Evaluating the Anti-Tumor Efficacy of a Bacterial Immunomodulatory Protein in a Murine Model

Overview

This video demonstrates a technique to assess the anti-tumor activity of Helicobacter pylori neutrophil-activating protein, or HP-NAP, in a mouse bladder cancer model. HP-NAP interacts with tumor-cell neutrophils to produce pro-inflammatory cytokines, inducing the differentiation of naïve T cells into type 1 T helper (Th1) and type 1 cytotoxic (Tc1) T cell phenotypes. The Th1 and Tc1 cells secrete interferon-gamma, inhibiting tumor angiogenesis and promoting cytotoxic T cell-mediated tumor destruction, reducing tumor volume.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animals

1. Grow C57BL6/J female mice to 8 weeks of age in individually ventilated cages with microisolation filters.
   NOTE: Females are preferred because the anatomic conformation of their external urogenital apparatus renders the catheterization easier than in males.

2. Cell Culture

1. Maintain the mouse urothelial carcinoma cell line MB49 in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 50 µg/ml gentamicin at 37 °C in humidified 5% carbon dioxide (CO₂).
2. Grow MB49 cells in T-75 flasks to 80% confluence. Trypsinize and resuspend at 0.5 × 10⁵ - 0.5 × 10⁶ cells per 150 μl of phosphate-buffered saline (PBS) before implantation in mice.

3. Intravesical Tumour Implant

1. Anesthetize mice using a mix of the anaesthetics zoletil and xylor (33 mg/kg and 20 mg/kg, respectively), injected intraperitoneally.
2. Place animals in a supine position and fix the hind legs to the pad with scotch tape. Paws must be sufficiently separated to render the urethral meatus well visible and easily accessible for catheter insertion.
3. NOTE: For catheterization, a 24 G pediatric venous catheter is suitable for 8 to 10 weeks-old mice. The choice of catheter is critical to avoid liquid leakage between the urethral wall and the catheter. Larger bore catheters must be used for bigger or older mice.
4. Using a small nipper, pinch one edge of the external part of the urethra and twist it very gently toward the "head end" of the mice. This action exposes the urethral meatus.
5. Remove the internal needle of the catheter and insert the latter in the urethra at an angle of 45°, slightly oriented towards the "tail end" of the mice. Do not force the entrance of the catheter; in case of resistance, just twist the catheter very gently until it slips inside the urethra. When about 0.5-0.8 cm of the catheter is in the urethra, carefully place the catheter parallel to the tail of the mice and insert it completely.
6. Using a 1 ml syringe, the needle of which has to be inserted in the catheter, wash the bladder from residual urine by injecting 200-300 μl of sterile PBS and aspirate all the liquid from the bladder.
7. NOTE: When injecting PBS, the bladder will fill up, and swelling will be visible between the hind legs of the mice; this confirms that the catheterization is correct. Most catheters have a dead volume that must be considered when calculating the injection volume.
8. Using a sterile 1 ml syringe, inject 200 μl of 0.1 N hydrochloric acid (HCl). Fill the bladder to burn its entire wall. After 10 sec, remove all the acid and immediately neutralize by injecting 200 μl of 0.1 N sodium hydroxide (NaOH) with a sterile syringe. In the case of injection of the highest number of cells (0.5 × 10⁶), the acidification step is not required.
9. Aspirate all the residual liquid and immediately flush the cavity with 300 µl of sterile PBS.
10. Empty the bladder by aspiration and inject 150 μl of PBS containing MB49 cells (range 0.5 × 10⁵ - 0.5 × 10⁶) into the bladder. Leave the syringe assembled to the catheter to avoid the leakage of cells. It is better to secure the syringe by fixing it to the pad with scotch tape.
11. After 1 hr, gently extract the catheter from the urethra and place the mice in the cage under an incandescent lamp until they awake: this usually occurs between 1 and 2 hr after the end of the treatment.

4. Intravesical Delivery of Helicobacter pylori neutrophil-activating protein (HP-NAP)

1. NOTE: At least 3 days after cell instillation, it is possible to begin the treatment.
2. Catheterize as described in steps 3.1 to 3.4. At this stage, it is not necessary to burn the bladder wall with HCl.
3. Using a 1 ml syringe, wash the bladder from residual urine by injecting 200-300 μl of sterile PBS and aspirate all the liquid from the bladder.
4. Inject 50 μg of HP-NAP per 150 μl PBS into the bladder. Leave the syringe attached to the catheter to avoid leakage of the protein solution. Secure the syringe by fixing it to the pad with scotch tape.
5. After 1 hr, gently extract the catheter from the urethra and place the mice in the cage under an incandescent lamp until they awake.

Materials

Name	Company	Catalog Number	Comments
C57BL/6J female mice	Harlan Italy (Udine, Italy)
MB49 Cells			Obtained from Prof. O'Donnel, University of Iowa Carver College of Medicine, Iowa, USA
RPMI	Sigma-Aldrich (St. Louis, Missouri, USA)	R8758
FBS	Sigma-Aldrich	F7524
PBS	Sigma-Aldrich	D1408	10X, to be diluted in apyrogenic water
Flask	Becton Dickinson (Franklin Lakes, New Jersey, USA)	353135
Syringe 1ml	Becton Dickinson	301358
Trypsin	Life Technologies (Waltham, Massachusetts, USA)
Gentamycin	Life Technologies	15710-049
Xilor	Bio 98 s.r.l. (Milano, Italy)		2% Xylazin
Zoletil	Virbac (Carros, France)	3.59713E+11	5% Zolazepam + 5% Tiletamine
24G Catheter	Terumo (Rome, Italy)	SR+DM2419PX
HCl	Carlo Erba Reagents (Milano, Italy)	403871	Liquid
NaOH	JT Baker (Center Valley, Pennsylvania, USA)	10095011	Powder