Mouse Orthotopic Bladder Research Articles

Establishment of Mouse Orthotopic Urinary Bladder Tumor Model and Its Analysis by Light and Electron Microscopy.

Mouse tumor models are an important tool in cancer research, and the orthotopic cancer cell transplantation model is the most widely used among them. Methods for establishing tumor models may differ in many ways, including the selection of cancer cell lines and the type of urinary bladder pretreatment. Here, we describe our mouse orthotopic bladder tumor model using a labeled MB49 urothelial cancer cell line and chemical pretreatment with the cationic polypeptide poly-L-lysine to traumatize the bladder epithelium. Double labeling of MB49 cancer cells by their transduction with GFP and internalization of metal nanoparticles allows the study of their implantation process from the first hours to several days after intravesical injection, as well as the analysis of developed tumors after 3weeks. Thus, our model provides a comprehensive analysis of the early and late stages of tumor development in the bladder at the light and electron microscopic level.

PD37-09 INTERMITTENT WST-11 VASCULAR TARGET PHOTODYNAMIC THERAPY IN A MOUSE ORTHOTOPIC BLADDER MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-09 INTERMITTENT WST-11 VASCULAR TARGET PHOTODYNAMIC THERAPY IN A MOUSE ORTHOTOPIC BLADDER MODEL Jie Chen, Jasmine Thomas, Karan Nagar, Lucas Nogueira, Peter Reisz, Kwanghee Kim, and Jonathan Coleman Jie ChenJie Chen More articles by this author , Jasmine ThomasJasmine Thomas More articles by this author , Karan NagarKaran Nagar More articles by this author , Lucas NogueiraLucas Nogueira More articles by this author , Peter ReiszPeter Reisz More articles by this author , Kwanghee KimKwanghee Kim More articles by this author , and Jonathan ColemanJonathan Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Vascular targeted photodynamic therapy (VTP) is a non-surgical tumor ablation approach used to treat early-stage prostate cancer and may also be effective for urothelial cancer based on preclinical data in heterotopic models. While orthotopic models are valuable as they reproduce factors such as local tissue environment and cancer progression pathogenesis compared to ectopic implantation, its implementation is technically difficult. Herein we describe our work in developing an orthotopic murine model for bladder urothelial cancer using intermittent laser pulses to ensure mouse survival and local tumor control. METHODS: The MB-49 murine urothelial cancer line was used for all experiments after establishing stable luciferase expression. Tumors were implanted in 16 1C57BL-6 mice by surgical approach by direct injection of 1x105 MB49-Luc cells into the bladder. Successful tumor implantation was assessed by IVIS images 72 hours after implantation. Mice were divided in two groups: control and VTP treated. VTP was performed 96 hours after cell injection at a power of 100mW in 30 seconds pulses, with 30 seconds interval. Mouse bladders were harvested 13 days post implantation. Safety, tumor control, and histology parameters were assessed. RESULTS: Adequate tumor growth was observed in all mice, and no major toxicity was observed in both groups. Final tumor size was 71% smaller, and histological viability was decreased in VTP treated tumors when compared to controls (p <0.001, figure 1). CD31, Ki67 expression were decreased 2.75 and 1.87-fold respectively, and tunnel staining was increased 3.2-fold in control tumors when compared to VTP treated. (p <0.05, figure 2). CONCLUSIONS: A mouse orthotopic bladder urothelial model via injection of MB-49 cell line is feasible and displays a very high tumor take rates. Intermittent VTP treatment has an effect on mouse bladder tumors as shown by the decrease in tumor size, weight and change in histology. Additional refinement to the model may provide a means to establish an appropriate timeline and utility for future studies. Source of Funding: NIH/NCI (P30 CA008748) and the Thompson Family Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e658-e659 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jie Chen More articles by this author Jasmine Thomas More articles by this author Karan Nagar More articles by this author Lucas Nogueira More articles by this author Peter Reisz More articles by this author Kwanghee Kim More articles by this author Jonathan Coleman More articles by this author Expand All Advertisement Loading ...

