The authors would like to acknowledge Eliana Beraldi for performing viral transduction of tumor cell lines and Ben Deeley for his instruction on use of the small animal ultrasound imaging platform.
This project was supported by the German Foundation System (DFG; JA 2117/1&#8211;1:1), the Canadian Cancer Society Research Institute and a Mentored Physician Scientist Award from Vancouver Coastal Health Research Institute. The ultrasound imaging platform was funded by the Canadian Foundation for Innovation.

All animal procedures were performed according to the guidelines of the Canadian Council on Animal Care (CCAC). The protocol was approved by the Animal Care Committee of the University of British Columbia (Protocol Number: A10-0192).
1. Preparation of Cell Lines
<ol>
	<li>Confirm the identity of the respective human bladder cancer cell lines by DNA fingerprinting7.</li>
	<li>For growth analysis of xenograft tumors by bioluminescence, transfect cell lines with a lentiviral construct carrying the firefly luciferase gene3.</li>
	<li>Thaw and expand the existing cell lines in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 &#176;C in a humidified 5% CO2 atmosphere. Passage the cells at least 3x but avoid culture times exceeding 3 months.</li>
</ol>
2. Preparation of Cell Suspension
<ol>
	<li>Thaw Matrigel. Keep the temperature below 4 &#176;C to avoid increased viscosity of the gel.</li>
	<li>Trypsinize the cells at a confluence of 70% and suspend in normal growth media.</li>
	<li>Count the cell number with a hemocytometer or automatic cell counter.</li>
	<li>Spin the cell suspension for 5 min at 200 x g. Remove the supernatant.</li>
	<li>Add the appropriate volume of DMEM (10% FBS) and Matrigel (1:1 ratio) in order to reach the desired cell concentration which is dependent on the utilized cell line and desired growth kinetics (8-15 x 106/ml). The injected volume of tumor cell suspension will be 40 &#181;l.</li>
	<li>Mix well by pipetting up and down (P1000), avoid creating air bubbles in the suspension.</li>
</ol>
3. Preparation of Animals
Note: Due to the potential need for transurethral catheterization in step 4.7, female mice are the preferred gender in this animal model.
<ol>
	<li>House mice according to institutional and national animal care guidelines. Obtain ethics committee approval for all experiments involving mice.</li>
	<li>Anesthetize the mice with 3% isoflurane/oxygen mixture. Confirm proper anesthetization of animals (e.g. unresponsiveness to toe pinches).</li>
</ol>
4. Experimental Setup
<ol>
	<li>Cut the bladder stabilization from any kind of flat rigid plastic material [Figure&#160;2 I]. Carefully inspect the strap and remove any sharp edges before application to the mice.</li>
	<li>Mount the animal on the heated imaging table [Figure&#160;1 I] of the small animal imaging platform with continuous monitoring of vital signs. Fix the lower limbs with a rubber band [Figure&#160;1 III].</li>
	<li>Disinfect the abdomen with 2% chlorhexidine gluconate and wipe the skin with a sterile cotton tip.</li>
	<li>Immobilize the bladder with the bladder stabilization strap [Figure&#160;2 II]. Thus, an evasion of the bladder during intramural injection in step 5.6 will be avoided.</li>
	<li>Apply sterile ultrasound gel to the lower abdomen.</li>
	<li>Slowly approach the ultrasound scanhead (frequency 40 MHz) [Figure&#160;1 IV] to the skin (longitudinal with a cranial angle of 45-70&#176;) and visualize the bladder on the ultrasound screen [Figure&#160;3 I].</li>
	<li>If the bladder is empty, fill it with 50 &#181;l&#160;sterile, warm phosphate-buffered saline (PBS) through a transurethral 24 G&#160;angiocatheter.</li>
</ol>
5. Separation of Bladder Wall Layers
<ol>
	<li>Attach a 1.0 ml&#160;syringe filled with PBS and connected to a 30 G, &#190; in needle (bevel directed anteriorly) to the syringe clamp.</li>
	<li>Position the needle towards the skin just above the pubic bone in a 30-45&#176; angle (80-90&#176; relative to the longitudinal axis of the ultrasound scanhead [Figure&#160;1]).</li>
	<li>Detect the needle on the ultrasound screen.</li>
	<li>Slowly perforate the skin and the abdominal wall muscles [Figure&#160;3 II].</li>
	<li>Turn the bevel of the needle 180&#176; (now directed posteriorly).</li>
	<li>Insert the tip of the needle into the bladder wall without penetrating the mucosa [Figure&#160;3 III]</li>
	<li>Slowly inject 50 &#181;l of PBS between the muscular layer and the mucosa in order to create an artificial space [Figure&#160;3 IV]. 
	Note: If the mucosa is accidently perforated during step 5.6, slowly pull back the needle and inject 50 &#181;l of PBS after the mucosal layer has flipped back over the needle tip.</li>
	<li>Withdraw the needle.</li>
</ol>
6. Intramural Inoculation of Bladder Cancer Cells
<ol>
	<li>Attach a second 1.0 ml&#160;syringe (filled with cancer cells suspended in Matrigel) with a 30 G, &#190; in needle to the syringe clamp.</li>
	<li>Guide the tip of the needle to the same PBS-filled space which was created in step 5.7.</li>
	<li>Inject 40 &#181;l&#160;of the cell suspension into this space [Figures&#160;3 V and&#160;VI].</li>
	<li>Withdraw the needle.</li>
</ol>
7. Post-interventional Supportive Care
<ol>
	<li>Dismount the mouse from the imaging platform.</li>
	<li>Keep the animal in a warm and comfortable environment under continuous surveillance while recovering from the anesthetic.</li>
	<li>After regaining consciousness and resuming normal ambulation, place the animal back in its home cage.</li>
</ol>

