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 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.</li>
	<li>Change the 0.05% BBN water twice per week.</li>
	<li>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.</li>
	<li>Expect the tumors to develop between 16 and 24 weeks of exposure18.</li>
</ol>
2. MRI setup
<ol>
	<li>Perform a subcutaneous injection of sterile saline (0.1&#8211;0.2 mL using a 25&#8211;27 G needle and 1 mL syringe) 10 min prior to MRI to facilitate bladder filling.</li>
	<li>Anesthetize each mouse with a gas mixture of 100% O2 and isoflurane (2%&#8211;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.&#160;</li>
	<li>Transfer the mouse to the imaging holder outfitted with a nosecone for delivery of inhaled isoflurane (0.5%&#8211;3%).</li>
	<li>Monitor body temperature and respiration using a rectal temperature probe connected to the physiological recording computer. 
	NOTE: Normal body temperature (36&#8211;37 &#176;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.</li>
</ol>
3. MRI image acquisition
<ol>
	<li>Utilize a quadrature body coil for excitation.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Acquire three sets of orthogonal-sliced images along the axial, coronal, and sagittal planes using radiological frames of reference.</li>
	<li>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 &#60;10 min per mouse.</li>
	<li>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.</li>
</ol>
4. MR image analysis
<ol>
	<li>Identify the set of slices of 0.5 mm thickness and in-plane resolution of 0.148 mm covering the whole bladder.</li>
	<li>Export to the medical image analysis software by selecting the folder with corresponding images in ANALYZE format.</li>
	<li>Select &#8220;representative axial view&#8221; 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.</li>
	<li>Carefully delineate the region of interest (ROI) by manually tracing the boundaries around the outer edge of the bladder (BLAout) and around the inner lumen (BLAin) of the bladder (see schematic and representative figures in Figure 2) in the selected representative axial view.</li>
	<li>Subtract the inner lumen from the outer edge to calculate the surface area of the bladder wall. 
	BLAwall = BLAout - BLAin 
	NOTE: The surface area of a control bladder with no tumor is expected to be less than that with a bladder tumor.</li>
</ol>
5. Euthanasia and dissection of bladder
<ol>
	<li>After 20 weeks of BBN exposure, euthanize the mice using standard operating procedures in accordance with local IACUC guidelines.</li>
	<li>Clean the area of incision with 70% ethanol, then grasp and lift the abdominal wall skin with forceps.</li>
	<li>Make a midline incision from the pubic symphysis to the xiphoid process.</li>
	<li>Sharply incise the peritoneal cavity by grasping with forceps and incising with scissors.</li>
	<li>Identify the bladder, which is located in the midline lower abdomen.</li>
	<li>Identify and cut the median umbilical ligament connecting the dome of the bladder to the umbilicus and abdominal wall.</li>
	<li>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.</li>
	<li>Identify the ureters entering the bladder and cut with scissors close to the bladder.</li>
	<li>Lifting the bladder cephalad, cut the urethra with scissors and remove the bladder.</li>
	<li>Immediately weigh the bladder after rinsing it with PBS.</li>
</ol>
6. Histologic examination of bladder tissue
<ol>
	<li>Fix the bladder tissue in 10% neutral buffered formalin for 36&#8211;48 h at room temperature (RT).</li>
	<li>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 previously19,20.</li>
	<li>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 neoplasms19,21.</li>
</ol>

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<li>Nagao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutagenicity+of+N-butyl-N-%284-hydroxybutyl%29nitrosamine%2C+a+bladder+carcinogen%2C+and+related+compounds%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Mutagenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine, a bladder carcinogen, and related compounds.</a> Cancer Research. 37, 399-407 (1977).</li>
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<li>Shin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+origin+of+bladder+neoplasia+and+tissue+dynamics+of+its+progression+to+invasive+carcinoma%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma.</a> Nature Cell Biology. 16 (5), 469-478 (2014).</li>
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</ol>

The authors have nothing to disclose.

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 &amp;amp; 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...

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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) ...

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JoVE Journal

Cancer Research

10.3K Views.  NorthShore University HealthSystem. Deze methode kan worden gebruikt om blaastumoren reproduceerbaar te genereren met behulp van de BBN-kankerverwekkende stof en om de grootte en uitbreiding van de blaastumorlast door MRI in beeld te brengen. Deze techniek maakt gebruik van een operator onafhankelijke snelle beeldvorming aanpak met korte acquisitie tijden op een hoge diagnostische kwaliteit om high-throughput testen van muizen uit te voeren in tussenliggende stadia van tumorontwikkeling. Om blaaskanker tumor vorming induceren, ...

Video: Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

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 ...

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Medicine

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

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