIS of the urinary bladder through enhanced migration on laminin.

Antitumor effects of recombinant human Interleukin-6 on mouse bladder carcinoma through Fas-mediated apoptosis

An apoptosis-inducing therapy is gradually becoming a new strategy for cancer treatment. The aim of this study was to investigate the mechanism of growth-inhibitory effects of recombinant human interleukin-6 (rhIL-6) on bladder tumor-bearing T739 mice in vivo. Murine bladder transitional carcinoma cells (BTT739) were inoculated subcutaneously into T739 mice as a tumor model for evaluating the antitumor effects of rhIL-6. Then the mice were divided randomly into 5 groups: A, B, C, D and E. Different doses (0, 2, 4, 8 x 10(6) IU/kg body weight) of rhIL-6 were injected intraperitoneally twice per day and administered for 14 days, and 1 mg/kg/d mitomycin-C(MMC) was used as control. Tumor size was measured and determined as the mean of the largest diameter and the diameter at right angle. Animals were killed by CO(2) inhalation on the 15th day after tumor cell inoculation. Then, tumors were removed, weighed and collected. The tumor growth inhibition rate of rhIL-6 was calculated. The morphological characteristic changes of tumor cells were observed under electron microscope, and cell cycle analysis was determined by flow cytometry. The expressions of Fas, FasL and Bcl-2 protein on tumor cells were qualitatively detected by immunofluorescence cell staining, and their relative contents (rate of positive cells, RPC) were quantitatively determined with flow cytometry. rhIL-6 could inhibit bladder tumor growth in a dose-dependent manner in vivo. The tumor growth-inhibitory rates of 2, 4, 8 x 10(6) IU/kg rhIL-6 and 1 mg/kg MMC were 11.8, 39.5, 39.7 and 68.8%, respectively. Flow cytometry results showed that a hypodiploid peak before G1 phase could be found in tumor cells treated with rhIL-6. Moreover, the cells treated with rhIL-6 displayed disappearance of nucleoli, chromatin gathering under the nuclear membrane in mass or ring-shape under transmission electron microscopy. The rates of Fas, FasL protein-positive cells estimated by flow cytometry in rhIL-6-treated mice were (12.57 +/- 0.83) and (20.1 +/- 0.87) %, respectively, significantly higher than that (4.66 +/- 0.17) and (14.1 +/- 0.83) % in control mice (P < 0.01). There was no significant difference in the rate of Bcl-2 protein-positive cells between the mice in these two groups (P > 0.05). rhIL-6 had obvious antitumor effects on mouse bladder carcinoma in vivo, and the Fas signaling pathway might play an important role in rhIL-6-induced bladder carcinoma cell apoptosis.

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

Combretastatin A‐4 inhibits cell growth and metastasis in bladder cancer cells and retards tumour growth in a murine orthotopic bladder tumour model

Bladder cancer is a highly recurrent cancer after intravesical therapy, so new drugs are needed to treat this cancer. Hence, we investigated the anti-cancer activity of combretastatin A-4 (CA-4), an anti-tubulin agent, in human bladder cancer cells and in a murine orthotopic bladder tumour model. Cytotoxicity of CA-4 was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, propidium iodide (PI) staining assay and clonogenic survival assay. In vivo microtubule assembly assay, cell cycle analyses, Western blot and cell migration assay were used to study the mechanism of CA-4. The effect of intravesical CA-4 therapy on the development of tumours was studied in the murine orthotopic bladder tumour model. CA-4 inhibited microtubule polymerization in vivo. Cytotoxic IC(50) values of CA-4 in human bladder cancer cells were below 4 nM. Analyses of cell-cycle distribution showed CA-4 obviously induced G(2)-M phase arrest with sub-G(1) formation. The analyses of apoptosis showed that CA-4 induced caspase-3 activation and decreased BubR1 and Bub3 in cancer cells. In addition to apoptosis, CA-4 was also found to induce the formation of multinucleated cells. CA-4 had a significantly reduced cell migration in vitro. Importantly, the in vivo study revealed that intravesical CA-4 therapy retarded the development of murine bladder tumours. These data demonstrate that CA-4 kills bladder cancer cells by inducing apoptosis and mitotic catastrophe. It inhibited cell migration in vitro and tumour growth in vivo. Hence, CA-4 intravesical therapy could provide another strategy for treating superficial bladder cancers.

