Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Summary

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.

Abstract

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 (r_s= 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.

Introduction

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 carcinoma^3,4,5. 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 treatments^6,7. 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 models^8,9,10,11. 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 analysis^9,12.

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.

Protocol

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

1. Obtain male C57/BL6 mice, each at least 6 weeks old.
   NOTE: Male mice develop bladder cancer more quickly and consistently than female mice^14,15.
2. Add N-nitrosobutyl(4-hydroxybutyl) amine (BBN) at a dose of 0.05% to the drinking water. Store it in an opaque container and provide it ad libitum as drinking water to mice¹⁶.
   NOTE: Storing the BBN solution in a clear container will degrade the carcinogen¹⁷.
3. Change the 0.05% BBN water twice per week.
4. Monitor the animals by inspecting for signs of distress associated with bladder tumors including hematuria, firm bladder, and masses. Inspect the mice twice per week or in accordance with local IACUC guidelines.
5. Expect the tumors to develop between 16 and 24 weeks of exposure¹⁸.

2. MRI setup

1. Perform a subcutaneous injection of sterile saline (0.1–0.2 mL using a 25–27 G needle and 1 mL syringe) 10 min prior to MRI to facilitate bladder filling.
2. Anesthetize each mouse with a gas mixture of 100% O₂ and isoflurane (2%–4% as necessary). Verify an adequate plane of anesthesia by testing the withdrawal reflex (toe pinch) before proceeding. Apply sterile eye ointment to the animal. 
3. Transfer the mouse to the imaging holder outfitted with a nosecone for delivery of inhaled isoflurane (0.5%–3%).
4. Monitor body temperature and respiration using a rectal temperature probe connected to the physiological recording computer.
   NOTE: Normal body temperature (36–37 °C) is maintained using the recirculating hot water circuit built into the animal MR holder. Temperature is measured through a rectal sensor and recorded on the physiological monitoring computer using dedicated physiological monitoring software. The same system is used to record the respiration and electrocardiogram signals measured through a pneumatic pillow placed under the rib cage and via 3-lead electrocardiogram electrodes. The respiration signal is also used for triggering MRI acquisition and reducing artifacts associated with respiration motion.

3. MRI image acquisition

1. Utilize a quadrature body coil for excitation.
2. Place a 4-channel receiver coil on the lower abdomen of the mouse being scanned to enable optimized detection of signals from the region of interest.
3. Initiate automatic adjustments through the integrated imaging software to acquire a tri-axial set of images of the whole mouse body. From this reference set of images, identify the region of interest (in this case, the bladder region).
4. Acquire three sets of orthogonal-sliced images along the axial, coronal, and sagittal planes using radiological frames of reference.
5. Utilize the true FISP imaging sequence (included as one of the features in the integrated imaging software) with the following MR parameters: TR = 900 msec, TE = 2 ms, FA = 70, 14 averages.
   NOTE: This set of parameters allows for rapid imaging with high diagnostic quality, including T1/T2 weighting in <10 min per mouse.
6. Spatial resolution and slice thickness are determined by geometric parameters selected by the user through the graphical interface of the integrated imaging platform. This results in a series of slices across the whole bladder of 0.5 mm thickness with an in-plane resolution of 0.148 mm.

4. MR image analysis

1. Identify the set of slices of 0.5 mm thickness and in-plane resolution of 0.148 mm covering the whole bladder.
2. Export to the medical image analysis software by selecting the folder with corresponding images in ANALYZE format.
3. Select “representative axial view” at the center of the bladder for quantitative analysis by scrolling through the generated images and identifying a slice at the midpoint of the bladder, which allows for visualization of the bladder wall and lumen.
   NOTE: The center slice should be the chosen one with the largest diameter.
4. Carefully delineate the region of interest (ROI) by manually tracing the boundaries around the outer edge of the bladder (BLA_out) and around the inner lumen (BLA_in) of the bladder (see schematic and representative figures in Figure 2) in the selected representative axial view.
5. Subtract the inner lumen from the outer edge to calculate the surface area of the bladder wall.
   BLA_wall = BLA_out - BLA_in
   NOTE: The surface area of a control bladder with no tumor is expected to be less than that with a bladder tumor.

5. Euthanasia and dissection of bladder

1. After 20 weeks of BBN exposure, euthanize the mice using standard operating procedures in accordance with local IACUC guidelines.
2. Clean the area of incision with 70% ethanol, then grasp and lift the abdominal wall skin with forceps.
3. Make a midline incision from the pubic symphysis to the xiphoid process.
4. Sharply incise the peritoneal cavity by grasping with forceps and incising with scissors.
5. Identify the bladder, which is located in the midline lower abdomen.
6. Identify and cut the median umbilical ligament connecting the dome of the bladder to the umbilicus and abdominal wall.
7. Grasp the dome of the bladder with forceps to provide countertraction and dissect the bladder away from surrounding structures, including the seminal vesicles, rectum, and fat.
8. Identify the ureters entering the bladder and cut with scissors close to the bladder.
9. Lifting the bladder cephalad, cut the urethra with scissors and remove the bladder.
10. Immediately weigh the bladder after rinsing it with PBS.

6. Histologic examination of bladder tissue

1. Fix the bladder tissue in 10% neutral buffered formalin for 36–48 h at room temperature (RT).
2. Embed the tissue in paraffin blocks, cut the slides for subsequent examination, and stain the slides with hematoxylin and eosin for microscopic examination as described previously^19,20.
3. Perform a microscopic examination of the mouse bladder at low (2.5x and 10x) and high (20x and 40x) magnifications, examining for macroscopic lesions, hyperplasia, carcinoma in situ, papillomas, papillary tumors, and invasive neoplasms^19,21.

Results

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...

Discussion

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 (BLA_wall) correlates significantly with ex vivo bladder weight and is associated with pathologic tumor stage.

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Materials

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