This work was supported by NIH R21 CA143505 to Christina Voelkel-Johnson.

All procedures involving animals have been reviewed and were approved by the Institutional Animal&#160;Care and Use Committee at the Medical University of South Carolina. &#160;The protocol was approved under USDA category D for pain.&#160;
1. Cell Implantation
<ol>
	<li>Two days before performing the procedure, plate 1 x 106 MB49 cells into T-25 flasks. Use high glucose DMEM supplemented with 10% FBS (and antibiotics, if desired). One flask is sufficient for each group of up to five mice that will be anesthetized and implanted in parallel.</li>
	<li>On the day of the procedure, prepare trypsin for instillation by diluting sterile 0.25% tissue culture grade trypsin to 0.125% with sterile DMEM base medium (1:2 dilution). 1.2 ml is sufficient for 10 mice. Place in a 37 &#176;C waterbath to equilibrate.</li>
	<li>Prewarm a heating pad and position it under the nosecones of the anesthesia system that will be used to deliver isoflurane. Place a clean absorbent pad over the heating pad.</li>
	<li>Place mice into the induction chamber of the anesthesia system and induce anesthesia with 3% isoflurane. When mice have lost consciousness, which is verified by loss of the toe-pinch reflex, transfer them to the nosecones. Make sure each animal is in a supine position and properly placed on the heating pad to maintain body temperature.</li>
	<li>Reduce the isoflurane concentration to 2% to maintain anesthesia.</li>
	<li>Apply ophthalmic ointment to both eyes to prevent drying.</li>
	<li>Optional step: Remove abdominal hair. Hair on the lower abdomen must be removed if in vivo bioluminescent imaging will be performed.
	<ol>
		<li>Apply a generous layer of depilatory cream, such as Veet, to the lowest 1 in&#160;square of fur on the abdomen, taking care not to contact the external genitalia. Wait 3 min.</li>
		<li>Remove hair by rubbing the area with a dry sponge, and then use a damp sponge to cleanse the skin. Repeat once, if needed.</li>
	</ol>
	</li>
	<li>Catheterize the bladder
	<ol>
		<li>Lubricate a new, sterile 24 G pediatric venous catheter with a nonirritating lubricating gel such as K-Y. Remove and discard the needle. If leaking of fluid around the catheter is a common problem, i.e. it occurs more frequently than in one of ten mice, larger bore catheters (20 G) can be used. This may be an important consideration in mice older than 8 weeks.</li>
		<li>Holding the hub of the catheter, use the thumb and index finger of your opposite hand to spread the hind legs of the mouse and expose the urethral meatus. Gently insert the catheter into the urethra at a 45&#176; angle, changing the angle to one parallel with the bench to insert it fully.</li>
		<li>After full insertion, lift the catheter gently, keeping it parallel to the bench, and visually confirm that it is placed in the urethra and not the vagina, which is below it. If correctly placed, the catheter will be able to be inserted fully since the vagina is shorter than the urethra.</li>
		<li>Reduce the isoflurane concentration to 1%.</li>
	</ol>
	</li>
	<li>Attach a tip to a P200 pipettor and remove urine from the bladder by applying suction to the external end of the catheter. Remove any remaining urine from the hub of the catheter. Discard urine and leave catheter in place.</li>
	<li>Carefully pipette 80 &#956;l&#160;0.125% trypsin solution into the hub of the catheter, avoiding air bubbles. Attach a 1 ml air-filled syringe to the catheter hub and slowly depress the plunger 0.1-0.2 ml&#160;to deliver the trypsin into the bladder.</li>
	<li>Leave the syringe and catheter assembly in place for 15 min. Repeat steps 1.8-1.11 for any of the remaining anesthetized mice (up to 4). Proceed to the next step during the waiting period. HINT: Initially it may be helpful to have someone else prepare the cells (step 1.12).</li>