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.

Abstract 1903: Enhanced antitumor effect of coincident combination intravesical MMC plus BCG therapy in an orthotopic bladder cancer model

Abstract Introduction and Objective: Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is currently the most successful adjuvant agent for the treatment and/or prophylaxis of superficial bladder tumors. However, superficial bladder tumors recur in 60 to 70% of all cases, and 30 % of these recurrent tumors present with a higher grade with invasive properties. We evaluated the combination of intravesical MMC plus BCG treatment against bladder tumors in a model of orthotopic murine bladder cancer. Methods: An orthotopic murine bladder cancer model was established using the simple instillation of 1 × 106 MBT-2 cells into the lumen of the bladder of a female C3H/HeN mouse through a urethral catheter. To assess the antitumoral effect of MMC, intravesical MMC therapy was administered at various dose-escalating concentrations: 0μg (control: PBS), 25μg, 50μg, 100μg, and 200μg, (n = 8 for each group; a total of 5 groups). Next, a comparative evaluation of tumor growth in a control group(PBS), a MCC-alone group, a BCG-alone group(100μg), and a combined MMC plus BCG (100μg) group were performed (n= 8 for each group; a total of 4 groups). Intravesical therapy was administered at 3-day intervals starting on day 5 and repeated 5 times. On day 60 after the initial implantation of the MBT-2 cells, all the surviving mice were sacrificed and necropsied. To evaluate macrophage infiltration in the tumor and the proliferative activity among the different groups, immunohistochemical staining analysis of the tumor cells utilizing CD68 and Ki-67 was performed in animal model. Results: In the MMC groups of 8 mice each treated with a dose of 0μg (survival; 26.4 ± 3.2 days), 25μg (34.8 ± 7.0 days) and 50μg (40.0 ± 13.2 days), 0, 0 and 2 of the mice failed to develop tumor and survived, respectively. In both the 100μg (17.3 ± 2.8 days) and 200μg (11.9 ± 3.0 days) groups, all eight mice died from adverse events. A significant survival advantage was observed in the combined MMC (50μg) plus BCG (100μg) group (51.5 ± 8.1 days), compared with that in the BCG-alone group (33.4 ± 16.8 days; p = 0.03) and the MMC-alone group (39.5 ± 13.5 days; p = 0.04). BCG significantly suppressed while MMC increased macrophage infiltration in the tumors, compared with in the control groups. The Ki-67 expression level, which is regarded as a proliferation index was significantly lower in the combined MMC (50μg) plus BCG (100μg) group (31.1 ± 5.2%) than in the BCG-alone group (50.9 ± 4.1%; p &amp;lt;0.001) and the MCC-alone group (39.2 ± 2.5%; p &amp;lt;0.05). Conclusion: Combining BCG plus MMC treatment decreased the tumor appearance rate, improved the survival period and reduced the proliferation rate in tumors, as compared with BCG-alone and MMC-alone treatment, possibly providing a greater therapeutic benefit over either agent alone. Immunochemotherapy with a combination of intravesical MMC plus BCG may be a promising alternative treatment for bladder tumors. 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 1903.