An improved mouse orthotopic bladder cancer model exhibiting progression and treatment response characteristics of human recurrent bladder cancer.

Nonmuscle-invasive (superficial) bladder cancer is generally treated via surgical removal, followed by adjuvant therapy (bacillus Calmette-Guerin). However, bladder cancer can often recur, and in a substantial number of recurrent cases, the cancer progresses and metastasizes. Furthermore, residual microtumors following excision may lead to an increased risk of recurrence. An in vivo model mimicking the pattern of urinary bladder microtumor regrowth may provide an effective experimental system for improving postsurgical treatment outcomes. A mouse bladder cancer model established using orthotopic transplant of UM-UC-3 human urinary bladder carcinoma cells has been established, however, to the best of our knowledge, no report has investigated sequential histological changes, including early-phase changes and treatment responses in bladder cancer. In the present study, the efficiency of the model was optimized and the sequential changes were examined using histopathology and in situ imaging. The therapeutic effects of cisplatin (CDDP) and gemcitabine (GEM) were also examined, which are drugs that are often used for follow-up chemotherapy. Tumor-seeding efficiency reached 90–100%, with muscle layer and bladder lumen invasion occurring in ~21 days, using the following modifications: i) Shallow catheter insertion to mitigate bladder wall damage; ii) bladder pretreatment using prewarmed trypsin, followed by light urethral clamping and body temperature maintenance for more efficient removal of transitional epithelium; and iii) seeding with UM-UC-3 cells (rather than HT1376, 5637 or T24 tumor cells) in a medium supplemented with Matrigel. Transplant with UM-UC-3 cells resulted in isolated microlesions that progressed into tumors, invading the bladder lumen and muscle layer to the serosal surface. Tumor growth was markedly reduced by weekly intravenous injections of CDDP and partially suppressed by GEM. Therefore, this model is reliable, and pathological progression and treatment responses recapitulate the features of recurrent human bladder cancer.

Anti-tumor effect of Radix Paeoniae Rubra extract on mice bladder tumors using intravesical therapy.

Radix Paeoniae Rubra (RPR) is the dried root of Paeonia lactiflora Pallas and Paeonia veitchii Lynch, and is a herbal medicine that is widely used in traditional Chinese medicine for the treatment of blood-heat and blood-stasis syndrome, similarly to Cortex Moutan. The present study identified the same three components in RPR and Cortex Moutan extracts. In addition, it has been reported that RPR has an anti-cancer effect. Bladder cancer is the seventh most common type of cancer worldwide. Due to the high recurrence rate, identifying novel drugs for bladder cancer therapy is essential. In the present study, RPR extract was evaluated as a bladder cancer therapy in vitro and in vivo. The present results revealed that RPR extract reduced the cell viability of bladder cancer cells with a half maximal inhibitory concentration of 1–3 mg/ml, and had an extremely low cytotoxic effect on normal urothelial cells. Additionally, RPR decreased certain cell cycle populations, predominantly cells in the G1 phase, and caused a clear sub-G increase. In a mouse orthotopic bladder tumor model, intravesical application of RPR extract decreased the bladder tumor size without altering the blood biochemical parameters of the mice. In summary, the present results demonstrate the anti-proliferative properties of RPR extract on bladder cancer cells, and its anti-bladder tumor effect in vivo. Compared to Cortex Moutan extract, RPR extract may provide a more effective alternative therapeutic strategy for the intravesical therapy of superficial bladder cancer.