	<li>Prepare MB49 cells for implantation. Cells were plated at 1 x 106 per T-25 flask 48 hr before implantation (step 1.1).
	<ol>
		<li>Remove media and add 500 &#181;l of 0.25% trypsin to the flask. When cells detach add 5 ml of complete DMEM to resuspend cells. Remove a 50 &#956;l&#160;aliquot to count, and centrifuge the remainder at 1,000 rpm for 5 min. During the centrifugation step continue to steps 1.12.2 and 1.12.3.</li>
		<li>Count the cells using a hemocytometer. Calculate the cells/ml. Then calculate the volume needed to bring the cell pellet to 4 x 106 cells/ml (2 x 105 cells per 50 &#956;l).</li>
		<li>Pour off supernatant from centrifuged cells and resuspend the pellet in DMEM base medium using the volume required to suspend cells to 4 x 106 cells/ml (as calculated in step 1.12.2). Keep cells at room temperature.</li>
	</ol>
	</li>
	<li>When the 15 min&#160;time point for trypsin treatment of bladders is reached, detach the air-filled syringe from the catheter leaving the catheter in place. Spent trypsin will normally flow back out through the catheter. Remove any remaining trypsin from the bladder by suction with the P200 pipettor as in step 1.9 above and discard.</li>
	<li>Immediately pipette 50 &#956;l&#160;of MB49 cell suspension into the hub of the catheter. Attach the 1 ml air-filled syringe to the hub of the catheter and deliver the cells to the bladder by slowly depressing the plunger 0.1-0.2 ml. Repeat this step up to four additional times to implant all five mice. Discard any unused cells.</li>
	<li>Leaving the catheter-syringe assembly in place, allow the cells to dwell in the bladder for 50 min.</li>
	<li>Gently withdraw the catheter-syringe assembly from the urethra. Turn the isoflurane concentration to zero and move each mouse an inch or two away from the nosecone to recover on the heating pad.</li>
	<li>When a mouse has regained its righting reflex, return it to its cage.</li>
</ol>
2. Intravesical Delivery of Adenovirus (8 Days after Implantation of Cells)
CAUTION: This part of the protocol uses adenovirus, which is an infectious agent and should be handled under strict BSL2 guidelines (http://oma.od.nih.gov/manualchapters/intramural/3035/).
All procedures performed with infectious agents were approved by the Institutional Biosafety Committee of the Medical University of South Carolina.
<ol>
	<li>Eight days after implanting mice with MB49 cells, check for hematuria. Pick up individual animals over a white piece of absorbent paper and gently press on the abdomen to blot drops of urine onto the paper. Urine that is pink to red indicates hematuria and is a sign that tumors have established. Tumor take rate is at least 80% and with excellent technique can be as high as 100%.</li>
	<li>Thaw adenoviral stock on ice and dilute to 109 PFU/50 &#956;l&#160;(2 x 1010 PFU/ml) in sterile, room temperature PBS.</li>
	<li>Anesthetize mice as described in steps 1.3-1.6 above.</li>
	<li>Catheterize mice as described in steps 1.8 and remove urine as in step 1.9</li>
	<li>After urine is removed, immediately pipette 50 &#956;l&#160;of virus suspension into the hub of the catheter. Attach the 1 ml air-filled syringe to the hub of the catheter and deliver the virus to the bladder by slowly depressing the plunger 0.1-0.2 ml.</li>
	<li>Leaving the catheter-syringe assembly in place, allow the virus to dwell in the bladder for 40 min.</li>
	<li>Gently withdraw the catheter-syringe assembly from the urethra, and discard into 10% bleach solution to inactivate any remaining virus.</li>
	<li>Turn the isoflurane concentration to zero and move each mouse an inch or two away from the nosecone to recover on the heating pad.</li>
	<li>When a mouse has regained its righting reflex, return it to its cage.</li>
	<li>Mark cages to indicate that mice have been instilled with adenovirus. Virus will be shed into the cage bedding for several days and all cages and bedding must be handled and disinfected appropriately.</li>
</ol>