316 SUCCESSFUL INTRAVESICAL THERAPY FOR BLADDER CANCER UTILIZING PACLITAXEL CONTAINING NANOPARTICLES

You have accessJournal of UrologyUrothelial Cancer: Medical & Surgical Therapy1 Apr 2010316 SUCCESSFUL INTRAVESICAL THERAPY FOR BLADDER CANCER UTILIZING PACLITAXEL CONTAINING NANOPARTICLES Mary Samplaski, Armine Smith, William Larchian, Vinod Labhasetwar, and Warren Heston Mary SamplaskiMary Samplaski More articles by this author , Armine SmithArmine Smith More articles by this author , William LarchianWilliam Larchian More articles by this author , Vinod LabhasetwarVinod Labhasetwar More articles by this author , and Warren HestonWarren Heston More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.380AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES About 70% of bladder cancers are superficial and amenable to intravesical therapy. Nanoparticles (NPs) provide a vehicle for localized drug delivery. They achieve rapid intracellular penetration and provide slow, regional release of encapsulated drug. The transferrin receptor (TfR) is overexpressed in bladder cancer cells. Paclitaxel (Tx) has activity against human bladder cancer. To optimize NP preparation, we evaluated Tf and poly-L-lysine (PLL) conjugated NP penetration. We then tested Tf conjugated NPs for Tx delivery to bladder cancer cells in a murine model. METHODS After bladder pretreatment with PLL, orthotopic MBT-2 bladder tumors were induced in C3H/HeJ female mice. Tumors were confirmed sonographically at 14 days. Coumarin-conjugated NPs were instilled intravesically in several groups: with & without PLL pretreatment, conjugated & unconjugated to PLL, and conjugated & unconjugated to Tf. Confocal microscopy of bladders was done after 1.5 hours of incubation. All subsequent experiments used Tf-conjugated NPs without PLL pretreatment. Cremophor (solvent for Tx) & Tx, Cremophor & saline, saline, and Tx loaded NPs (8 mg/kg) were instilled intravesically for 1.5 hours. Ultrasound was performed starting on post-instillation day (PID) 5. Bladders were examined on PID 15. RESULTS Tf conjugation led to enhanced NP accumulation in the tumors. PLL conjugation and PLL pre-wash enhanced overall penetration but negated the selective tumor binding. By PID 5 the Cremophor, Cremophor & saline, and saline groups had developed enlarging bladder tumors > 1mm. On PID 5 and 7, tumors in the Tx-NP group were comparatively smaller, 0.682mm and 0.878mm, respectively. By PID 9 tumors in the Tx-NP group were also enlarging (Fig 1). Histolology confirmed invasive tumors in all groups on PID 15. CONCLUSIONS Tf-NP conjugation allows for selective uptake of the NP by cancer cells. A single dose of NP encapsulated Tx suppresses murine superficial bladder tumor growth for 7 days. Further studies will focus on repeated dosing of Tx-NPs and increasing the dose of Tx. Cleveland, OH© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e125 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mary Samplaski More articles by this author Armine Smith More articles by this author William Larchian More articles by this author Vinod Labhasetwar More articles by this author Warren Heston More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Response of the Murine Urinary Bladder Microcirculation to Lipopolysaccharide from Escherichia coli and Pseudomonas aeruginosa

There is in vitro evidence demonstrating the inflammatory response to lipopolysaccharide (LPS) differs depending on the bacterial source. The objective of our study was to examine the in vivo inflammatory response to LPS from two common uropathogens, Escherichia coli (Ec) and Pseudomonas aeruginosa (Pa).LPS or saline was injected transurethrally into bladders of female C57Bl/6 mice at varying doses. The bladder was exteriorized, and the bladder microcirculation examined by intravital microscopy. Leukocyte adhesion at 4 hrs post challenge with 5 mg/kg Pa LPS was 2.9±0.6 cells/field of view and at 7 mg/kg it was 3.9±0.8 cells/field of view, a significant increase from saline control. In contrast, leukocyte adhesion 4 hours post 5 mg/kg Ec LPS challenge was 17.4±3.9 and with 7 mg/kg 19.7±2.9 cells/field of view. However, at 4.5 hours adhesion was similar with either LPS (5 mg/kg) with 18.4±3.0 and 21.2±4.0 cells/field of view, respectively. The flux of rolling leukocytes was significantly increased above baseline levels only after Ec LPS challenge. There was also a significant increase in the number of neutrophils with Ec LPS stimulation.In support of the in vitro observations, these in vivo results demonstrate that Ec LPS is more potent and induces a more rapid inflammatory response than Pa LPS.Supported by a Collaborative Health Research Projects grant from CIHR and NSERC.