Anti-Bladder-Tumor Effect of Baicalein fromScutellaria baicalensisGeorgi and Its ApplicationIn Vivo

Some phytochemicals with the characteristics of cytotoxicity and/or antimetastasis have generated intense interest among the anticancer studies. In this study, a natural flavonoid baicalein was evaluated in bladder cancer in vitro and in vivo. Baicalein inhibits 5637 cell proliferation. It arrests cells in G1 phase at 100 μM and in S phase below 75 μM. The protein expression of cyclin B1 and cyclin D1 is reduced by baicalein. Baicalein-induced p-ERK plays a minor role in cyclin B1 reduction. Baicalein-inhibited p65NF-κB results in reduction of cell growth. Baicalein-induced pGSK(ser9) has a little effect in increasing cyclin B1/D1 expression instead. The translation inhibitor cycloheximide blocks baicalein-reduced cyclin B1, suggesting that the reduction is caused by protein synthesis inhibition. On the other hand, neither cycloheximide nor proteasome inhibitor MG132 completely blocks baicalein-reduced cyclin D1, suggesting that baicalein reduces cyclin D1 through protein synthesis inhibition and proteasomal degradation activation. In addition, baicalein also inhibits cell invasion by inhibiting MMP-2 and MMP-9 mRNA expression and activity. In mouse orthotopic bladder tumor model, baicalein slightly reduces tumor size but with some hepatic toxicity. In summary, these results demonstrate the anti-bladder-tumor properties of the natural compound baicalein which shows a slight anti-bladder-tumor effect in vivo.

Cortex Moutan Induces Bladder Cancer Cell Death via Apoptosis and Retards Tumor Growth in Mouse Bladders

Cortex Moutan is the root bark of Paeonia suffruticosa Andr. It is the herbal medicine widely used in Traditional Chinese Medicine for the treatment of blood-heat and blood-stasis syndrome. Furthermore, it has been reported that Cortex Moutan has anticancer effect. In this study, the Cortex Moutan extract was evaluated in bladder cancer therapy in vitro and in vivo. Cortex Moutan extract reduces cell viability with IC50 between 1~2 mg/ml in bladder cancer cells, and it has lower cytotoxicity in normal urotheliums. It arrests cells in G1 and S phase and causes phosphatidylserine expression in the outside of cell membrane. It induces caspase-8 and caspase-3 activation and poly(ADP-ribose) polymerase degradation. The pan caspase inhibitor z-VAD-fmk reverses Cortex Moutan-induced cell death. Cortex Moutan also inhibits cell invasion activity in 5637 cells. In mouse orthotopic bladder cancer model, intravesical application of Cortex Moutan decreases the bladder tumor size without altering the blood biochemical parameters. In summary, these results demonstrate the antiproliferation and anti-invasion properties of Cortex Moutan in bladder cancer cells and its antibladder tumor effect in vivo. Cortex Moutan may provide an alternative therapeutic strategy for the intravesical therapy of superficial bladder cancer.

Sunitinib Malate Provides Activity Against Murine Bladder Tumor Growth and Invasion in a Preclinical Orthotopic Model

To evaluate the effects of sunitinib on localized bladder cancer in a mouse orthotopic bladder tumor model. We used an established orthotopic mouse bladder cancer model in syngeneic C3H/He mice. Treatment doses of 40 mg/kg of sunitinib or placebo sterile saline were administrated daily by oral gavage. Tumor volume, intratumoral perfusion, and in vivo vascular endothelial growth factor receptor-2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The findings were correlated with the total bladder weight, tumor stage, and survival. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by immunostaining of CD31 and Ki-67. Significant inhibition of tumor growth was seen after sunitinib treatment compared with the control. The incidence of extravesical extension of the bladder tumor and hydroureter in the sunitinib-treated group (30% and 20%, respectively) was lower than the incidence in the control group (66.7% and 55.6%, respectively). Sunitinib therapy prolonged the survival in mice, with statistical significance (log-rank test, P = .03). On targeted contrast-enhanced micro-ultrasound imaging, in vivo vascular endothelial growth factor receptor-2 expression was reduced in the sunitinib group and correlated with a decrease in microvessel density. The results of our study have demonstrated the antitumor effects of sunitinib in the mouse localized bladder cancer model. Sunitinib inhibited the growth of bladder tumors and prolonged survival. Given that almost 30% of cases in our treatment arm developed extravesical disease, sunitinib might be suited as a part of a multimodal treatment regimen for bladder cancer.

