Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

The overall goal of this procedure is to establish a tumor in the bladder cavity of female mice and to subsequently deliver therapeutic agents. This is accomplished by first harvesting tumor cells from a culture and resus suspending them in PBS. Next mice are catheterized and tumor cells are injected into the bladder cavity.

Three days after tumor implant therapeutics are injected into the mice and the injections are repeated every three days for a total of 13 days. Finally, the bladder is excised and histological analysis is performed. Ultimately, histological analysis and immune histochemical analysis are used to verify the efficacy of the injected therapeutics.

The main advantage of this technique over existing methods like intraperitoneal or subcutaneous tumors model is that orthotopic model mimic the naturally occurring environment of the tumor. No.After raising C 57 BL six J, female mice to eight weeks of age, according to the text protocol grow MB 49 cells in RPMI 1640, medium supplemented with FBS and Gentamycin to 80%confluence. In T 75 flasks tryps the cells and then resuspend them at 0.5 times 10 to the fifth to 0.5 times 10 to the six cells per 150 microliters of PBS following the anesthetization of a mouse with an intraperitoneal injection of Zola, te and Xor.

Place the animal in a supine position on a pad and use tape to fix the hind legs to the pad, ensuring that the paws are sufficiently separated for inserting the catheter using a small nipper pinch, one edge of the external part of the urethra and gently twisted toward the head end of the mouse. This will expose the urethral atu. Remove the internal needle of the catheter and gently insert the ladder in the urethra at a 45 degree angle, slightly oriented towards the tail end of the mouse.

Do not force the catheter, but gently twist, twisted if resistance is felt. When approximately 0.5 to 0.8 centimeters of the catheter is in the urethra, carefully position the catheter parallel to the tail of the mouse and insert it completely. Insert a one milliliter syringe with 200 to 300 microliters of sterile PBS into the catheter and wash residual urine from the bladder before aspirating all the fluid from the bladder.

Next, using a sterile one milliliter syringe, inject 200 microliters of 0.1 NHCL. Fill the bladder in order to burn the entire wall. After 10 seconds, remove all the acid and immediately neutralize by injecting 200 microliters of 0.1 NNAH.

Then aspirate all the residual fluid and immediately use 300 microliters of sterile PBS to flush the cavity. After emptying the bladder by aspiration, inject 150 microliters of M 49 cell suspension. Leave the syringe assembled to the catheter to prevent cells from leaking and use tape to secure it.

One hour later, gently extract the catheter from the urethra and place the mouse in a cage under a heating lamp to recover at least three days. After injecting the MB 49 cells after anesthetizing the animal catheterized as just demonstrated, and using a one milliliter syringe with 200 to 300 microliters of sterile PBS washed the residual urine from the bladder aspirating all the liquid inject 50 micrograms of helicobacter pylori neutrophil activating protein, or H-P-N-A-P per 150 microliters of PBS into the bladder, leaving the syringe attached to the catheter to avoid leakage. After an hour, gently remove the catheter from the urethra and place the mouse in a cage under a heating lamp until it awakens.

Mice implanted at day zero. With MB 49, cells were treated with 50 micrograms of H-P-N-A-P or PBS on days 3, 6, 9, and 12. On day 13, the animals were sacrificed and the bladders collected for histological and immune histochemical analysis.

As shown in this figure, the tumors from the bladders of H-P-N-A-P treated mice were significantly smaller than those from vehicle treated mice. Interestingly, while in vehicle treated animals, the tumors invade the underlying fat tissue. In seven of the eight HP NAP treated animals, the tumors are confined to within the bladder wall.

Finally, as demonstrated here, vascularity is reduced and the extent of necrosis is increased in HP NAP treated mice compared to control animals suggesting that HP NAP provides strong anti-tumor potential against bladder cancer Deeper, the profile of these cells can be characterized by fax analysis in order to define the immune response. Solicited by therapeutics.