The overall goal of this non-surgical method is to consistently produce orthotopic non-muscle-invasive bladder tumors in mice that can be easily monitored for the evaluation of intravesical therapies. This method can help answer key questions in the field of bladder cancer. Such as the evaluation of novel intravesical therapies.
The main advantage of this technique is that it is easy to implant tumors in mice and monitor their growth, which helps to accurately determine responses to therapies. One day before the implantation, when the cells are about 80%confluent passage to the MB49 PSA tumor cells in complete DMEM media with a one to two split. On the day of the procedure, collect and count the cells, making sure they are sufficiently healthy.
Mix in equal volume of cell suspension and 4%Trypan Blue, and count the blue-stained live cells using a hemocytometer. The cells should have at least 80%viability. Next, resuspend the cells at two million per milliliter in a 15 milliliter centrifuge tube, and wash them three times in DMEM.
Then, keep them chilled until they are to be injected. Once a female mouse has been anesthetized, provide it hydration by an intraperitoneal injection of Hartmanns Solution at 1 milliliters per 10 grams of body weight. Repeat this every one or two hours during the anesthesia.
Next, apply sterile ophthalmic ointment to both eyes, reapply as needed. Then, position the mouse supine on paper towels over a heat pad to maintain body tempurature. Next, secure the hind legs with tape.
Now apply gentle pressure to the lower abdominal region, and collect urine into a 1.5 milliliter tube to measure the basal level of urinary PSA. Next, load a one-milliliter syringe with sterile PLL and attach a 24-gauge IV catheter with the needle stylet removed. Apply lubricant to the tip of the catheter.
Then, insert the catheter into the urethra, and using forceps, guide it to the bladder. Stop when resistance is felt. Then, slowly eject 50 microliters of PLL at a rate of 10 microliters every 20 seconds to avoid vesicoureteral reflux.
Now leave the catheter in the bladder for 20 minutes, with a stopper to prevent outflow. After 20 minutes, remove the catheter and vacate the bladder of any contents, by gently pressing on the lower abdomen. Then, using a one-milliliter syringe, flush any remaining contents out of the catheter.
Now mix the MB49 PSA cells thoroughly by pipetting, and load them into a 1-mL syringe. Attach the catheter and lubricate it as before. Insert the catheter into the bladder as before.
And commence with injecting the cells, using a gradual ejection of 10 microliters per 20 seconds. Then wait one hour. After an hour, remove the catheter and vacate the bladder of any contents.
Then, release the mouse, positioning on its ventral side and revive it with an injection of atipamezole. Now, monitor the mouse until it regains sternal recumbency, and then return it to its home cage. PSA secretion of MB49 cells was found to vary with the growth media.
DMEM media is used because it resulted in the most PSA secretion. In order to determine the sensitivity of the PSA ELISA, different numbers of MB49 PSA secreting cells were mixed with MB49 parent cells. The assay detected at least 100, 000 PSA-secreting cells per million cells.
The PLL method of tumor implantation results in multiple tumors developing in the bladder. Both overnight collections of urine using metabolic cages and spot urine samples could be used to measure PSA by ELISA. For all animal studies, mouse weight was recorded as a measure of health.
And urinary PSA is monitored on a weekly basis as a measure of tumor growth. Different therapies were used on each mouse, resulting in different tumor sizes. Two weeks after implantation, the bladders were harvested and processed.
Tumor sizes varied, and the PSA measurements predicted this. After watching this video, you should have a good understanding of how to produce orthotropic bladder tumors in mice, and monitor their growth. Once mastered, this technique can be done in two hours per mouse.
While attempting this procedure, it's important to remember to ensure cells are mixed before implantation, and to perform the implantation at a slow rate. Following this procedure, other methods, like flow cytometric analysis of Uvm or ELISA can be performed in order to investigate immune activation post-therapy.