<ol>
	<li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+orthotopic+models+for+bladder+cancer+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+models+of+human+cancer+xenografted+in+the+nude+mouse:+the+importance+of+orthotopic+transplantation.">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>Hadaschik, B. A., 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=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU Int. 100, 1377-1384 (2007).</li><li>Kang, M. 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=An+Orthotopic+Bladder+Tumor+Model+and+the+Evaluation+of+Intravesical+saRNA+Treatment.">An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment.</a> J. Vis. Exp. (65), (2012).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder+Cancer.">An Orthotopic Model of Murine Bladder Cancer.</a> J. Vis. Exp. (48), (2011).</li><li>Dinney, C. P., 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=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> J. Urol. 154, 1532-1538 (1995).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), (2011).</li><li>Xiao, Z., 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=Characterization+of+a+novel+transplantable+orthotopic+rat+bladder+transitional+cell+tumour+model.">Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model.</a> Br. J. Cancer. 81, 638-646 (1999).</li><li>Iinuma, S., Bachor, R., Flotte, T., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+and+phototoxicity+of+5-aminolevulinic+acid-induced+PpIX+in+an+orthotopic+rat+bladder+tumor+model.">Biodistribution and phototoxicity of 5-aminolevulinic acid-induced PpIX in an orthotopic rat bladder tumor model.</a> J. Urol. 153, 802-806 (1995).</li><li>J&#228;ger, W., 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=Hiding+in+plain+view:+Genetic+profiling+reveals+decades+old+cross-contamination+of+bladder+cancer+cell+line+KU7+with+HeLa.">Hiding in plain view: Genetic profiling reveals decades old cross-contamination of bladder cancer cell line KU7 with HeLa.</a> J. Urol. (13), (2013).</li><li>Horiguchi, Y., Larchian, W. A., Kaplinsky, R., Fair, W. R., Heston, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravesical+liposome-mediated+interleukin-2+gene+therapy+in+orthotopic+murine+bladder+cancer+model.">Intravesical liposome-mediated interleukin-2 gene therapy in orthotopic murine bladder cancer model.</a> Gene Ther. 7, 844-851 (2000).</li><li>Dinney, C. P., 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=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> J. Urol. 154, 1532-1538 (1995).</li><li>Black, P. 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=Validating+bladder+cancer+xenograft+bioluminescence+with+magnetic+resonance+imaging:+the+significance+of+hypoxia+and+necrosis.">Validating bladder cancer xenograft bioluminescence with magnetic resonance imaging: the significance of hypoxia and necrosis.</a> BJU Int. 106, 1799-1804 (2010).</li></ol>

Open access for this video-article is sponsored by&#160;FUJIFILM VisualSonics, Inc.

