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

We present a method for establishing an orthotopic bladder tumor model in immunocompromised mice. Treating these tumors with intravascularly delivered small double stranded RNA and monitoring the tumors with both bladder ultrasound and bioluminescence imaging. General anesthesia is induced with vaporized isof, fluorine, and sterile ophthalmic ointment is applied.

The mouse is then placed in supine position, and anesthesia is maintained with isof fluorine via nose cone. Next, the mouse must be catheterized. We use a 24 gauge intravenous catheter with the needle stylet removed.

Catheterization is often the most difficult and unpredictable step of the protocol. Ample lubrication with KY jelly and correct technique are keys to successful catheterization. Shown here is the area of the mouse vulva.

The urethra lies just below the vulvar folds. After lubricating, the catheter forceps are used to gently grasp and stabilize the vulva and urethra. Initially, a steeper 45 degree angle was used to cannulate the urethra, but once in the urethra, a more shallow angle is taken to pass smoothly into the bladder.

We will show this process here. Once again, gentle pelvic pressure to straighten the urethra can often help and care must be taken to avoid pushing too hard against resistance, as this can lead to urethra or bladder perforation. Urine seen here within the catheter confirms this location within the bladder lumen.

Urine within the catheter is then removed with the stylet needle and the catheter is now left in place. Approximately five millimeters of catheter should remain visible outside the urethra so that the tip of the catheter remains in the bladder lumen and not up against the bladder wall. If the catheter is placed too far in the catheter, tip may embed in the bladder wall and prevent injections.

For all subsequent injections. A syringe loaded onto the stylet needle is placed through the catheter. This allows for injection of accurate volumes by eliminating the dead space within the catheter and it makes repeated catheterizations unnecessary to create a burn injury to the bladder wall.

10 microliters of 0.5 molar silver nitrate is injected and allowed to dwell for 10 seconds. The silver nitrate can form a white precipitate with residual urine within the bladder. This precipitate and any remaining solution is removed from the catheter.

We then proceed to wash the bladder with 100 microliters of sterile water four times each time removing fluid from the catheter lumen. After this first wash, the white precipitate is still visible within the catheter, but with each wash, the fluid will become more clear. A four oh silk tie is used to occlude the urethra and we are now ready to inject the cancer cells.

The cells are loaded into a syringe attached to the stylet needle, and the cells are injected into the bladder. The catheter is removed and the urethra will remain tied with the mouse under anesthesia for two hours. At preset intervals, tumor progression is monitored.

Using in vivo bioluminescence imaging, the mice are first injected with Lucifer and substrate using intraperitoneal injection. After general anesthesia is established with vaporized isof fluorine, the mice are placed in supine position within the ivus machine. Bioluminescence is measured in units of photons per second.

The presence of KU seven cells within the mouse bladders is readily apparent and the bioluminescent intensity can be followed over time. The presence and progression of bladder tumors can also be monitored with ultrasound imaging. After general anesthetic is induced with vaporized isof, fluorine and sterile ophthalmic ointment is applied.

The mouse is placed in supine position on the ultrasound table. Maintenance anesthetic is achieved with isof fluorine via nose cone. The mouse's hind legs can be secured to the table to avoid excess movement of the body while manipulating the ultrasound probe.

Once this is complete, ultrasound gel is applied to the midline portion of the animal's pelvis. After a gel is applied, a 40 megahertz probe is secured to the holding device and slowly lowered onto the animal's body. The bladder is readily visible as a round midline hypoechoic structure.

If the animal has not undergone significant irritation prior to anesthetized, the bladder is typically already distended and we have found it unnecessary to catheterize the mouse to achieve bladder distension. Both transverse and longitudinal images can be obtained here. A tumor is easily seen within the lumen of the bladder.

If a tumor is found, images can be saved for measurement of tumor dimensions. After establishment of bladder tumor, the mice are treated intravesical with small double stranded RNA formulated in lipid nanoparticles. General anesthetic is established and ophthalmic ointment is once again applied.

The mouse is then placed in supine position and urethral catheterization is performed. As we previously described, a four oh silk suture is used to occlude the urethra around the catheter. Typically, two knots are tied as shown here.

An empty syringe loaded onto a stylet needle is used to evacuate the bladder completely. 50 microliters of small double stranded RNA are instilled intravesical for a total dose of three milligrams per kilogram. The needle and catheter are then removed.

The mouse will remain anesthetized with the urethra occluded for a total of two hours. After watching this video, you should have a good understanding of a method for creating an orthotopic mouse bladder tumor model monitoring the tumor with bioluminescence imaging and ultrasound, and treating the tumor with intravesical delivered small RNA. There are some nuances to the technique, which takes some practice, especially with the catheterization, but overall, it's a protocol that should be easily replicable.

We've had good success in planting the bladder tumors into mice, and treatment effects with the small RNA are very promising. However, what's most exciting about this protocol is that while we treated the mice with small RNA, the same technique can be used to test any intravesical therapy for bladder cancer.I.