Name: 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 (Video) | JoVE Journal
Uploaded: 2015-05-29T15:00:00.000+00:00
Duration: 6 min 12 s
Description: Learn to establish an orthotopic bladder tumor model in mice for evaluating HP-NAP therapeutic efficacy against bladder cancer. This method enables histological assessment of treatment outcomes in vivo.

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5 minutes

This work was supported by Associazione Italiana per la Ricerca sul Cancro, Italian Ministry of University and Research, Prin projects and Progetti di Ricerca di Ateneo, grant N&#176; CPDA137871, Fondazione Cariplo, grant N&#176; 2011-0485 to MdB, and by Finanziamento Giovani Studiosi, University of Padua, to Gaia Codolo.

All the procedures for animal handling have been approved by the Italian Ministry of Health (D.M. 204/2011-B).
1. Animals
<ol>
	<li>Grow&#8203; 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.</li>
</ol>
2. Cell Culture
<ol>
	<li>Maintain the mouse urothelial carcinoma cell line MB49 in RPMI 1,640 medium supplemented with 10% heat-inactivated foetal bovine serum and 50 &#181;g/ml gentamicin at 37 &#176;C in humidified 5% CO2.</li>
	<li>Grow MB49 cells in T-75 flasks to 80% confluence. Trypsinize and resuspend at 0.5 &#215; 105 - 0.5 &#215; 106 cells per 150 &#956;l of PBS before implantation in mice.</li>
</ol>
3. Intravesical Tumour Implant
<ol>
	<li>Anesthetize mice using a mix of the anaesthetics zoletil and xylor (33 mg/kg and 20 mg/kg, respectively), injected intraperitoneally.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using a small nipper, pinch one edge of the external part of urethra and twist it very gently toward the &#8220;head end&#8221; of the mice. This action exposes the urethral meatus.</li>
	<li>Remove the internal needle of the catheter and insert the latter in the urethra at an angle of 45&#176;, slightly oriented towards the &#8220;tail end&#8221; 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.</li>
	<li>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 &#956;l of sterile PBS and aspirate all the liquid from the bladder.</li>
	<li>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.</li>
	<li>Using a sterile 1 ml syringe, inject 200 &#956;l 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 &#956;l of 0.1 N NaOH with a sterile syringe. In case of injection of the highest number of cells (0.5 &#215; 106), the acidification step is not required.</li>
	<li>Aspirate all the residual liquid and immediately flush the cavity with 300 &#181;l of sterile PBS.</li>
	<li>Empty the bladder by aspiration, and inject 150 &#956;l of PBS containing MB49 cells (range 0.5 &#215; 105 - 0.5 &#215; 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.</li>
	<li>After 1 hr, gently extract the catheter from the urethra, and place the mice in the cage, under an incandescent&#160;lamp until they awake: this usually occurs between 1 and 2 hr after the end of the treatment.</li>
</ol>
4. Intravesical Delivery of HP-NAP
<ol>
	<li>Note: At least 3 days after cell instillation, it is possible to begin the treatment.</li>
	<li>Catheterize as described in steps 3.1 to 3.4. At this stage, it is not necessary to burn the bladder wall with HCl.</li>
	<li>Using a 1 ml syringe, wash the bladder from residual urine by injecting 200-300 &#956;l of sterile PBS and aspirate all the liquid from the bladder.</li>
	<li>Inject 50 &#956;g of HP-NAP per 150 &#956;l 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.</li>
	<li>After 1 hr, gently extract the catheter from the urethra and place the mice in the cage, under an incandescent&#160;lamp, until they awake.</li>
</ol>
5. Tumour Explants and Histological Analysis
<ol>
	<li>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.</li>
	<li>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 &#8220;head end&#8221; of the mice. To minimize the risk of damaging the bladder, pay particular attention to the depth of the cut.</li>
	<li>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.</li>
	<li>For histological and immune histochemical analysis, prepare serial sections of the bladder (46 &#181;m thickness) using a standard microtome.</li>
	<li>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.</li>
	<li>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&#215;) to identify the area of highest vascular density. Within this region, count individual microvessels in five separate random fields at high power (20&#215;).</li>
</ol>

