Name: An Orthotopic Model of Murine Bladder Cancer (Video) | JoVE Journal
Uploaded: 2011-02-06T17:00:00.000+00:00
Duration: 9 min 7 s
Description: Learn to establish an orthotopic bladder cancer model in female C57BL/6 mice using catheterization, electrocautery, and tumor cell adhesion. This method enables reliable in vivo oncology research.

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5 minutes

This work was funded by the National Science Foundation CAREER award (CBET-0846395) and the Department of Comparative Medicine, Tulane University.

1. Animals
<ol>
	<li>Female C57BL/6J mice (Mus musculus) are verified free of contagious ectoparasites, helminth endoparasites, and antibodies to 17 murine viruses by the vendor prior to shipping. Mice are housed in the AAALAC-accredited animal facility of Tulane University in accordance with Guide for the Care and Use of Laboratory Animals.1 </li>
	<li>Specifically, mice are group housed (4-5 mice/cage) in individually ventilated polycarbonate cageswith microisolation filter tops on hardwood bedding. They are provided ad libitum pelleted rodent food and tap water.</li>
	<li>Mice are allowed 7 days to acclimate before the start of the study.</li>
</ol>
2. Cells
<ol>
	<li>The murine transitional cell carcinoma cell line MB49 is used to produce the bladder tumors.</li>
	<li>Cells are cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin.</li>
	<li>Cultures are maintained at 37&deg;C and 5% CO2 with media changes every two to three days.</li>
</ol>
3. Animal Orthotopic Tumor Model
The instillation of the bladder tumor model described here is a modification of the procedure described by Gunther et al.2
<ol>
	<li>Anesthesia is induced with 2% isoflurane (MWI/VetOne, Meridian, ID, Cat# 501017) in oxygen and maintained at 0.75-1.25%. (Multiple animals can be treated concurrently, which would necessitate an increase in isoflurane levels.) To prevent corneal drying, eyes are lubricated with an artificial tears ophthalmic ointment. Body heat is maintained with a heated pad under a paper drape.</li>
	<li>The fur of the lower back is clipped to promote contact with the metal grounding plate, and the shaved area is coated with electroconduction gel.</li>
	<li>Each mouse is then individually placed in dorsal recumbancy onto a metal grounding plate, and the bladder is catheterized transurethrally using a lubricated 24-gauge, 3/4 inch, over-the-needle IV catheter with the stylet removed.</li>
	<li>The urinary bladder is drained of urine, and a platinum wire is inserted through the catheter into the urinary bladder leaving exposed wire on each end of the catheter - approximately 2 mm on the lumenal end of the catheter. The wire is made of platinum because of the resistance of the material to oxidation.</li>
	<li>A 2.5 W current is then applied to the bladder for approximately 0.5 second by touching the exterior portion of the wire with an electrocautery unit set to coagulation.</li>
	<li>The wire is then moved to a second location in the bladder and another 2.5 W charge applied.</li>
	<li>The wire is removed, and 100 &mu;l of MB49 cells at a concentration of 1x106 cells/ml is instilled through the catheter into the bladder via a 1ml syringe.</li>
	<li>The syringes are left attached to the catheters to prevent efflux of the cell suspensions, and the catheterized mice are maintained under anesthesia for 90 minutes to allow the cells to attach to the burn sites.</li>
	<li>100-200 &mu;l of sterile normal saline is administered by IP injection to each mouse prior to recovery.</li>
	<li>At the end of the incubation period the catheters are removed and the animals are allowed to recover on the heated pad until righting reflexes return, at which point the mice are returned to their home cages.</li>
</ol>
4. Ultrasound
<ol>
	<li>Tumors are allowed to grow for 3-5 days before the mice undergo ultrasound examination of the urinary bladder to monitor tumor growth.</li>
	<li>For the initial ultrasound examinations, animals are anesthetized as described above. Mice are placed in dorsal recumbancy, and abdominal skin is prepared by clipping the fur.</li>
	<li>A 24 gauge IV catheter is inserted transurethrally into the urinary bladder. At this point, the bladder can be drained and the urine collected for further analysis.</li>
	<li>100 &mu;l of sterile normal saline is instilled into the bladder through the catheter to improve bladder visibility on ultrasound.</li>
	<li>Ultrasound conduction gel is applied to the abdomen, and ultrasonography is performed to assess the bladder wall and lumen.</li>
</ol>
5. Representative Results:
Tumor tissue will begin to appear 3-5 days after the innoculation. In our experience, in a group of 12 mice, 50% or more routinely have a detectable tissue mass within the bladder (0.5 - 1.0 &mu;l, using V=&pi;/6&middot;L&middot;W&middot;H) by day five. When using MB 49 cells, tumors will continue to grow and euthanasia will be required by day 24 if the mice go untreated. Euthanasia is recommended when mice become lethargic or cachectic, display ruffled fur, hunching, and non-responsive behavior, or when they lose 20% of their initial body weight.
The tumors grow quickly and will have necrotic centers after they reach a mass of 200 mg or more,3 ostensibly because the cells in the developing tumor proliferate faster than what can be serviced by the angiogenic blood supply. One can also indirectly monitor tumor progression via the level of hematurea, which is presumably related to rapid angiogenesis within developing tumors. It was consistently noted that the urine of untreated tumor-bearing mice contained blood by day 14. The concentration of red blood cells appeared to increase over time in these mice, and the volumes of urine collected diminished. Tumors in excess of 50 mg can also be palpated with relative ease, typically by 14 days post innoculation.

