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This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.
This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.
Bladder cancer represents a significant global health burden, with notable sex-specific disparities in incidence and prognosis. This malignancy is characterized by distinct molecular subtypes, each associated with diverse pathogenic pathways. The molecular and pathological features differ significantly between non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC)1,2. NMIBC accounts for approximately 75% of cases, featuring tumors of transitional epithelial origin that remain localized without metastasis or spread. Conversely, MIBC is characterized by the infiltration of cancer cells into the bladder's muscle layer, accompanied by a high risk of dissemination, frequently necessitating cystectomy in clinical practice3.
In preclinical research, models that accurately represent the superficial stage of the disease are essential for evaluating drug therapies. The establishment of a superficial bladder carcinoma in situ model is, therefore, of paramount importance, serving as a critical research tool for the development of clinical drugs and innovative instillation therapies.
The development of murine orthotopic bladder cancer models has traditionally faced challenges. Chemically induced models often begin as superficial tumors but may evolve into invasive forms, with variability that limits their utility in large-scale experiments4. Traditional orthotopic implantation models, which rely on the injection of tumor cells through the bladder wall5, struggle to faithfully reproduce superficial bladder cancer. Alternative methods, including mucosal injury combined with tumor cell instillation6,7,8,9,10, have been attempted but are limited by high mortality rates and low tumor take rates, hindering their broader application.
This study aims to develop a new method for constructing a superficial bladder cancer model that demonstrates greater stability, lower mortality, and fixed tumor positioning compared to existing models. By achieving a more precise representation of the disease in its early stages, the current model is poised to enhance the rigorous evaluation of preventive and therapeutic interventions, thereby advancing bladder cancer treatment.
All animal experimental protocols and procedures were approved by the Ethical Review Committee for Animal Experimentation of Tianjin Medical University, Tianjin, China (approval number SYXK: 2020-0010). Six- to eight-week-old female C57BL/6J mice were used for this study. The animals were housed under controlled environmental conditions, including a 12-h light-dark cycle, temperatures ranging from 21-25 °C, adjustable humidity levels between 30%-70%, and unrestricted access to food and water unless otherwise specified. The details of the reagents and equipment used are listed in the Table of Materials.
1. Cell preparation
2. Animal preparation
3. Animal orthotopic tumor model generation
4. Post-operative monitoring
The efficacy of submucosal injections was initially assessed through the administration of Trypan Blue. Post-injection, the dye's distribution within the submucosal layer was clearly visualized, confirming the precise and controlled delivery of the injected substances (Figure 1).
Tumor development was meticulously tracked. Approximately 14 days post-implantation, a notable incidence of hematuria was observed, a critical symptom indicative of bladder neoplasia....
Research on bladder cancer relies on animal models, which are indispensable for both basic and applied research. To better mimic the tumor growth environment, orthotopic bladder tumor models provide a superior approach compared to subcutaneous tumor models11.
Currently, orthotopic bladder cancer models can be categorized based on experimental purposes into two main types: those utilizing immunodeficient mice, such as patient-derived xenograft (PDX) models
The authors declare that they have no competing interests.
This study was supported by the Tianjin Municipal Health Industry Key Project fund (grant no. TJWJ2022XK014), the Scientific Research Project fund of Tianjin Municipal Education Commission (grant no. 2022ZD069), the Tianjin Institute of Urology Talent Funding Program (grant no. MYSRC202310), the Clinical Medicine Research Project fund of the Second Hospital of Tianjin Medical University (grant no. 2023LC03), the Youth Fund of the Second Hospital of Tianjin Medical University (grant no. 2022ydey15), and the Talent Cultivation Project of the Department of Urology, the Second Hospital of Tianjin Medical University (grant no. MNRC202313). The sponsors played a role in the preparation, review, and approval of the manuscript.
Name | Company | Catalog Number | Comments |
34 G dispensing needle | Suzhou Haorun Fluid Technology Co., Ltd., Suzhou, China | SGL-362 | |
30 G dispensing needle | Suzhou Haorun Fluid Technology Co., Ltd., Suzhou, China | SGL-362 | |
Avertin | Sigma-Aldrich LLC | T48402 | |
Trypan blue | Sigma-Aldrich LLC | 302643 |
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