This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.

All animal work adhered to the Institutional Animal Care and Use Committee (IACUC) guidelines on animal use and handling (Protocol number 084/12) at the National University of Singapore.
1. Growing MB49-PSA Cells In Vitro and Measuring PSA Secretion
<ol>
	<li>Maintain murine bladder cancer cells MB49-PSA15 in complete Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 0.05 mg/mL Penicillin-Strepto...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+characterization+of+a+syngeneic+orthotopic+murine+bladder+cancer+model.">Histopathological characterization of a syngeneic orthotopic murine bladder cancer model.</a> Int Braz J Urol. 34, 220-226 (2008).</li><li>Chan, E. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-interleukin-10R1+monoclonal+antibody+in+combination+with+bacillus+Calmette--Guerin+is+protective+against+bladder+cancer+metastasis+in+a+murine+orthotopic+tumour+model+and+demonstrates+systemic+specific+anti-tumour+immunity.">Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette--Guerin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity.</a> Clin Exp Immunol. 177, 261-268 (2014).</li><li>Patel, A. 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The authors have nothing to disclose.

The most critical steps in the protocol are 1) successfully maintaining the tumorigenicity of the cell line; 2) ensuring measurable PSA secretion before tumor cell implantation in mice; 3) generating a single-cell suspension for implantation so as to reduce variation in tumor size; and 4) instilling cells at a slow rate to prevent vesico-ureteral reflux, resulting in tumor cell implantation in the kidney.
After prolonged passage in vitro, MB49/MB49-PSA cells can lose their tumorigenic...

The goal of this method is to generate murine orthotopic bladder tumors and to monitor the implanted tumors as accurately as possible, so that mice without tumor implantation are not thought to have been cured at end-point analysis. Overall, the method shown will reduce the need for large numbers of mice for experimental analysis and ensure greater accuracy in determining therapeutic outcomes.
The development of an orthotopic model for cancer is important, as implanting tumor cells subcutaneously does not recapitulate the environment of the clinical disease or enable the development of therapeutic strategies. The architecture of the bladder permits the instillation of bladder cancer therapies directly into the bladder with minimal systemic effects. Thus, animal models that recapitulate this environment, such as an orthotopic model, are important to evaluate new therapies. The conclusions drawn from any experimental set-up are dependent upon the limitations of the model.
Several techniques have been developed for the production of orthotopic bladder tumors in mice. These rely on damaging the glycosaminoglycan layer of the bladder, enabling tumor cells to be implanted. The techniques used include electrocautery, which results in a single point of damage in the bladder wall, leading to tumor development at one site in the bladder1,2. However, the success rate of tumor implantation using electrocautery is operator-dependent varying from 10 - 90%, and it includes the danger that the bladder wall will be punctured, leading to tumors developing in the peritoneal cavity. Chemical cautery is performed using silver nitrate, which damages the bladder wall3. Similarly, acid has been used to damage the bladder wall4. Trypsin has also been used to damage the bladder as well5. These methods may result in the development of more than one tumor in the bladder. Furthermore, there is a danger of severe damage to the bladder if the chemicals are left in contact with the bladder wall for too long. The method developed by Ninalga et al. uses the positively charged poly-L-lysine (PLL)6 molecules to coat the bladder wall; this enables the negatively charged tumor cells to stick to the glycosaminoglycan layer of the bladder. This method generally results in more than one tumor developing in the bladder, but tumor implantation is at 80 - 100%4,7. Technically, it is also the easiest procedure to perform. To ensure that the tumors that develop are fairly even in size, it is important that the tumor cells are not grouped in large clumps before implantation.
In order to evaluate therapeutic efficacy, it is best to perform this study on mice with fairly similar-sized tumors. Thus, a good detection system that can quantify tumor size soon after implantation is important. Several strategies have been used to evaluate tumors. These include magnetic resonance imaging (MRI)8-10, fluorescence11, bioluminescence12,13, ultrasound14, and enzyme-linked immunosorbent assay (ELISA)15,16. While MRI and ultrasound do not require modifications of the tumor cells, there is a need for sensitive equipment and contrast agents for MRI. The fluorescence-, luminescence-, and ELISA-based assays require modification of the tumor cells to express marker proteins that can be detected by these methods. For luminescence, a substrate is required for the detection of the luciferase activity; thus, there is an added step and increased cost. Both luminescence and fluorescence require specialized equipment. To produce fluorescence, green fluorescent protein (GFP) cyclization, which is catalyzed by molecular oxygen, is required. Thus, GFP expression may be variable within a tumor mass depending on access to oxygen, making this a rather unreliable marker17. Modification of the murine bladder cancer cell line MB49 to secrete human Prostate Specific Antigen (PSA)15,16 as a surrogate marker is another strategy. These markers also provide an alternative means of confirming tumor presence at the termination of the experiment, making them an alternative to immunohistochemistry. This study reports the PLL method of orthotopic tumor implantation and presents a comparison of tumor detection systems, namely ELISA, fluorescence, and ultrasound imaging.