Intravesical Interleukin-15 Gene Therapy in an Orthotopic Bladder Cancer Model

Interleukin-15 (IL-15) is known to stimulate the proliferation of CD8(+) T-cells and natural killer cells, and also to help to maintain memory CD8(+) T cells, suggesting that it may be of value in cytokine treatment of bladder cancer. In this experiment, we tested the efficiency of intravesical liposomal IL-15 gene delivery and its antitumor effect in a mouse orthotopic bladder cancer model. We established an orthotopic bladder cancer model by implanting 5×10(5) MBT-2 cells into female C3H/HeN mice through the urethra. The mice received repeated intravesical gene delivery injected with liposome-mediated plasmids (5 μg) transurethrally. On day 23, the bladder weights in the group receiving medium alone, the beta-galactosidase gene delivery control group, and the IL-15 gene therapy group were 196±36 mg, 201±35 mg, and 96±29 mg, respectively (p<0.05), demonstrating the antitumor effect of intravesical IL-15 gene therapy in this model. In the bladders treated with IL-15 gene plasmid instillation, histological analysis revealed that many inflammatory cells were induced around the tumors. Immunohistochemical analysis confirmed that there was predominant infiltration of CD8(+) T cells around the tumor nest. After the intravesical IL-15 gene therapy, the growth of rechallenged subcutaneous MBT-2 cells in surviving mice was inhibited again via tumor-specific cytotoxic T lymphocytes, although newly implanted FM3A cells in the same mice were not rejected. The present findings indicate that IL-15 gene therapy may be a promising new adjuvant therapy for bladder cancer.

In Vivo Targeted Contrast Enhanced Micro-Ultrasound to Measure Intratumor Perfusion and Vascular Endothelial Growth Factor Receptor 2 Expression in a Mouse Orthotopic Bladder Cancer Model

We evaluated the feasibility of using targeted contrast enhanced micro-ultrasound imaging to assess intratumor perfusion and vascular endothelial growth factor receptor 2 expressions in a mouse orthotopic bladder cancer model. We created an orthotopic mouse model by implanting MBT-2 murine bladder cancer cell lines in the bladder of syngeneic C3H/He mice (Jackson Laboratory, Bar Harbor, Maine). Successful tumor implantation was confirmed by transabdominal micro-ultrasound imaging on post-implantation day 11. Contrast enhanced micro-ultrasound imaging was done on days 14 and 21. Vascular endothelial growth factor receptor 2 targeted contrast agent was prepared by adding biotinylated anti-vascular endothelial growth factor receptor 2 monoclonal antibodies to streptavidin coated microbubbles. The targeted contrast agents were injected via the retro-orbital route. We quantified intratumor perfusion, vascular endothelial growth factor receptor 2 endothelial expression and blood volume in real time. In the initial study intratumor perfusion data and vascular endothelial growth factor receptor 2 expression could only be measured in 10 of 14 mice (71%) due to motion artifact. We modified our technique by applying an elastic band over the lower abdomen to minimize body wall movement. After the modification complete images were acquired in all mice at 2 consecutive imaging sessions. Measurements were made of intratumor perfusion and in vivo vascular endothelial growth factor receptor 2 expression. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. Targeted contrast enhanced micro-ultrasound imaging enables investigators to detect and monitor vascular changes in orthotopic bladder tumors. It may be useful for direct, noninvasive, in vivo evaluation of novel anti-angiogenesis therapeutic agents. With the modified technique target enhanced contrast ultrasound can be applied in an orthotopic bladder cancer model.