441 EMBRYONIC SMOOTH MUSCLE INDUCES BLADDER SMOOTH MUSCLE DIFFERENTIATION

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2010441 EMBRYONIC SMOOTH MUSCLE INDUCES BLADDER SMOOTH MUSCLE DIFFERENTIATION Gregory Tasian, Mei Cao, Gerald Cunha, and Laurence Baskin Gregory TasianGregory Tasian More articles by this author , Mei CaoMei Cao More articles by this author , Gerald CunhaGerald Cunha More articles by this author , and Laurence BaskinLaurence Baskin More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.512AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelium-derived signals are necessary to induce smooth muscle differentiation from mesenchyme during murine bladder development. We tested the hypothesis that fetal bladder smooth muscle without urothelium can induce smooth muscle differentiation from bladder mesenchyme. METHODS Fetal intact bladders (IB) were harvested from timed pregnant FVB and transgenic GFP mice at the time points shown below. Bladder mesenchyme (BLM) was isolated by incubating IB in 0.02M EDTA and then removing the urothelium by microdissection. Embryonic day (E) 12 BLM from GFP mice was recombined with E12 BLM, E12 IB, and E16 BLM from FVB mice and incubated overnight on agar at 37°C. The recombined tissues were grafted beneath the kidney capsule of male nude mice, maintained for two weeks, and then were harvested and processed. H&E staining was performed and immunohistochemistry and immunofluoresence staining was used to detect smooth muscle α-actin, uroplakin and GFP. RESULTS At the time of harvest, E12 BLM does not express α-actin; E16 BLM does express α-actin. E12 BLM (GFP) recombined with E12 BLM (FVB) did not survive. E12 BLM (GFP) expressed smooth muscle α-actin when recombined with E12 IB (FVB). The α-actin positive cells derived from E12 BLM (GFP) diffusely migrated through the smooth muscle of the FVB bladder wall. When recombined with E16 BLM (FVB), E12 BLM (GFP) also demonstrated smooth muscle differentiation, as shown by the expression of smooth muscle α-actin; however, these cells did not migrate (see Figure). CONCLUSIONS Urothelium is necessary for initial induction of smooth muscle differentiation from bladder mesenchyme. After smooth muscle has formed, BLM alone can induce further smooth muscle differentiation. This may be due to an intrinsic property of smooth muscle, presence of a diffusible factor within the mesenchyme that induces smooth muscle differentiation, or initiation of signaling pathways by the urothelium prior to its removal that result in mesenchymal smooth muscle differentiation. San Francisco, CA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e174 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gregory Tasian More articles by this author Mei Cao More articles by this author Gerald Cunha More articles by this author Laurence Baskin More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1150 ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL BCG PLUS MMC THERAPY IN AN ORTHOTOPIC MURINE BLADDER CANCER MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101150 ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL BCG PLUS MMC THERAPY IN AN ORTHOTOPIC MURINE BLADDER CANCER MODEL Minoru Horinaga, Masashi Matsushima, Ryuichi Fukuyama, Hitoshi Yanaihara, Masahiro Iida, Yoko Nakahira, and Hirotaka Asakura Minoru HorinagaMinoru Horinaga Saitama, Japan More articles by this author , Masashi MatsushimaMasashi Matsushima Saitama, Japan More articles by this author , Ryuichi FukuyamaRyuichi Fukuyama Konan, Japan More articles by this author , Hitoshi YanaiharaHitoshi Yanaihara Saitama, Japan More articles by this author , Masahiro IidaMasahiro Iida Saitama, Japan More articles by this author , Yoko NakahiraYoko Nakahira Saitama, Japan More articles by this author , and Hirotaka AsakuraHirotaka Asakura Saitama, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.649AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is currently the most successful adjuvant agent for the treatment and/or prophylaxis of superficial bladder tumors. However, superficial bladder tumors recur in 60 to 70% of all cases, and 30 % of these recurrent tumors present with a higher grade with invasive properties. We evaluated the combination of intravesical BCG plus MMC treatments against bladder tumors in a model of orthotopic murine bladder cancer. METHODS An orthotopic murine bladder cancer model was established using the simple instillation of one million MBT-2 cells into the lumen of the bladder of a female C3H/HeN mouse. To assess the antitumoral effect of MMC, intravesical MMC therapy was administered at various dose-escalating concentrations: 0ug(control: PBS), 25ug, 50ug, 100ug and 200ug (n = 8 for each group). Next, a comparative evaluation of tumor growth in a control group (PBS), a BCG-alone group(100ug), a MCC-alone group, and a combined BCG (100ug) plus MMC group was performed (n= 8 for each group; total 4 groups). Intravesical therapy was administered at 3-day intervals starting on day 5 and repeated 5 times. On day 60 after the initial implantation of the MBT-2 cells, all the surviving mice were sacrificed and necropsied. To evaluate the macrophage infiltration in the tumor and the proliferative activity among the different groups, CD-68 and Ki-67 staining of the tumor cells was performed in animal model. RESULTS In the MMC groups of 8 mice each treated with a dose of 0ug (survival; 26.4 days), 25ug (34.8 days) and 50ug (40.0 days), 0, 0 and 2 of the mice survived, respectively. In both the 100ug (survival;17.3 days) and 200ug (11.9 days) groups, all eight mice died from adverse events. A significant survival advantage was observed in the combined BCG (100ug) plus MMC (50ug) group (51.5 days), compared with that in the BCG-alone (100ug) group (33.4 days p = 0.03) and the MMC-alone (50ug) group (39.5 days p = 0.04). BCG significantly suppressed while MMC increased macrophage infiltration in the tumor, compared with in the control group. The Ki-67 expression level, which is regarded as a proliferation index, was significantly lower in the BCG plus MCC group (31.1 ± 5.2%) than in the BCG-alone group (50.9 ± 4.1%; p <0.001) and the MCC-alone group (39.2 ± 2.5%; p <0.05). CONCLUSIONS Combining BCG plus MMC treatment decreased the tumor appearance rate, improved the survival period, and reduced the proliferation rate in tumors, as compared with BCG-alone and MMC-alone treatment, possibly providing a greater therapeutic benefit over either agent alone. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e445 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Minoru Horinaga Saitama, Japan More articles by this author Masashi Matsushima Saitama, Japan More articles by this author Ryuichi Fukuyama Konan, Japan More articles by this author Hitoshi Yanaihara Saitama, Japan More articles by this author Masahiro Iida Saitama, Japan More articles by this author Yoko Nakahira Saitama, Japan More articles by this author Hirotaka Asakura Saitama, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Biological similarities between murine chemical-induced and natural human bladder carcinogenesis