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:404px;">MB49-PSA cells</td>
			<td style="width:188px;">N/A</td>
			<td style="width:188px;">N/A</td>
			<td style="width:113px;">ref (Wu QH, 2004)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640 media</td>
			<td>HyClone</td>
			<td>SH30027.01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) media</td>
			<td>Biowest</td>
			<td>L0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American, Premium</td>
			<td>Biowest</td>
			<td>S181B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30088.03&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine</td>
			<td>Biowest</td>
			<td>X0550</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin-Streptomycin</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hygromycin B</td>
			<td>Invitrogen</td>
			<td>10687-010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">free PSA (Human) ELISA kit</td>
			<td>Abnova</td>
			<td>KA0209</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TRIzol Reagent for RNA extraction&#160;</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>4374967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Mouse Actb</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Mm00607939_s1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Human KLK3</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Hs00426859_g1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">C57BL/6J female mice</td>
			<td>In Vivos</td>
			<td></td>
			<td>4-6 wk old</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia (75mg/kg Ketamine and 1mg/kg Medetomidine)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reversal drug (1mg/kg Atipamezole)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ear punch</td>
			<td>Electron Microscopy Sciences</td>
			<td>72893-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hartmann&#39;s solution or Compound sodium lactate</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ophthalmic ointment - Duratears sterile ocular lubricant ointment</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat pack - HotHands handwarmers</td>
			<td>Heatmax Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Introcan Certo IV catheter</td>
			<td>B Braun</td>
			<td>4251300</td>
			<td>24G x 3/4&#8243;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aquagel Lubricating jelly</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;Poly-L-lysine solution, 0.01%,</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Quantichrom Creatinine Assay Kit</td>
			<td>BioAssay Systems</td>
			<td>DICT-50&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluorescent dye - VivoTrack 680</td>
			<td>Perkin Elmer</td>
			<td>NEV12000&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNAlater-ICE Frozen Tissue Transition Solution</td>
			<td>Ambion</td>
			<td>4427575</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Realtime PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Software v2.3</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Metabolic Cage</td>
			<td>Tecniplast&#160;</td>
			<td>Vertical type rack for 12 cages</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">BD FACSCanto I system&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSDiva software v7</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IVIS SpectrumCT in vivo imaging system&#160;</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Living Image Software v3.1</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vevo 2100 imaging system&#160;</td>
			<td>VisualSonics&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

PSA secretion from MB49 cells was found to vary with the growth media. MB49-PSA is grown in DMEM media because this results in increased PSA secretion (Figure 1A). In order to determine the sensitivity of the PSA ELISA and real-time PCR, different numbers of MB49-PSA-secreting cells were mixed with MB49 parental cells. PSA ELISA detects a minimum of 1 x 105 PSA-secreting cells/1 x 106 cells (Figure 1B), while real-time PCR analysis d...

Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

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

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 ...