A novel immunotherapy for superficial bladder cancer by intravesical immobilization of GM‐CSF

In situ gene therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) was demonstrated to successfully inhibit tumour cell growth in a mouse orthotopic bladder cancer model, but suffered from several disadvantages, such as limited efficiency for gene delivery, low expression efficiency of the transgene and the safety concern resulting from viral vector. In order to address the limits, a novel immunotherapy was developed attentively through immobilization of streptavidin-tagged bioactive GM-CSF on the biotinylated mucosal surface of bladder wall on the basis of both the unique property of streptavidin (SA) to bind rapidly and almost irreversibly to any biotin-linked molecule and the outstanding ability of biotin to be incorporated easily into the proteins on the cell surface. The mouse orthotopic model of MB49 bladder cancer was used to evaluate the feasibility and efficacy of the novel immunotherapy performed twice a week for 3 weeks. Briefly, 1 day after intravesical implantation of 1 × 106 MB49 tumour cells in C57BL/6 mouse, 100 μl of 1 mg/ml NHS-PEO4-biotin was instilled and allowed to incubate in the bladder for 30 min., followed by intravesical instillation of 100 μl of 0.15 mg/ml SA-GM-CSF bifunctional fusion protein and incubation for 1 hr. SA-GM-CSF fusion protein was shown to be immobilized efficiently and durably on the biotinylated mucosal surface of bladder wall. The bladder cancer incidence was dramatically decreased from 100% in the control group to 37.5% in the SA-GM-CSF group. Importantly, 70% of the SA-GM-CSF-cured mice were protected against a second intravesical wild-type MB49 tumour challenge, indicating that an effective anti-tumour immunity was generated against MB49 bladder cancer. Thus, the novel immunotherapy may be an attractive therapeutic alternative and should be evaluated in bladder cancer patients.

Role of syndecan‐1 (CD138) in cell survival of human urothelial carcinoma

Heparan sulfate proteoglycan syndecan-1, CD138, is well known to be associated with cell proliferation, adhesion, and migration in various types of malignancies. In the present study, we focused on the role of syndecan-1 in human urothelial carcinoma of the urinary bladder. Silencing of syndecan-1 by siRNA transfection down-regulated transcriptional factor junB and the long isoform of FLICE-inhibitory protein (FLIP long), resulting in the induction of apoptosis in the urothelial carcinoma cell lines UMUC2 and UMUC3. Knockdown of junB and FLIP long as well as syndecan-1 silencing mediated apoptosis that was inhibited by pan-caspase inhibitors. Transurethral injection of syndecan-1 siRNA into the urinary bladder significantly reduced syndecan-1 gene expression and growth of red fluorescent-labeled KU-7/RFP bladder cancer cells in the mouse orthotopic bladder cancer model. Immunohistochemical examination showed high syndecan-1 protein expression in high-grade, superficial, and deep invasive carcinomas (pT1 and >or=pT2) as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes (pTa). In addition, the percentage of cancer cells positive for syndecan-1 at initial diagnosis was statistically associated with the frequency of bladder cancer recurrence after transurethral resection. In conclusion, syndecan-1 might contribute to urothelial carcinoma cell survival and progression; therefore, this molecule could be a new therapeutic target in human urinary bladder cancer.

INTRAVESICAL INTERLEUKIN-15 GENE THERAPY IN A MOUSE ORTHOTOPIC BLADDER CANCER MODEL

You have accessJournal of Urology1 Apr 2009INTRAVESICAL INTERLEUKIN-15 GENE THERAPY IN A MOUSE ORTHOTOPIC BLADDER CANCER MODEL Kazuhiro Matsumoto, Eiji Kikuchi, Minoru Horinaga, Akira Miyajima, and Mototsugu Oya Kazuhiro MatsumotoKazuhiro Matsumoto More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Minoru HorinagaMinoru Horinaga More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(09)60993-9AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "INTRAVESICAL INTERLEUKIN-15 GENE THERAPY IN A MOUSE ORTHOTOPIC BLADDER CANCER MODEL." The Journal of Urology, 181(4S), p. 349 © 2009 by American Urological AssociationFiguresReferencesRelatedDetails Volume 181Issue 4SApril 2009Page: 349 Advertisement Copyright & Permissions© 2009 by American Urological AssociationMetricsAuthor Information Kazuhiro Matsumoto More articles by this author Eiji Kikuchi More articles by this author Minoru Horinaga More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement PDF downloadLoading ...

