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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.
Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.
Bladder cancer is the fifth most common cancer overall, responsible for approximately 80,000 new cases and 16,000 deaths in the United States in 20171. After about 30 years without significant advances in the systemic treatment of bladder cancer2, recent anti-PD-1 and anti-PD-L1 checkpoint inhibitor trials have demonstrated exciting and occasionally durable responses in patients with advanced urothelial carcinoma3,4,5. However, only approximately 20% of patients show an objective response to these treatments, and further studies are needed to expand the effective use of immunotherapy in patients with bladder cancer.
Murine bladder cancer models are critical tools in preclinical evaluation of novel treatments6,7. In order to control for tumor size when randomizing mice to different treatments, tumor burden must be assessed and controlled between treatment groups. Previous studies have used ultrasound or bioluminescence to evaluate orthotopic cell line-based bladder cancer models8,9,10,11. However, both techniques present several disadvantages. Ultrasound measurements can be influenced by skills of the operator and lack three-dimensional features and high spatial resolution. Bioluminescence methods can only provide semi-quantitative evaluation of the tumor cells and do not allow for visualization of bladder anatomy and morphology. Furthermore, bioluminescence can only be used with cell line-based models, which express bioluminescent genes in hairless mice or mice with white coats.
Magnetic resonance imaging (MRI), on the other hand, offers unique flexibility in the acquisition of high-resolution anatomical images, exhibiting a broad range of tissue contrast that enables accurate visualization and quantitative assessment of tumor burden without the need to express bioluminescent properties. MR images are more easily reproducible with the appropriate analysis pipelines and guaranteed 3-D visualization of the bladder. The biggest limitations of MRI are the length of time necessary for an examination and associated high costs that limit high throughput assays. However, several studies have shown that MR sequences can provide high-quality diagnostic images that can be used to effectively detect and monitor cell line-based bladder tumors; thus, they may be used for high throughput analysis9,12.
Here, we describe a non-invasive MR-based method to reliably and efficiently characterize carcinogen-induced bladder tumors in mice. To accomplish this, we use a fast imaging with steady state precession MR technique (true FISP), which guarantees short scanning sessions while still providing high quality and high spatial resolution (~100 microns) for the detection and measurement of bladder tumors13. Furthermore, to confirm the accuracy of this non-invasive MRI assay, we describe the correlation between MRI-derived parameters and ex vivo bladder weight as well as pathologically-confirmed tumor stage.
All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Northwestern University.
1. Induction of tumors with BBN
2. MRI setup
3. MRI image acquisition
4. MR image analysis
5. Euthanasia and dissection of bladder
6. Histologic examination of bladder tissue
Using the protocol described (Figure 1), bladder tumors were induced in C57/B6 male mice. MRI was performed at 16 weeks, and mice were euthanized at 20 weeks. Ex vivo bladder weights (BW) for each mouse were recorded. Slides were stained with hematoxylin and eosin, and all histology slides were reviewed for tumor stage.
To analyze the tumor burden using MR, the bladder wall inner lumen (BLA...
Accurate imaging of tumor models is necessary for appropriate pre-euthanasia staging and animal randomization prior to initiation of experimental treatment. Using the procedure presented here, we demonstrate methodology to (1) generate bladder tumors using the BBN carcinogen and (2) stratify bladder tumor burden through the use of MR. An MR-derived area measurement (BLAwall) correlates significantly with ex vivo bladder weight and is associated with pathologic tumor stage.
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The authors have nothing to disclose.
J. J. M. is funded by the Veterans Health Administration Merit grant BX0033692-01. J. J. M. is also supported by the John P. Hanson Foundation for Cancer Research at the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. We thank the Center for Translational Imaging for providing the MRI acquisition and processing. Funding sources had no role in writing of the manuscript or the decision to submit for publication.
Name | Company | Catalog Number | Comments |
C57BL/6 mice | The Jackson Laboratory | 664 | Mice |
N-butyl-N-(4-hydroxybutyl)nitrosamine carcinogen (BBN) | TCI American | B0938 | Carcinogen |
0.9% normal saline | Hospira, Inc | NDC 0409-488-02 | |
Isoflurane | Piramal HealthCare | 60307-120-25 | Anesthetic |
7Tesla ClinScan MRI | Bruker | NA | Dedicated Small Animal Imaging MRI |
Syngo | Siemens | NA | MR Integrated Imaging Software |
Model 1030 Monitoring & Gating System | Small Animal Instruments, Inc. (SAII) | NA | Small animal physiologic monitoring |
Formalin, Neutral Buffered, 10% | Sigma | HT501128 | Fixative |
Eosin Y | Fisher Scientific | NC1093844 | Histologic staining agent |
Hematoxylin | Fisher Scientific | 23-245651 | Histologic staining agent |
Jim7 | Xinapse Systems | NA | Medical image analysis software |
GraphPad Prism v7.04 | Graphpad | NA | Graphing software |
R v3.4.2 | The R Project for Statistical Computing | NA | Statistical software |
R package pROC v1.10.0. | The R Project for Statistical Computing | NA | ROC analysis |
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