<ol><li>Siegel, R., Ma, J., Zou, Z., Cancerstatistics Jemal, A. CA Cancer J Clin. 64, 9-29 (2014).</li>
<li>Saint, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prognostic+value+of+a+T+helper+1+urinary+cytokine+response+after+intravesical+bacillus+Calmette-Guerin+treatment+for+superficial+bladder+cancer%5BTitle%5D)+AND+%22J+Urol%22%5BJournal%5D)">Prognostic value of a T helper 1 urinary cytokine response after intravesical bacillus Calmette-Guerin treatment for superficial bladder cancer.</a> J Urol. 167, 364-367 (2002).</li>
<li>Shahin, O., Thalmann, G. N., Rentsch, C., Mazzucchelli, L., Studer, U. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+retrospective+analysis+of+153+patients+treated+with+or+without+intravesical+bacillus+Calmette-Guerin+for+primary+stage+T1+grade+3+bladder+cancer%3A+recurrence%2C+progression+and+survival%5BTitle%5D)+AND+%22J+Urol%22%5BJournal%5D)">A retrospective analysis of 153 patients treated with or without intravesical bacillus Calmette-Guerin for primary stage T1 grade 3 bladder cancer: recurrence, progression and survival.</a> J Urol. 169, 96-100 (2003).</li>
<li>Lamm, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complications+of+bacillus+Calmette-Guerin+immunotherapy%5BTitle%5D)+AND+%22Urol+Clin+North+Am%22%5BJournal%5D)">Complications of bacillus Calmette-Guerin immunotherapy.</a> Urol Clin North Am. 19, 565-572 (1992).</li>
<li>De Jager, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+complete+remission+in+bladder+carcinoma+in+situ+with+intravesical+TICE+bacillus+Calmette+Guerin.+Overview+analysis+of+six+phase+II+clinical+trials%5BTitle%5D)+AND+%22Urology%22%5BJournal%5D)">Long-term complete remission in bladder carcinoma in situ with intravesical TICE bacillus Calmette Guerin. Overview analysis of six phase II clinical trials.</a> Urology. 38, 507-513 (1991).</li>
<li>Dovedi, S. J., Davies, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+targeted+therapies+for+bladder+cancer%3A+a+disease+waiting+for+a+drug%5BTitle%5D)+AND+%22Cancer+Metastasis+Rev%22%5BJournal%5D)">Emerging targeted therapies for bladder cancer: a disease waiting for a drug.</a> Cancer Metastasis Rev. 28, 355-367 (2009).</li>
<li>Evans, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+Helicobacter+pylori+neutrophil-activating+protein%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Characterization of a Helicobacter pylori neutrophil-activating protein.</a> Infect Immun. 63, 2213-2220 (1995).</li>
<li>Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R., Hogle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+crystal+structure+of+Dps%2C+a+ferritin+homolog+that+binds+and+protects+DNA%5BTitle%5D)+AND+%22Nat+Struct+Biol%22%5BJournal%5D)">The crystal structure of Dps, a ferritin homolog that binds and protects DNA.</a> Nat Struct Biol. 5, 294-303 (1998).</li>
<li>Amedei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neutrophil-activating+protein+of+Helicobacter+pylori+promotes+Th1+immune+responses%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses.</a> J Clin Invest. 116, 1092-1101 (2006).</li>
<li>Bernard, M., D'Elios, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immune+modulating+activity+of+the+Helicobacter+pylori+HP-NAP%3A+Friend+or+foe%5BTitle%5D)+AND+%22Toxicon%22%5BJournal%5D)">The immune modulating activity of the Helicobacter pylori HP-NAP: Friend or foe.</a> Toxicon. 56, 1186-1192 (2010).</li>
<li>Codolo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neutrophil-activating+protein+of+Helicobacter+pylori+down-modulates+Th2+inflammation+in+ovalbumin-induced+allergic+asthma%5BTitle%5D)+AND+%22Cell+Microbiol%22%5BJournal%5D)">The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma.</a> Cell Microbiol. 10, 2355-2363 (2008).</li>
<li>Gunther, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+syngeneic+orthotopic+murine+bladder+cancer+%28MB49%29%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59, 2834-2837 (1999).</li>
<li>Codolo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HP-NAP+inhibits+the+growth+of+bladder+cancer+in+mice+by+activating+a+cytotoxic+Th1+response%5BTitle%5D)+AND+%22Cancer+Immunol+Immunother%22%5BJournal%5D)">HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response.</a> Cancer Immunol Immunother. 61, 31-40 (2012).</li>
<li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+orthotopic+models+for+bladder+cancer+research%5BTitle%5D)+AND+%22BJU+Int%22%5BJournal%5D)">Mouse orthotopic models for bladder cancer research.</a> BJU Int. 104, 1286-1291 (2009).</li>
<li>Kubota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metastatic+models+of+human+cancer+xenografted+in+the+nude+mouse%3A+the+importance+of+orthotopic+transplantation%5BTitle%5D)+AND+%22J+Cell+Biochem%22%5BJournal%5D)">Metastatic models of human cancer xenografted in the nude mouse: the importance of orthotopic transplantation.</a> J Cell Biochem. 56, 4-8 (1994).</li>
<li>Loi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+the+orthotopic+model+to+validate+antivascular+therapies+for+cancer%5BTitle%5D)+AND+%22Int+J+Dev+Biol%22%5BJournal%5D)">The use of the orthotopic model to validate antivascular therapies for cancer.</a> Int J Dev Biol. 55, 547-555 (2011).</li>
<li>Miyazaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preconditioning+methods+influence+tumor+property+in+an+orthotopic+bladder+urothelial+carcinoma+rat+model%5BTitle%5D)+AND+%22Mol+Clin+Oncol%22%5BJournal%5D)">Preconditioning methods influence tumor property in an orthotopic bladder urothelial carcinoma rat model.</a> Mol Clin Oncol. 2, 65-70 (2014).</li>
<li>Horiguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+orthotopic+mouse+superficial+bladder+tumor+model+for+studies+on+intravesical+treatments%5BTitle%5D)+AND+%22Hum+Cell%22%5BJournal%5D)">Establishment of orthotopic mouse superficial bladder tumor model for studies on intravesical treatments.</a> Hum Cell. 21, 57-63 (2008).</li>
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The authors declare that they have no competing financial interests.