MICRO-ULTRASOUND IMAGING FOR BLADDER CANCER IN A MURINE MODEL

4534 A validated mouse model for superficial bladder cancer is warranted to enable preclinical studies on intravesical anticancer agents. We sought to improve upon our previously described orthotopic superficial bladder cancer mouse model with validation using in vivo ultrasound imaging. Using the murine bladder tumor (MBT-2) cell line, 11 C3H/HeJ female mice were inoculated with 5 x 105 MBT-2 cells in 50 uL serum-free RPMI via transurethral catheterization without electrocautery. Using the VisualSonicsTM Vevo 770® ultrasound device, we performed transabdominal micro-ultrasound imaging 3, 6, and 15 days after tumor instillation. Imaging was also performed in all mice prior to tumor cell instillation. Tumors were measured using linear 2-dimensional measurements using sagittal and transverse imaging planes. Mouse bladders were subsequently harvested, formalin-fixed, embedded in paraffin, sectioned and stained with hematoxylin and eosin for confirmation of bladder tumors. Superficial bladder tumors were evident on micro-ultrasound imaging starting on day 3 post-MBT-2 cell instillation in all mice. Multifocal tumors were seen in 27% of mice on day 3, 91% on day 6, and 100% on day 15. Average number of tumors found on days 3, 6, and 15 was 1.3, 2.4, and 3.5. Mean tumor size (max dimension of largest tumor) on days 3, 6, and 15 was 0.7mm, 1.1mm, 1.3mm. Histology sections on all mice confirmed presence of bladder tumors in all mice. Using abdominal micro-ultrasound, we validate the presence of superficial bladder tumors in our MBT-2 mouse orthotopic bladder cancer model. Our model is reproducible and can be used to evaluate the efficacy of various intravesical anti-cancer agents. Tumors can be followed longitudinally for progression or regression with micro-ultrasound imaging.

GFP image analysis in the mouse orthotopic bladder cancer model

Precise and objective measurements of tumor response have yet to be standardized in the mouse orthotopic bladder cancer model. In this study, we used image analysis and green fluorescent protein (GFP) to objectively measure tumor size in response to chemotherapy. KU-7 human bladder cancer cells transfected with GFP were intravesically inoculated into 8-week-old female nude mice. Fourteen days after tumor cell inoculation, the mice were assigned into a control (PBS) group or a doxorubicin (conc. 1.0 mg/ml) treatment group and received a single instillation of treatment. Fourteen days after treatment, the bladders were surgically exposed and fluorescent images were captured and later analyzed using image analysis. Bladders were processed for histological examination. Tumor incidence determined by GFP expression and histology was 100 and 80%, respectively, in the doxorubicin treatment group. A 9-fold (histology) vs. 12-fold (GFP expression) difference in tumor regression measured by tumor area (P<0.05) and a 5-fold (histology) vs. 9-fold (GFP expression) difference in tumor regression measured by the percent of tumor area in the bladder (P<0.001) were observed in the doxorubicin treatment group. Our findings suggest that using image analysis provides a precise, sensitive and objective means to measure tumor growth and treatment response in the mouse orthotopic bladder cancer model in lieu of histological methods. Consequently, the number of mice required in an experiment can be reduced since tissue samples are not needed for histology, thus making tissue samples readily available for additional assays in both a labor-effective and cost-effective manner.