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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animals
<ol>
	<li>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.</li>
</ol>
2. Cell Culture
<ol>
	<li>Maintain the mouse urothelial carcinoma cell line MB49 in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 50 &#181;g/ml gentamicin at 37 &#176;C in humidified 5% carbon dioxide (CO2).</li>
	<li>Grow MB49 cells in T-75 flasks to 80% confluence. Trypsinize and resuspend at 0.5 &#215; 105&#160;- 0.5 &#215; 106&#160;cells per 150 &#956;l of phosphate-buffered saline (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 must be sufficiently separated to render the urethral meatus well visible and easily accessible for catheter insertion.</li>
	<li>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.</li>
	<li>Using a small nipper, pinch one edge of the external part of the urethra and twist it very gently toward the &#34;head end&#34; 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 &#34;tail end&#34; 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.</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 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 hydrochloric acid (HCl). Fill the bladder to burn its entire wall. After 10 sec, remove all the acid and immediately neutralize by injecting 200 &#956;l of 0.1 N sodium hydroxide (NaOH) with a sterile syringe. In the 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&#160;- 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 Helicobacter pylori neutrophil-activating protein (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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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&#39;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>3.59713E+11</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>
	</tbody>
</table>

evaluating the anti-tumor efficacy of a bacterial immunomodulatory protein in a murine model

Source: Codolo, G. et al. <a href="https://app.jove.com/v/52743">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.</a>&#160;J. Vis. Exp. (2015)
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 na&#239;ve 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.

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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with an anesthetized female mouse in a supine position, bearing urothelial tumors in its bladder epithelium.
Secure the hind legs, locate the urethral opening, and insert a catheter into the mouse&#39;s bladder. Wash the bladder to remove urine.
Inject Helicobacter pylori neutrophil-activating protein, or HP-NAP &#8212; an immunomodulatory bacterial protein.
In the tumor microenvironment, HP-NAP interacts with Toll-like receptors on neutrophils and initiates a signaling cascade, increasing proinflammatory cytokine expression.
The cytokines trigger the differentiation of na&#207;ve T lymphocytes into type 1 T helper, or Th1, and type 1 cytotoxic, or Tc1, lymphocytes that produce interferon-gamma.
Interferon-gamma inhibits new blood vessel formation, limiting nutrient and oxygen availability to the tumor.
Additionally, it upregulates tumor-antigen-presenting MHC molecules on the tumor cells, facilitating interaction with Tc1 cells.
This interaction results in the release of cytotoxic molecules that induce tumor cell death, demonstrating the anti-tumor efficacy of HP-NAP.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

53 visualizações. Fonte: Codolo, G. et al. Avaliação da eficácia da proteína HP-NAP de H. pylori como ferramenta terapêutica para o tratamento do câncer de bexiga em um modelo murino ortotópico.J. Vis. Exp. (2015) Este vídeo demonstra uma técnica para avaliar a atividade antitumoral da proteína ativadora de neutrófilos Helicobacter pylori, ou HP-NAP, em um modelo de câncer de bexiga de camundongo. O HP-NAP interage com neutrófilos de células tumorais para produzir citocinas pró-inflamatórias, ind...

Vídeo: Avaliação da eficácia antitumoral de uma proteína imunomoduladora bacteriana em um modelo murino - Conceito

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

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

Um modelo de tumor de murino de bexiga ortotópico e Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

JoVE Journal

Pesquisa do câncer

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

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.

Ressonância magnética avaliação dos tumores de bexiga murino induzida por substância cancerígena

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

Cultura, Manipulação e Transplante Ortotópico de Organoides tumorais da bexiga de camundongo

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

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

Transcrição

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