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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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=Atezolizumab+in+patients+with+locally+advanced+and+metastatic+urothelial+carcinoma+who+have+progressed+following+treatment+with+platinum-based+chemotherapy:+a+single-arm,+multicentre,+phase+2+trial.">Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.</a> The Lancet. 387 (10031), 1909-1920 (2016).</li><li>Sharma, P., 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=Nivolumab+monotherapy+in+recurrent+metastatic+urothelial+carcinoma+(CheckMate+032):+a+multicentre,+open-label,+two-stage,+multi-arm,+phase+1/2+trial.">Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial.</a> The Lancet Oncology. 17 (11), 1590-1598 (2016).</li><li>Bellmunt, 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=Pembrolizumab+as+Second-Line+Therapy+for+Advanced+Urothelial+Carcinoma.">Pembrolizumab as Second-Line Therapy for Advanced Urothelial Carcinoma.</a> New England Journal of Medicine. 376 (11), 1015-1026 (2017).</li><li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+orthotopic+models+for+bladder+cancer+research.">Mouse orthotopic models for bladder cancer research.</a> BJU International. 104 (9), 1286-1291 (2009).</li><li>Zhang, N., Li, D., Shao, J., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+bladder+cancer:+The+model+establishment+and+evaluation+(Review).">Animal models for bladder cancer: The model establishment and evaluation (Review).</a> Oncology Letters. 9 (4), 1515-1519 (2015).</li><li>Patel, A. 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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. 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=Biodistribution+of+gold+nanoparticles+in+BBN-induced+muscle-invasive+bladder+cancer+in+mice.">Biodistribution of gold nanoparticles in BBN-induced muscle-invasive bladder cancer in mice.</a> International Journal of Nanomedicine. 12, 7937-7946 (2017).</li><li>Dai, Y. C., 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=The+interaction+of+arsenic+and+N-butyl-N-(4-hydroxybutyl)nitrosamine+on+urothelial+carcinogenesis+in+mice.">The interaction of arsenic and N-butyl-N-(4-hydroxybutyl)nitrosamine on urothelial carcinogenesis in mice.</a> PLoS One. 12 (10), e0186214 (2017).</li><li>Williams, P. D., Lee, J. K., Theodorescu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Credentialing+of+Rodent+Bladder+Carcinogenesis+Models.">Molecular Credentialing of Rodent Bladder Carcinogenesis Models.</a> Neoplasia. 10 (8), (2008).</li><li>Fantini, D., 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=A+Carcinogen-induced+mouse+model+recapitulates+the+molecular+alterations+of+human+muscle+invasive+bladder+cancer.">A Carcinogen-induced mouse model recapitulates the molecular alterations of human muscle invasive bladder cancer.</a> Oncogene. 37 (14), 1911-1925 (2018).</li><li>. 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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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Cancer Research

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

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 multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

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 tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

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 particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

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

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

Patient-derived organoids (PDOs) are a powerful tool in translational cancer research, reflecting both the genetic and phenotypic heterogeneity of the disease and response to personalized anti-cancer therapies. Here, a consolidated protocol to generate human primary bladder cancer PDOs in preparation for the evaluation of phenotypic analyses and drug responses is detailed.