The overall goal of this non-surgical method is to consistently produce orthotopic non-muscle-invasive bladder tumors in mice that can be easily monitored for the evaluation of intravesical therapies. This method can help answer key questions in the field of bladder cancer. Such as the evaluation of novel intravesical therapies.The main advantage of this technique is that it is easy to implant tumors in mice and monitor their growth, which helps to accurately determine responses to therapies. One day before the implantation, when the cells are about 80%confluent passage to the MB49 PSA tumor cells in complete DMEM media with a one to two split. On the day of the procedure, collect and count the cells, making sure they are sufficiently healthy.Mix in equal volume of cell suspension and 4%Trypan Blue, and count the blue-stained live cells using a hemocytometer. The cells should have at least 80%viability. Next, resuspend the cells at two million per milliliter in a 15 milliliter centrifuge tube, and wash them three times in DMEM.Then, keep them chilled until they are to be injected. Once a female mouse has been anesthetized, provide it hydration by an intraperitoneal injection of Hartmanns Solution at 1 milliliters per 10 grams of body weight. Repeat this every one or two hours during the anesthesia.Next, apply sterile ophthalmic ointment to both eyes, reapply as needed. Then, position the mouse supine on paper towels over a heat pad to maintain body tempurature. Next, secure the hind legs with tape.Now apply gentle pressure to the lower abdominal region, and collect urine into a 1.5 milliliter tube to measure the basal level of urinary PSA. Next, load a one-milliliter syringe with sterile PLL and attach a 24-gauge IV catheter with the needle stylet removed. Apply lubricant to the tip of the catheter.Then, insert the catheter into the urethra, and using forceps, guide it to the bladder. Stop when resistance is felt. Then, slowly eject 50 microliters of PLL at a rate of 10 microliters every 20 seconds to avoid vesicoureteral reflux.Now leave the catheter in the bladder for 20 minutes, with a stopper to prevent outflow. After 20 minutes, remove the catheter and vacate the bladder of any contents, by gently pressing on the lower abdomen. Then, using a one-milliliter syringe, flush any remaining contents out of the catheter.Now mix the MB49 PSA cells thoroughly by pipetting, and load them into a 1-mL syringe. Attach the catheter and lubricate it as before. Insert the catheter into the bladder as before.And commence with injecting the cells, using a gradual ejection of 10 microliters per 20 seconds. Then wait one hour. After an hour, remove the catheter and vacate the bladder of any contents.Then, release the mouse, positioning on its ventral side and revive it with an injection of atipamezole. Now, monitor the mouse until it regains sternal recumbency, and then return it to its home cage. PSA secretion of MB49 cells was found to vary with the growth media.DMEM media is used because it resulted in the most PSA secretion. In order to determine the sensitivity of the PSA ELISA, different numbers of MB49 PSA secreting cells were mixed with MB49 parent cells. The assay detected at least 100, 000 PSA-secreting cells per million cells.The PLL method of tumor implantation results in multiple tumors developing in the bladder. Both overnight collections of urine using metabolic cages and spot urine samples could be used to measure PSA by ELISA. For all animal studies, mouse weight was recorded as a measure of health.And urinary PSA is monitored on a weekly basis as a measure of tumor growth. Different therapies were used on each mouse, resulting in different tumor sizes. Two weeks after implantation, the bladders were harvested and processed.Tumor sizes varied, and the PSA measurements predicted this. After watching this video, you should have a good understanding of how to produce orthotropic bladder tumors in mice, and monitor their growth. Once mastered, this technique can be done in two hours per mouse.While attempting this procedure, it's important to remember to ensure cells are mixed before implantation, and to perform the implantation at a slow rate. Following this procedure, other methods, like flow cytometric analysis of Uvm or ELISA can be performed in order to investigate immune activation post-therapy.

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Research

JoVE Journal

Cancer Research

14.4K Views.  National University of Singapore, National University Health System. The overall goal of this non-surgical method is to consistently produce orthotopic non-muscle-invasive bladder tumors in mice that can be easily monitored for the evaluation of intravesical therapies. This method can help answer key questions in the field of bladder cancer. Such as the evaluation of novel intravesical therapies.The main advantage of this technique is that it is easy to implant tumors in mice and monitor their growth, which helps to accurately determine responses to the...