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

Podsumowanie

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Streszczenie

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Guérin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.

The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.

In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Wprowadzenie

Bladder cancer is one of the most common cancers of the urogenital tract, with nearly 75,000 new cases every year in the USA¹. High rates of recurrence require lifelong follow-up, which makes bladder cancer one of the costliest cancers to treat. The gold standard treatment for the high-grade NMIBC is trans-urethral resection, followed by intravesical immunotherapy with BCG. Although the precise mechanism of the anti-tumour activity of BCG remains to be fully elucidated, it is accepted that the activation of a cell-mediated immune response enriched in T helper (Th)1 and cytotoxic T (Tc)1 cells is crucial for the success of the therapy².

Despite the fact that BCG remains the treatment of choice for NMIBC, a high proportion of patients do not respond to the therapy; moreover, it can cause a number of side effects: about 70% of treated tumours recur after some time and ~15% progress to the muscle-invasive form of the disease. Other side effects associated with BCG treatment include dysuria, cystitis and kidney infection^3-6.

The development of novel therapeutic strategies must take into consideration the use of preclinical models, following initial in vitro assessment; this is particularly relevant in tumours whose microenvironment can significantly influence their development and responsiveness to treatment.

Over the last decade, we have addressed multiple aspects of the immune modulating activity of HP-NAP, a protein produced by the bacterium Helicobacter pylori, initially identified as capable of promoting endothelial adhesion of polymorphonuclear cells (PMNs)⁷. Structurally, HP-NAP belongs to the DNA-protecting protein under starved conditions (Dps) family⁸ and consists of 12 identical subunits arranged in a dodecameric shell.

We have demonstrated that HP-NAP is a Toll-like receptor (TLR)2 agonist, with a strong immuno-modulating activity responsible for driving the differentiation of T lymphocytes towards the Th1 phenotype, both in vitro and in vivo^9-10. In virtue of this activity, HP-NAP is able to redirect the Th2 immune response into the more beneficial Th1 response in a murine model of allergic asthma¹¹.

To evaluate the Th1-dependent anti-tumour potential of HP-NAP, we took advantage of a mouse model of bladder cancer developed several years ago by O’Donnel and colleagues¹² for evaluating the impact of the BCG administration.

With this protocol, we demonstrate that HP-NAP has a strong anti-tumour potential against bladder cancer and that the efficacy of HP-NAP administration parallels the significant accumulation, within tumour and regional lymph nodes, of both Th1 and Tc1 lymphocytes producing Interferon (IFN)-γ¹³. Tumours isolated from HP-NAP-treated mice showed more necrosis and less vascularization than the untreated counterpart.

The present report provides a stepbystep protocol, detailing the preparation of the animals, their urethral catheterization, the chemical burning required for the attachment of cells to the bladder mucosa, and the injection of the tumour cells. We also describe the topical administration of HP-NAP that can be considered as a prototype of any therapeutic agents developed for the treatment of bladder cancer. The evidence obtained by comparing tumours isolated from control and HP-NAP-treated animals emphasizes not only the fact that HP-NAP could be a good candidate for bladder cancer immunotherapy, but also the general effectiveness of the experimental set up.

Protokół

All the procedures for animal handling have been approved by the Italian Ministry of Health (D.M. 204/2011-B).

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 uro-genital apparatus renders the catheterization easier than in males.

2. Cell Culture

1. Maintain the mouse urothelial carcinoma cell line MB49 in RPMI 1,640 medium supplemented with 10% heat-inactivated foetal bovine serum and 50 µg/ml gentamicin at 37 °C in humidified 5% 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 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 have to be sufficiently separated to render the urethral meatus well visible and easily accessible for the insertion of the catheter.
3. Note: For the catheterization, a 24 G paediatric venous catheter is suitable for 8 to 10 weeks-old mice. The choice of catheter is very important in order to avoid leakage of liquid 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 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 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 a 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 HCl. Fill the bladder in order to burn its entire wall. After 10 sec, remove all the acid and immediately neutralize by injecting 200 μl of 0.1 N NaOH with a sterile syringe. In 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 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.

5. Tumour Explants and Histological Analysis

1. At the end of the treatment, sacrifice the animals, place them in supine positions and fix the hind legs to the pad with scotch tape.
2. Using a surgical scalpel, make a vertical incision about 1.52 cm long through the skin and the peritoneum, starting just above external genitalia to the “head end” of the mice. To minimize the risk of damaging the bladder, pay particular attention to the depth of the cut.
3. The bladder will be positioned in the centre-left of the cut and the tumour will be visible as a dark pink/violet mass. Upon its excision, place the entire bladder in a histological cage and fix in 10% buffered formalin before embedding in paraffin.
4. For histological and immune histochemical analysis, prepare serial sections of the bladder (46 µm thickness) using a standard microtome.
5. Stain the sections with haematoxylin/eosin and proceed with the calculation of tumour size (D x d²) and determining the percentage of necrosis per cross-sectional area using a computer assisted image analyser.
6. For vessel counting, proceed by staining the sections for the endothelial marker CD31/PECAM, using a standard immunoperoxidase technique. Scan the sections at low power (4×) to identify the area of highest vascular density. Within this region, count individual microvessels in five separate random fields at high power (20×).

Wyniki

Figure 1 shows the catheterization technique; the mouse is catheterized and instilled with 0.5 × 10⁶ MB49 cells. Treatment with HP-NAP is started 3 days after the injection of tumour cells, to enable them to attach to the bladder wall and proliferate. All the animals belonging to the control group develop the tumour; some of them may die before the end of the 13 day period due to the occlusion of the urethra.

After the first injection with HP-NAP, the animals a...

Dyskusje

The majority of advances in cancer therapy require tests in animal models before starting clinical trials. The possibility of studying tumour biology in vivo, taking advantage of animal models, represents a crucial tool for researchers investigating cancer pathogenesis, enabling the assessment of different therapeutic approaches. Orthotopic models remain the gold standard^14-15, both because of the huge amount of cell lines available to set up a tumour model and because they mimic the environment of th...

Materiały

Name	Company	Catalog Number	Comments
Materials
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)	359713301992	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
Equipment
Surgical Scalpel	Albion Surgical Limited (Sheffield, England)
Microtome	Leica Microsystem ( Wetzlar, Germany)	RM2235
Microscope Slides	VWR International (Radnor, Pennsylvania, USA)	631-0108
Image Analyzer	Zeiss (Jena, Germany)	Cyres System