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The authors declare that they have no competing financial interests.

The primary methodology described in this protocol is catheterization of mouse bladders, which has broad applications for instillation of cells or any agent intended for local delivery to the bladder epithelium. The specific protocol outlined above has been optimized for short-term studies (~10 days). Implanting the accurate number of cells is critical, since a higher cell number will result in more rapid tumor growth and possibly loss of animals due to large tumor burden. Using 200,000 MB49 cells for instillation may re...

Bladder cancer is the second most common cancer of the urogenital tract with nearly 75,000 new cases and 15,000 deaths expected in 20121. High rates of recurrence require lifelong follow-up, which makes bladder cancer one of the costliest cancers to treat. Bladder cancer that has invaded the muscle layer may metastasize to liver, lung or bone via the lymphatic system. Multimodal therapy of advanced tumors results in only 20-40% survival after 5 years. Therefore, effective treatment strategies aimed at reducing the recurrence and progression of superficial bladder cancer as well as improving therapeutic outcome in patients with advanced disease are urgently needed.
Development of novel therapeutics requires preclinical models to evaluate efficacy following initial in vitro assessment. The tumor microenvironment can significantly influence cancer development and responsiveness, which highlights the need for preclinical models in which tumors arise or can be established in the organ of origin. One approach is the development of transgenic models in which tumors arise spontaneously or can be induced in an organ-specific manner. An excellent protocol of a transgenic bladder cancer model has recently been published2. The drawback of transgenic models is that tumors tend to develop slowly and with less uniformity than desired. In addition, the cost of maintaining a breeding colony has to be considered. An alternative to transgenic models is orthotopic implantation of tumor cells, which has the benefit of short time frames for tumor establishment in commercially available mice. While some human bladder cancer cell lines can be grown orthotopically (we have successfully used UM-UC-3), it may be desirable to establish tumors in immunocompetent mice. Two murine bladder cancer cell lines, which grow orthotopically are MBT-2 and MB493. Since MBT-2 cells are contaminated with replicating type C retrovirus4, we have chosen MB49 cells for our studies. It is important to note that MB49 cells were isolated from a male mouse and orthotopic implantations are for anatomical reasons performed in female mice. This has the benefit of easy identification of the implanted cells by markers of the Y chromosome, but the gender mismatch can be a drawback for immunological studies.
The bladder epithelium is lined by a glycosaminoglycan (GAG) layer, which functions as a barrier for infection by microorganisms. This barrier can also interfere with implantation of tumor cells and several methods have been developed to overcome this difficulty (Table 1). Electrocautery has been used extensively as a physical means to disrupt the GAG layer 5-13 and a protocol demonstrating electrocautery has recently been published in JoVE14. However, should an electrocautery unit not be available, chemical means to destroy the GAG layer such as silver nitrate or poly-L-lysine can also be used15-24. Tumors are established effectively by a brief exposure of the bladder to a small volume of silver nitrate (5-10 &#956;l, 0.15-1.0 M, ~10 sec) or longer contact with poly-L-lysine (100 &#956;l of 0.1 mg/ml for 20 min) (Table 1). Here we describe a method that uses trypsin to facilitate implantation of MB49 cells.
In an attempt to improve therapeutic approaches for bladder cancer, gene therapy has garnered significant attention. From a clinical point of view, bladder cancer is an ideal target for gene therapy due to easy accessibility of the organ and the ability to locally deliver the payload. Viral vectors that have been explored for bladder cancer gene therapy include an oncolytic herpes simplex virus25, retrovirus26, canarypox virus27, vaccinia virus, AAV, and adenovirus28. In the second part of our protocol, we describe a method for viral delivery that is virtually identical to instillation of the tumor cells. Of interest in our lab is the development of novel approaches to gene delivery, which we assess via bioluminescence using an adenoviral vector that expresses a luciferase transgene. However, the methodology of bladder catheterization can be used for delivery of various agents and therefore has broad applicability.

<table border="1" cellpadding="0" cellspacing="0">
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			<td style="height:25px;">
			Name of reagent
			</td>
			<td style="height:25px;">
			Company
			</td>
			<td style="width:188px;height:25px;">
			Catalog number
			</td>
			<td style="width:193px;height:25px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="height:40px;">
			6-8 week old female mice
			</td>
			<td style="height:40px;">
			Jackson Laboratories
			</td>
			<td style="width:188px;height:40px;">
			Strain Name: C57BL/6J
			Stock Number: 000664
			</td>
			<td style="width:193px;height:40px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			Trypsin*
			</td>
			<td style="height:25px;">
			MediaTech
			</td>
			<td style="width:188px;height:25px;">
			MT25-053-CI
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			DMEM*
			</td>
			<td style="height:25px;">
			MediaTech
			</td>
			<td style="width:188px;height:25px;">
			MT10-017-CV
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			FBS
			</td>
			<td style="height:25px;">
			Hyclone
			</td>
			<td style="width:188px;height:25px;">
			SH30071.03
			</td>
			<td style="width:193px;height:25px;">
			Heat-inactivated
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			T25 flasks*
			</td>
			<td style="height:25px;">
			Corning Costar
			</td>
			<td style="width:188px;height:25px;">
			Corning No.:3056
			Fisher: 07-200-63
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:42px;">
			MB49 cells
			</td>
			<td style="height:42px;">
			N/A
			</td>
			<td style="width:188px;height:42px;">
			N/A
			</td>
			<td style="width:193px;height:42px;">
			Obtained from Dr. Boehle (see reference11)
			</td>
		</tr>
		<tr>
			<td style="height:46px;">
			Puralube Vet Ointment*
			</td>
			<td style="height:46px;">
			Pharmaderm
			</td>
			<td style="width:188px;height:46px;">
			Henry Schein Company
			No.:036090-6050059
			Fisher: NC9676869
			</td>
			<td style="width:193px;height:46px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:53px;">
			Depilatory cream: Veet
			</td>
			<td style="height:53px;">
			local pharmacy
			</td>
			<td style="width:188px;height:53px;">
			