The present study investigated the similarities between rodent and human urothelial carcinogenesis models using DNA content, p53 and Ki-67 immunoexpression as surrogate markers of bladder carcinogenesis. Following N-butyl-N-(4-hydroxybutyl)-nitrosamine exposure, 49 human cystectomy specimens of bladder cancer and 53 rat bladder specimens were studied. All of the tumours and adjacent mucosa present in each specimen were evaluated. High similarities were observed between the rodent urothelium carcinogenesis process and the corresponding process in humans, in regards to the histopathological features and biological alteration profile: DNA aneuploidy, p53 overexpression and high proliferative index measured by Ki-67 immunoexpression. Despite these similarities, a higher frequency of alterations was observed in earlier stages in the rat chemical-induced carcinogenesis, namely in 5c aneuploid cells, p53 overexpression and higher Ki-67 labelling index. These results confirm that this experimental animal model is a suitable and reproducible model of bladder carcinogenesis, particularly in regards to high-risk non-invasive and invasive urothelial carcinomas. These features mandate its use in the identification of new molecular targets and evaluation of tumour response to new cytotoxic drugs or drug combinations in bladder cancer therapeutic intervention.

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.

Role of TREK-1 Potassium Channel in Bladder Overactivity After Partial Bladder Outlet Obstruction in Mouse

Mouse models of partial bladder outlet obstruction cause bladder hypertrophy. Expression of a number of ion channels is altered in hypertrophic detrusor muscle, resulting in bladder dysfunction. We determined whether mechanosensitive TREK-1 channels are present in the murine bladder and whether their expression is altered in partial bladder outlet obstruction, resulting in abnormal filling responses. Partial bladder outlet obstruction was surgically induced in CD-1 mice and the mice recovered for 14 days. Cystometry was done to evaluate bladder pressure responses during filling at 25 microl per minute in partial bladder outlet obstruction mice and sham operated controls. TREK-1 channel expression was determined at the mRNA and protein levels by quantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively, and localized in the bladder wall using immunohistochemistry. Obstructed bladders showed about a 2-fold increase in weight vs sham operated bladders. TREK-1 channel protein expression on Western blots from bladder smooth muscle strip homogenates was significantly decreased in obstructed mice. Immunohistochemistry revealed a significant decrease in TREK-1 channel immunoreactivity in detrusor smooth muscle in obstructed mice. On cystometry the TREK-1 channel blocker L-methioninol induced a significant increase in premature contractions during filling in sham operated mice. L-methioninol had no significant effect in obstructed mice, which showed an overactive detrusor phenotype. TREK-1 channel down-regulation in detrusor myocytes is associated with bladder overactivity in a murine model of partial bladder outlet obstruction.