Name: magnetic resonance imaging assessment of carcinogen-induced murine bladder tumors
Uploaded: 2019-03-29T13:00:00
Description: Watch this Scientific Journal Video about Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors at JoVE.com

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.

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
<ol>
	<li>Obtain male C57/BL6 mice, each at least 6 weeks old. 
	NOTE: Male mice develop bladder cancer more quickly and consistently than female mice14,15.</li>
	<li>Add N-nitrosobutyl(4-hydroxybutyl) amine (BBN) at a dose of 0.05% to the drinking water. Store it in...

<ol>
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S., Karlik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+for+detecting+and+treatment+monitoring+of+orthotopic+murine+bladder+tumor+implants.">Magnetic resonance imaging for detecting and treatment monitoring of orthotopic murine bladder tumor implants.</a> The Journal of Urology. 145 (6), 1297-1301 (1991).</li><li>Jurczok, A., Fornara, P., Soling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+to+monitor+bladder+cancer+cell+adhesion+in+vivo:+a+new+approach+to+optimize+a+syngeneic,+orthotopic,+murine+bladder+cancer+model.">Bioluminescence imaging to monitor bladder cancer cell adhesion in vivo: a new approach to optimize a syngeneic, orthotopic, murine bladder cancer model.</a> BJU International. 101 (1), 120-124 (2008).</li><li>Vandeveer, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+Immunotherapy+of+Non-Muscle+Invasive+Mouse+Bladder+Cancer+with+Avelumab,+an+Anti-PD-L1+Immune+Checkpoint+Inhibitor.">Systemic Immunotherapy of Non-Muscle Invasive Mouse Bladder Cancer with Avelumab, an Anti-PD-L1 Immune Checkpoint Inhibitor.</a> Cancer Immunology Research. 4 (5), 452-462 (2016).</li><li>Kikuchi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantitative+analysis+of+early+stage+orthotopic+murine+bladder+tumor+using+in+vivo+magnetic+resonance+imaging.">Detection and quantitative analysis of early stage orthotopic murine bladder tumor using in vivo magnetic resonance imaging.</a> Journal of Urology. 170, 1375-1378 (2003).</li><li>Chung, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T2-weighted+fast+MR+imaging+with+true+FISP+versus+HASTE:+comparative+efficacy+in+the+evaluation+of+normal+fetal+brain+maturation.">T2-weighted fast MR imaging with true FISP versus HASTE: comparative efficacy in the evaluation of normal fetal brain maturation.</a> American Journal of Roentgenology. 175 (5), 1375-1380 (2000).</li><li>Miyamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+bladder+cancer+development+and+progression+by+androgen+receptor+signals.">Promotion of bladder cancer development and progression by androgen receptor signals.</a> Journal of the National Cancer Institute. 99 (7), 558-568 (2007).</li><li>Bertram, J. S., Craig, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+induction+of+bladder+cancer+in+mice+by+butyl-(4-hydroxybutyl)-nitrosamine+and+the+effects+of+hormonal+modifications+on+the+sex+difference+in+response.">Specific induction of bladder cancer in mice by butyl-(4-hydroxybutyl)-nitrosamine and the effects of hormonal modifications on the sex difference in response.</a> European Journal of Cancer. 8 (6), 587-594 (1972).</li><li>Nagao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenicity+of+N-butyl-N-(4-hydroxybutyl)nitrosamine,+a+bladder+carcinogen,+and+related+compounds.">Mutagenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine, a bladder carcinogen, and related compounds.</a> Cancer Research. 37, 399-407 (1977).</li><li>Hirose, M., Fukushima, S., Hananouchi, M., Shirai, T., Ogiso, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+susceptibilities+of+the+urinary+bladder+epithelium+of+animal+species+to+three+nitroso+compounds.">Different susceptibilities of the urinary bladder epithelium of animal species to three nitroso compounds.</a> Gan. Gann; The Japanese Journal of Cancer Research. 67 (2), 175-189 (1976).</li><li>Shin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+origin+of+bladder+neoplasia+and+tissue+dynamics+of+its+progression+to+invasive+carcinoma.">Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma.</a> Nature Cell Biology. 16 (5), 469-478 (2014).</li><li>Epstein, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17:+Immunohistology+of+the+Bladder,+Kidney,+and+Testis.">Chapter 17: Immunohistology of the Bladder, Kidney, and Testis.</a> Diagnostic Immunohistochemistry. , 624-661 (2019).</li><li>Cohen, S. M., Ohnishi, T., Clark, N. M., He, J., Arnold, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+of+rodent+urinary+bladder+carcinogens:+collection,+processing,+and+evaluation+of+urine+and+bladders.">Investigations of rodent urinary bladder carcinogens: collection, processing, and evaluation of urine and bladders.</a> Toxicologic Pathology. 35 (3), 337-347 (2007).</li><li>Wood, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+of+the+bladder.">Tumors of the bladder.</a> Campbell-Walsh Urology. 11 (92), 2184-2204 (2016).</li><li>Zitvogel, L., Pitt, J. M., Daillere, R., Smyth, M. J., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+in+oncoimmunology.">Mouse models in oncoimmunology.</a> Nature Reviews Cancer. , (2016).</li><li>Kaneko, S., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+protects+against+bladder+cancer+in+females+via+a+KDM6A-dependent+epigenetic+mechanism.">X chromosome protects against bladder cancer in females via a KDM6A-dependent epigenetic mechanism.</a> Science Advances. 4 (6), eaar5598 (2018).</li><li>Smilowitz, H. 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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.
...

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.

<table>
	<tbody>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td>664</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>N-butyl-N-(4-hydroxybutyl)nitrosamine carcinogen (BBN)</td>
			<td>TCI American</td>
			<td>B0938</td>
			<td>Carcinogen</td>
		</tr>
		<tr>
			<td>0.9% normal saline</td>
			<td>Hospira, Inc</td>
			<td>NDC 0409-488-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal HealthCare</td>
			<td>60307-120-25</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>7Tesla ClinScan MRI</td>
			<td>Bruker</td>
			<td>NA</td>
			<td>Dedicated Small Animal Imaging MRI</td>
		</tr>
		<tr>
			<td>Syngo</td>
			<td>Siemens</td>
			<td>NA</td>
			<td>MR Integrated Imaging Software</td>
		</tr>
		<tr>
			<td>Model 1030 Monitoring &#38; Gating System</td>
			<td>Small Animal Instruments, Inc. (SAII)</td>
			<td>NA</td>
			<td>Small animal physiologic monitoring</td>
		</tr>
		<tr>
			<td>Formalin, Neutral Buffered, 10%</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Fisher Scientific</td>
			<td>NC1093844</td>
			<td>Histologic staining agent</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Fisher Scientific</td>
			<td>23-245651</td>
			<td>Histologic staining agent</td>
		</tr>
		<tr>
			<td>Jim7</td>
			<td>Xinapse Systems</td>
			<td>NA</td>
			<td>Medical image analysis software</td>
		</tr>
		<tr>
			<td>GraphPad Prism v7.04</td>
			<td>Graphpad</td>
			<td>NA</td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>R v3.4.2</td>
			<td>The R Project for Statistical Computing</td>
			<td>NA</td>
			<td>Statistical software</td>
		</tr>
		<tr>
			<td>R package pROC v1.10.0.</td>
			<td>The R Project for Statistical Computing</td>
			<td>NA</td>
			<td>ROC analysis</td>
		</tr>
	</tbody>
</table>

magnetic resonance imaging assessment of carcinogen-induced murine bladder tumors

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.

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...

Watch this Scientific Journal Video about Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors at JoVE.com

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

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) ...

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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Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...