<ol><li>Institute of Laboratory Animal Resources. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Guide+for+the+Care+and+Use+of+Laboratory+Animals%22">Guide for the Care and Use of Laboratory Animals</a>. , National Academies Press. Washington, DC. (1996).</li>
<li>Gunther, J. H., Jurczok, A., Wulf, T., Brandau, S., Deinert, I., Jocham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+syngeneic+orthotopic+murine+bladder+cancer+%28MB49%29%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59, 2834-2837 (1999).</li>
<li>Zhang, X., Atala, A., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression-targeted+gene+therapy+for+the+treatment+of+transitional+cell+carcinoma%5BTitle%5D)+AND+%22Cancer+Gene+Ther%22%5BJournal%5D)">Expression-targeted gene therapy for the treatment of transitional cell carcinoma.</a> Cancer Gene Ther. 15, 543-552 (2008).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Care and Use Committee of Tulane University.

This minimally invasive method of establishing orthotopic bladder tumors in female mice can also be applied for cell types other than MB49, although differing results should be anticipated. When less-aggressive tumor cells are used, increasing the wattage slightly (using 0.5 W increments to establish the new optimum) will yield an area more amenable to tumor cell attachment, and longer bladder incubation times for the instilled cells will yield a greater amount of cell attachment at the burn sites (unpublished data). A...

<table><tbody><tr><td>C57Bl/6J mice</td><td>Jackson Laboratory</td><td>000664</td><td>3-5-wk-old</td></tr><tr><td>Polycarbonate cages and ventilated rack</td><td>Lab Products</td><td>Super Mouse 750</td><td>327mm x&lt;br/&gt;190mm x&lt;br/&gt;143mm</td></tr><tr><td>Hardwood Bedding</td><td>Harlan Laboratories</td><td>Laboratory &amp;#8211;grade SaniChips</td><td></td></tr><tr><td>Rodent diet</td><td>Harlan Laboratories</td><td>Rodent Diet 2019</td><td></td></tr><tr><td>IV Catheter</td><td>MWI Veterinary Supply</td><td>009231</td><td>24 G x &amp;#190; in. </td></tr><tr><td>Electrocautery unit</td><td>Bovie Medical Corporation</td><td>Aaron 900</td><td></td></tr><tr><td>Ultrasound unit</td><td>Sonosite</td><td>180 Plus (Vet)</td><td></td></tr><tr><td>MB49 cell line</td><td>Wake Forest University Baptist Medical Center</td><td>N.A.</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>21063-029</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini Bio Products</td><td>100106 500A57B</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15140-163</td><td></td></tr></tbody></table>

an orthotopic model of murine bladder cancer

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 ...