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 standard14-15, both because of the huge amount of cell lines available to set up a tumour model and because they mimic the environment of th...

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 infection3-6.
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 vivo9-10. 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&#8217;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)-&#947;13. 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.

<table><tbody><tr><td colspan="4">Materials</td></tr><tr><td>C57BL/6J female mice</td><td>Harlan Italy (Udine, Italy)</td><td></td><td></td></tr><tr><td>MB49 Cells</td><td></td><td></td><td>Obtained from Prof. O&#039;Donnel, University of Iowa Carver College of Medicine, Iowa, USA</td></tr><tr><td>RPMI</td><td>Sigma-Aldrich (St. Louis, Missouri, USA)</td><td>R8758</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D1408</td><td>10X, to be diluted in apyrogenic water</td></tr><tr><td>Flask</td><td>Becton Dickinson (Franklin Lakes, New Jersey, USA)</td><td>353135</td><td></td></tr><tr><td>Syringe 1ml</td><td>Becton Dickinson</td><td>301358</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies (Waltham, Massachusetts, USA)</td><td></td><td></td></tr><tr><td>Gentamycin</td><td>Life Technologies</td><td>15710-049</td><td></td></tr><tr><td>Xilor</td><td>Bio 98 s.r.l. (Milano, Italy)</td><td></td><td>2% Xylazin</td></tr><tr><td>Zoletil</td><td>Virbac (Carros, France)</td><td>359713301992</td><td>5% Zolazepam + 5% Tiletamine</td></tr><tr><td>24G Catheter</td><td>Terumo (Rome, Italy)</td><td>SR+DM2419PX</td><td></td></tr><tr><td>HCl</td><td>Carlo Erba Reagents (Milano, Italy)</td><td>403871</td><td>Liquid</td></tr><tr><td>NaOH</td><td>JT Baker (Center Valley, Pennsylvania, USA)</td><td>10095011</td><td>Powder</td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>Surgical Scalpel</td><td>Albion Surgical Limited (Sheffield, England)</td><td></td><td></td></tr><tr><td>Microtome</td><td>Leica Microsystem ( Wetzlar, Germany)</td><td>RM2235</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR International (Radnor, Pennsylvania, USA)</td><td>631-0108</td><td></td></tr><tr><td>Image Analyzer</td><td>Zeiss (Jena, Germany)</td><td>Cyres System</td><td></td></tr></tbody></table>

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

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

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (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.

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 are tr...

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.

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

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.

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Preparation of Mouse Bladder Tissue for Visualization of Bacterial Infection

In this video, we describe the intravesical procedure of delivering cancer cells into the bladder of a mouse model. This procedure helps generate an orthotopic bladder tumor model that can be used to develop therapeutic strategies.

Modeling Orthotopic Bladder Tumor in Mouse Model: A Procedure for Intravesical Administration of Cancer Cells into Murine Bladder

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...

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An Orthotopic Bladder Cancer Model for Gene Delivery Studies

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy.  After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Learn to establish an orthotopic bladder tumor model in mice for evaluating HP-NAP therapeutic efficacy against bladder cancer. This method enables histological assessment of treatment outcomes in vivo.

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 (Video) | JoVE Journal

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

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