			</td>
			<td style="width:193px;height:53px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:53px;">
			Lubricant:
			K-Y Jelly
			</td>
			<td style="height:53px;">
			local pharmacy
			</td>
			<td style="width:188px;height:53px;">
			
			</td>
			<td style="width:193px;height:53px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:72px;">
			Catheters*
			</td>
			<td style="height:72px;">
			Exel International
			</td>
			<td style="width:188px;height:72px;">
			Exel International
			No.:26751;
			Fisher: 14-841-21
			</td>
			<td style="width:193px;height:72px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:45px;">
			Isoflurane
			</td>
			<td style="height:45px;">
			Terrell
			</td>
			<td style="width:188px;height:45px;">
			NDC 66794-011-25
			</td>
			<td style="width:193px;height:45px;">
			Obtained though hospital pharmacy
			</td>
		</tr>
		<tr>
			<td style="height:52px;">
			1 ml slip tip TB syringes
			</td>
			<td style="height:52px;">
			Becton Dickinson
			</td>
			<td style="width:188px;height:52px;">
			BD309659
			Fisher:14-823-434
			</td>
			<td style="width:193px;height:52px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:27px;">
			D-Luciferin
			</td>
			<td style="height:27px;">
			Gold Biotechnologies
			</td>
			<td style="width:188px;height:27px;">
			L-123-1
			</td>
			<td style="width:193px;height:27px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:76px;">
			Ad-CMV-Luc
			</td>
			<td style="height:76px;">
			VectorBiolabs
			</td>
			<td style="width:188px;height:76px;">
			1000; Request large scale amplification and CsCl purification for in vivo use
			</td>
			<td style="width:193px;height:76px;">
			Infectious agent that requires BSL2 containment
			</td>
		</tr>
		<tr>
			<td style="height:76px;">
			Steady-Glo&#160;Luciferase Assay System
			</td>
			<td style="height:76px;">
			Promega
			</td>
			<td style="width:188px;height:76px;">
			E2510 (10 ml), E2520 (100 ml), or E2550 (10 x 100 ml)
			</td>
			<td style="width:193px;height:76px;">
			
			</td>
		</tr>
	</tbody>
</table>
*available through multiple vendors



EQUIPMENT
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:97px;">
			Material name
			</td>
			<td style="width:114px;">
			Company
			</td>
			<td style="width:330px;">
			Catalog number
			</td>
			<td style="width:97px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			Anesthesia system
			</td>
			<td style="width:114px;">
			E-Z Systems, Euthanex Corporation
			</td>
			<td style="width:330px;">
			Anesthesia system: EZ7000
			5-port mouse rebreathing device: EZ109
			</td>
			<td style="width:97px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			Xenogen IVIS 200
			</td>
			<td style="width:114px;">
			Caliper Life Sciences
			</td>
			<td style="width:330px;">
			http://www.caliperls.com/products/preclinical-imaging/ivis-imaging-system-200-series.htm
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			FLUOstar Optima
			</td>
			<td style="width:114px;">
			BMG Labtech
			</td>
			<td style="width:330px;">
			http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
	</tbody>
</table>

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.

Hematuria is observed in nearly all mice within 8 days after implantation of 200,000 MB49 cells. As shown in Figure 1, bladder weight more than doubles from 34.7&#177;3.3 mg (range 31-37 mg, n=4) in nontumor bearing mice to 87.5&#177;19.2 mg (range 77-120 mg, n=10) in mice that have been implanted with MB49 cells. In terms of gene delivery, we found that imaging mice 24 hr after viral instillation yields a stronger signal than after 48 hr (Figure&#160;2). Adenoviral delivery is highly va...

Watch this Scientific Journal Video about An Orthotopic Bladder Cancer Model for Gene Delivery Studies at JoVE.com

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

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 ...

an-orthotopic-bladder-cancer-model-for-gene-delivery-studies

Research

JoVE Journal

Medicine

12.3K Views.  Medical University of South Carolina. 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.