This is Professor WT Godbe and along with Dr.Georgina Dobe, we'd like to present a method for establishing an orthotopic model of mure bladder carcinoma. Let us begin with the steps that we take to prepare our animals for this procedure. The mice that we're showing today are female C 57 blacks purchased from the Jackson Laboratories.We chose female because of the ease of catheterization. These miser between three and five weeks of age, and they typically weigh in the range of 16 to 20 grams, the miser and ize using a ventilator that's set to deliver isof fluorine at 2%in oxygen at a flow rate of 0.8 to one liter per minute. Respiration rates are constantly monitored to prevent corneal drying.During this procedure that takes in excess of one and a half hours, the eyes are lubricated with an artificial tears ophthalmic ointment that's applied directly over the eyes and then rolled down into the eyes the fur, the lower back is clipped to promote contact with the metal grounding plate, which will be used during the electrocautery procedure that will produce a burn injury within the bladder. The burn sites themselves will serve as adhesion points for the delivered tumor cells after they're introduced into the bladder lumen. The remainder of the techniques used in this protocol rely heavily on catheterization for which we use 24 gauge IV catheters.With the stylus safely removed and discarded, the sheaths of the catheters are lubricated with the substance such as KY jelly or astroglide to help produce or prevent trauma to the urethra. For catheterization, the vulva is held steady with forceps. While the catheter is inserted in, the urethra care has to be taken for the catheterization techniques, so this will be shown in detail.We recommend the use of surgical loops to help make the field more clear. Here we see the external pelvic anatomy of the mouse. We're now indicating the vulva which fold around the external urethral opening.The tendency for catheterization is to try to insert the catheter into the middle of the structure, but the urethral opening is at the bottom. We have found it easier to grasp the ulfa with forceps than to gently roll the structure out and up to make the urethra more easily accessible. Take care not to catheterize the vagina, which is immediately below the urethral opening.Another parameter to be aware of is the angle of catheterization. Now the natural tendency is to go in straight parallel to the longitudinal axis of the animal. However, a steeper angle makes insertion of the catheter a little easier after it's initially inserted.You can then reduce the angle of catheterization to help prevent trauma to the animal. Here, Dr.Beck is coming in from a steep angle. Notice the catheter will be inserted at the bottom of the vulva.Twisting of the catheter sometimes helps to lubricate the urethra and aid with insertion, and after it is initially inserted, the angle is made less steep and we can insert directly into the bladder. Notice how long it takes a skilled user. However, not every insertion is going to take that long.Here's how long the procedure takes in the best case scenario. Now that we're able to efficiently catheterize a mouse, let's return to the tumor cell installation protocol. The next step is to drain the bladder of most of the urine, and we do so by applying general pressure to the pelvic area where the urine is gonna be forced out through the catheter and the end of the catheter serves as a collection area.The catheter will then be removed and drained. Next, we prepare a platinum wire, which will be used to conduct a current into the bladder to induce the burden injury. And notice that this platinum wire has a hook on the end, and that's for measurement purposes.Following insertion completely through the catheter, we want two to three millimeters of wire exposed on the internal side of the catheter. It is this exposed portion that will come into contact with the bladder wall and deliver the burn injury. After catheterization of the mouse, we will separately insert the wire.The catheter is pulled out slightly to allow extra space for the wire to protrude from the end of the catheter without piercing the bladder. We deliver the current via an electrocautery unit. We use a Bovie electrocautery unit set for 2.5 watts and coagulation, and we deliver the current via tapping.The exterior portion of the wire. Electro electric conduction gel has been placed between the mouse and the grounding plate. This is an example of the amount of time we actually spend delivering the current.It's less than 0.5 seconds. Because of the short duration, we will deliver a second burn injury. We repositioned the wire in the catheter and then tap the wire again.Following installation of the burn injury, the M will immediately receive the tumor cells and this will be performed through yet another catheterization. Except this time the catheter will be attached to a syringe that's loaded with a hundred microliters of cell suspension. The cells that we'll be using here are MB 49 cells at a concentration of 1 million cells per milliliter for a total of 100, 000 cells to be delivered.We pull up an excess of 100 microliters and gauge the amount that we deliver through the marks on the side of the syringe. Following loading the syringe with the cells, we lubricate the catheter and re catheterize the animal. As you can see, not every catheterization will go as smoothly as the optimal conditions we showed before.Care must be taken not to injure the animal. Following successful catheterization of the mouse, a hundred microliters of the cell suspension will be delivered through the syringe, through the catheter, and into the lumen of the bladder. The entire unit will be kept in place for 90 minutes during which time the isof fluorine administration rate will be reduced to 1%as a maintenance dose.Tumor cells are allowed to develop into tumors for three to five days before we commence ultrasound examinations To monitor tumor cell growth, the fur on the abdomen is clipped to improve contact between the ultrasound probe and the subject. At this point, the mice will be catheterized and a hundred microliters of sterile normal saline will be instilled into the bladders to improve bladder visibility and tumor visualization. Upon ultrasound ultrasonic conduction gel is then applied to the abdomen and we found that swabbing it in with wet gauze helps to keep it from rolling off.During the ultrasound procedure itself, a liberal amount of ultrasonic conduction gel is also applied to the head of the probe. Ultrasounds are then performed and monitored in real time. This is an example of the data that can be obtained during the ultrasound procedure.In this case, the probe was in a longitudinal aspect which permits visualization of the catheter within the bladder. We're now indicating some tumor tissue that's developed during the incubation time. The entire bladder is usually easy to see and is delineated by a sharp border as indicated here.Here we can see evidence of the establishment of the tumor model.

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