Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors
March 29th, 2019
Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.
This methodology can be used to reproducibly generate bladder tumors using the BBN carcinogen and to image the size and extension of the bladder tumor burden through MRI. This technique uses an operator independent rapid imaging approach with short acquisition times at a high diagnostic quality to conduct high-throughput assays of mice at intermediate stages of tumor development. To induce bladder cancer tumor formation, add 0.05%BBN to an opaque drinking water container and provide the water to 6-week old C57 black six mice ad libitum.
Then monitor the animals two times a week for sign of distress associated with bladder tumors in accordance with the local IACUC guidelines. 16 to 24 weeks after exposure, load a one milliliter syringe equipped with a 25 to 27 gauge needle with 100 to 200 microliters of sterile saline and inject the saline subcutaneously into a BBN-treated animal. After 10 minutes, transfer the mouse into the imaging holder of a magnetic resonance imager and insert a rectal temperature probe connected to the physiological recording computer to monitor the animal's body temperature.
Next, place a 4-channel quadrature receiver coil over the lower abdomen of the mouse and initiate automatic adjustments in the integrated imaging software to acquire a triaxial set of images of the whole mouse body. From this reference set of images, identify the region of interest and acquire three sets of orthogonal sliced images along the axial, coronal, and sagittal planes. Using the true fast imaging with steady-state progression imaging sequence, set the TR to 900 milliseconds, the TE to two milliseconds, and the FA to 70, 14 averages.
Then select the appropriate geometric parameters within the imaging platform software to obtain a series of slices across the whole bladder of 0.5 milliliter thickness and an in-plane resolution of 0.148 millimeters. When all of the images have been obtained, export the set of slices covering the whole bladder to an appropriate medical image analysis software program and scroll through the generated images to locate a slice at the midpoint of the bladder that allows visualization of the bladder wall and lumen to select the representative axial view at the center of the bladder for quantitative analysis. Then trace the boundaries around the outer edge and inner lumen of the bladder to carefully delineate the region of interest within the selected representative axial view and subtract the inner lumen from the outer edge to calculate the surface area of the bladder wall.
20 weeks after BBN exposure, clean the area of incision with 70%ethanol and use forceps to lift the abdominal wall skin. Make a midline incision from the pubic symphysis to the xiphoid process and grasp the peritoneum to allow a sharp incision of the peritoneal cavity. Identify the bladder in the midline lower abdomen, using forceps to grasp the dome of the bladder.
Dissect the bladder away from the surrounding structures including the seminal vesicles, rectum, and fat. Cut the median umbilical ligament connecting the dome of the bladder to the umbilicus and abdominal wall. Cut the ureters close to the bladder and lifting the bladder cephalad, cut the urethra to remove the bladder.
Then rinse the excised bladder with PBS and immediately weigh the tissue on a balance. Here, representative true fast imaging with steady-state progression magnetic resonance images of bladder wall 3-D reconstructions and pathologic images of a mouse with a large tumor and of a control mouse without a tumor are shown. The bladder wall thickness as derived from the magnetic resonance imaging correlates weekly with the ex-vivo obtained bladder weight measurements.
Examination of the MRI-derived bladder wall thickness and ex-vivo obtained bladder weight data also demonstrate an association with the tumor stage as well as an association when stratifying the pathology by non-muscle invasive bladder cancer and muscle invasive bladder cancer. Keep in mind that it is best to acquire the images in mice when they have a full bladder. Following MR imaging of the bladder tumors, mice can be split into cohorts with similar tumor characteristics, as appropriate.
This technique allows researchers to explore new immunotherapies and other treatments to improve outcomes for our patients. It is important to remember that BBN is a carcinogen and that the appropriate precaution should always be taken when handling this compound.
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Overview
This study presents a reliable MRI protocol for assessing bladder tumor size and stage in mice induced by the BBN carcinogen. The methodology allows for high-throughput imaging of tumor burden with minimal distress to the animals.
Key Study Components
Area of Science
- Oncology
- Imaging Techniques
- Animal Models
Background
- Bladder tumors can be induced using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN).
- Accurate assessment of tumor burden is crucial for experimental treatment.
- Current imaging methods may not provide the necessary speed and reliability.
- This study aims to address these limitations with a new MRI protocol.
Purpose of Study
- To develop a fast and reliable MRI protocol for bladder tumor assessment.
- To enable high-throughput assays of tumor development in mice.
- To ensure minimal distress to the animals during imaging.
Methods Used
- Induction of bladder tumors in C57 black six mice using 0.05% BBN in drinking water.
- Monitoring of animals for signs of distress bi-weekly.
- Subcutaneous injection of saline prior to MRI imaging.
- Use of a magnetic resonance imager with physiological monitoring.
Main Results
- The MRI protocol allows for accurate imaging of tumor size and extension.
- High diagnostic quality achieved with rapid imaging techniques.
- Methodology supports reproducibility in tumor generation and assessment.
- Facilitates efficient monitoring of tumor development stages.
Conclusions
- The developed MRI protocol is effective for assessing bladder tumors in mice.
- It provides a reliable method for high-throughput imaging.
- This approach can enhance experimental treatment evaluations in oncology.
Frequently Asked Questions
Executive Industry Relevance
High-throughput, operator-independent MRI assessment of BBN-induced murine bladder tumors enables precise, quantitative tumor burden evaluation prior to experimental randomization. This imaging workflow supports robust preclinical modeling for immunotherapy and therapeutic candidate evaluation, reducing biological variability and enhancing predictive confidence at the discovery-to-preclinical inflection point. Reliable tumor stratification ensures translational continuity and risk-adjusted portfolio advancement.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rigorous quantification of tumor burden for hypothesis-driven target validation.
- Supports biological de-risking by correlating imaging outputs with ex vivo pathology and tumor stage.
- Facilitates predictive confidence in model selection for immunotherapy research.
Screening & Assay Development
- Provides standardized, reproducible imaging outputs for downstream cohort randomization.
- Delivers quantitative, operator-independent measurements suitable for high-throughput screening.
- Ensures assay readiness and platform scalability for compound evaluation in heterogeneous tumor models.
Translational & Preclinical Research
- Aligns preclinical tumor burden assessment with human disease-relevant imaging endpoints.
- Enables stratification of non-muscle invasive versus muscle invasive disease for translational biomarker studies.
- Supports continuity from discovery through preclinical validation by linking imaging and pathological data.
Pipeline & Workflow Integration
This MRI-based assessment integrates at the pre-randomization stage, bridging early discovery, lead identification, and preclinical validation in immuno-oncology pipelines.
- Discovery Biology: Quantitative imaging supports null hypothesis testing and mechanistic de-risking in murine models.
- Screening: Operator-independent MRI outputs enable reproducible cohort selection and assay standardization.
- Analytics: Provides quantitative area and thickness measurements correlated with ex vivo bladder weight and tumor stage.
- Translational Research: Imaging endpoints facilitate alignment with clinical staging and biomarker strategies.
- Enterprise Reuse: The protocol is adaptable for repeated use across diverse bladder cancer research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in tumor model evaluation.
- Operational Value: Delivers standardized, scalable, and reproducible imaging workflows for high-throughput studies.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by enabling robust cohort stratification.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of immuno-oncology assets.
Implementation Considerations
- Requires expertise in small animal MRI operation and image analysis.
- Needs access to high-resolution MRI instrumentation and compatible analysis software.
- Demands cross-team standardization for imaging acquisition and data interpretation.
- Adaptable to other carcinogen-induced or genetically engineered murine models with protocol optimization.
- Practical limitations include the need for full bladder imaging and adherence to animal welfare protocols.
