Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

Summary

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

Abstract

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.

Introduction

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 instillation^6,7,8,9,10, 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.

Protocol

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

1. Culture the murine bladder cancer cell line MB49 in complete Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) to support optimal growth.
2. Maintain the cell cultures under standard conditions at 37 °C in a humidified incubator with 5% CO₂. Refresh the media every 2-3 days to ensure cell health and viability.
3. Harvest the cells using a standardized trypsin digestion procedure. Gently pipette to dislodge the cells from the culture flask after applying trypsin and incubating for a brief period.
4. Count the harvested cells using a hemocytometer to determine the exact cell density. Prepare a single-cell suspension by resuspending cells to 2 x 10³/µL in phosphate-buffered saline (PBS). Keep this suspension on ice to maintain cell viability.

2. Animal preparation

1. Group house the mice (5 mice per cage) in individually ventilated polycarbonate cages.
2. Allow the mice to acclimate for 7 days before the start of the study.

3. Animal orthotopic tumor model generation

1. Administer anesthesia to the mice via intraperitoneal injection using a 2.5% solution of Avertin (following institutionally approved protocols).
2. Wait until the mice reach an appropriate level of anesthesia for surgery, as indicated by a decrease in respiratory rate with an increase in depth, the absence of eyelid and corneal reflexes, reduced muscle tone and reflex responses, and no reaction to a tail pinch.
3. Position the anesthetized mouse in a supine position to facilitate the procedure.
4. Use a 24 G intravenous catheter with the needle stylet removed for the urethral catheterization procedure. Apply ample lubrication to the catheter to minimize discomfort and facilitate smooth insertion.
5. Identify the urethra, situated immediately posterior to the vulvar folds and anterior to the vagina, while keeping the mouse in a supine position.
   1. Begin the catheterization by approaching the urethra at a 45-degree angle to navigate beneath the pubic bone. Adjust to a more shallow angle to guide the catheter through the urethra and into the bladder.
6. Avoid applying excessive force against resistance, as this can lead to urethral or bladder perforation. Confirm the catheter's location within the bladder lumen by checking for urine within the catheter.
7. Gently apply pressure to the lower abdomen of the mouse to facilitate the drainage of urine from the bladder. Ensure the bladder is completely emptied to prepare for the subsequent steps of the procedure.
8. Gently push the 24 G intravenous catheter toward the top of the bladder. Ensure the tip of the catheter makes contact with the bladder's apex.
   1. Once contact is confirmed, release the catheter, allowing it to retract slightly. Maintain the catheter in a fixed position relative to the mouse to ensure stability and prevent dislodgement.
9. Insert the modified 34 G dispensing needle along the 24 G catheter. Advance the needle until it reaches the top of the bladder.
   1. Once positioned, rotate the needle six times to create a localized mucosal injury at the bladder's apex. After completing the rotation, carefully withdraw the needle along the catheter.
10. Connect the 30 G dispensing needle to the top of a 1 mL syringe. Ensure a secure connection between the needle and the syringe.
    1. Once connected, draw the pre-prepared 2 x 10³/µL MB49 cell suspension into the syringe by gently pulling back the plunger. Verify that the cell suspension is aspirated into the syringe without any air bubbles.
11. Insert the 30 G dispensing needle along the 24 G catheter. Ensure the needle is properly aligned and secured within the catheter.
    1. Once positioned, apply pressure to the plunger of the 1 mL syringe to inject the contents (prepared in step 1). Maintain steady pressure on the plunger for 60 s to ensure the contents are delivered without significant volume reduction.
    2. After 60 s, carefully withdraw the syringe and the 30 G dispensing needle from the catheter.

4. Post-operative monitoring

1. Place the anesthetized mouse in a supine position on a heating pad until the righting reflex returns.
2. Ensure the animal is not left unattended until it has regained sufficient consciousness.
3. Do not return the animal to the company of other animals until it has fully recovered.

النتائج

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....

Discussion

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

Materials

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