Almost all major advances in cancer therapy will require testing in animal models before initiating clinical trials. Animal models of cancer are essential tools that enable researchers to study tumor biology in vivo. Orthotopic xenograft models remain the gold standard1,2 and continue to offer the most flexibility (in terms of selection of cell lines) and have the most practical utility.
The illustrated procedure is a minimally invasive modification of the orthotopic model previously described by Dinney et al.12 We established xenograft tumors by ultrasound guided percutaneous injection of three different cell lines with a technical success rate of 100%. During continuous follow-up, 98% of mice demonstrated constant increase in tumor volume.
By performing a minimally-invasive technique we were able to address existing limitations of the intramural model. Besides respecting animal welfare, the reduced invasiveness of this procedure also contributes to reproducibility of in vivo experiments by decreasing the number of surgical complications. It is highly time effective to avoid an abdominal laparotomy and associated need for wound closure. We were able to decrease significantly the procedure time per animal to 3.4 min (&#177;1.6). However, the chief advantage of our novel approach is its accuracy. High-resolution ultrasound allows us to visualize the space created by saline injection under the mucosa of the bladder wall. This first step injection facilitates tumor cell injection in a second step and minimizes the risk of tumor cell spillage. This contrasts to the technique of intramural injection after laparotomy, where it is impossible to visualize needle placement and there is always an element of uncertainty regarding the exact depth of injection. Also, as we are inoculating tumor cells strictly into the anterior bladder wall, tumor growth on the posterior bladder wall is avoided. Subsequently the rate of obstructive complications due to tumor growth in proximity of the ureteral orifices is extremely rare. This accompanying effect allows longer growth and treatment periods.
The main limitation of the ultrasound-guided tumor inoculation is the need for adequate technical equipment. Therefore the performance of this procedure will likely be restricted to centers which are specialized in animal models of human cancer. This should encourage collaborations between research groups outside of these institutions and the groups with expertise in such novel animal modeling.
Although dependent on familiarity with ultrasound imaging and some manual dexterity, this model is easy to learn under competent instruction. The key step in the procedure is the creation of an artificial space submucosally in the bladder wall with saline. Once this space is created without perforation of the mucosal layer, it remains stable for several minutes. The guidance of the second needle into this space in order to inoculate the tumor cells is relatively simple. The main complication during the creation of the submucosal space is perforation of the needle into the bladder lumen. The creation of a submucosal space, however, remains feasible. The needle has to be withdrawn slowly into the bladder wall and the saline injected just when the mucosal layer flips over the tip of the needle. After this maneuver the submucosal space is less stable (saline will escape to the bladder lumen within 30-60 sec) and the injection of the tumor cells has to be performed quickly. Spillage of tumor cells into the bladder lumen can occur in these cases with perforation of the mucosa. Although the loss of tumor cells from the intramural space might lead to a decreased tumor volume during follow-up, we have never observed any intravesical tumor uptake.
Another potential complication is the spillage of tumor cells to the peritoneal cavity through the injection channel. We observed only one intraperitoneal tumor cell dissemination in 50 animals, and this occurred in one of our first attempts. We attribute this to the injection of too large a tumor cell suspension volume. This was supported by the fact that reducing the volume from 50-40 &#181;l&#160;resulted in no further intraperitoneal spillage.
This minimally invasive inoculation of murine orthotopic bladder cancer xenograft represents an innovative modification of the existing &#8220;intramural model&#8221;, benefitting both the investigator and the animals equally. The advantages of this model encourage its adaptation to other organs such as kidney, prostate and liver in order to establish orthotopic xenograft tumors in a minimally-invasive fashion.

Cancer research is dependent on animal models of human cancer using cell lines derived from patient tumors in order to deepen our understanding of tumor biology. For in vivo growth analysis under different treatment strategies murine orthotopic bladder cancer models remain the reference standard1,2. The inoculation of human bladder cancer cells in immunocompromised mice (xenograft model) relies on intravesical instillation (&#8220;intravesical model&#8221;)3,4,5 or direct injection into the bladder wall (&#8220;intramural model&#8221;)6,7. Both techniques can also be performed in rats8,9.
Intravesical instillation induces the formation of tumors on the urothelial surface of the bladder which then are amenable to subsequent intravesical instillation of novel treatment agents. However, the number of cell lines which are reliably tumorigenic when delivered via this method is limited and one of those cell lines, KU7, has recently been demonstrated to be HeLa4,10. Intravesical instillation is also time consuming due to necessary dwell times, and it frequently induces tumor growth in adjacent elements of the urinary tract, including urethra, ureter, and renal pelvis11. Furthermore, intravesical instillation often leads to tumor growth on the floor of the bladder where the ureters enter the bladder, and this can cause upper tract obstruction and concomitant renal failure.
Primary invasive bladder cancer xenografts which are suitable for systemic treatments are created by direct injection of tumor cells into the bladder wall12. Although numerous cell lines grow adequately in this model, its limitation is the invasiveness of the model related to the need for an abdominal incision13. The model is also challenging to learn due to the technical difficulty of injecting cells precisely into the muscle wall of the bladder.
A novel approach to establish orthotopic primary invasive bladder cancer xenografts in mice has been developed in our department in order to address existing shortcomings of the &#8220;intramural model&#8221;. We were able to optimize the percutaneous, ultrasound-guided injection of bladder cancer cells into the anterior bladder wall resulting in this novel technique to successfully replace the established invasive model. Moreover we have potentially enhanced the accuracy and reproducibility of the &#8220;intramural model&#8221;.

<table><tbody><tr><td>Chlorhexidine gluconate (2%)</td><td>Aplicare</td><td>82-319</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Thermo Scientific</td><td>SH3008101</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Scientific</td><td>SH3007103</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter Corporation</td><td>402-069-02</td><td></td></tr><tr><td>Trypsin (0.25%)</td><td>Thermo Scientific</td><td>SH3004202</td><td></td></tr><tr><td>Syringe (1 ml)</td><td>BD Bioscience</td><td>309659</td><td></td></tr><tr><td>Hypodermic needle (30 G; &amp;frac34; in)</td><td>Kendall</td><td>830340</td><td></td></tr><tr><td>Angiocatheter (24 G)</td><td>BD Bioscience</td><td>381112</td><td></td></tr><tr><td>Vevo 770 small animal imaging platform</td><td>VisualSonics</td><td></td><td></td></tr><tr><td>RMV 706 ultrasound scanhead</td><td>VisualSonics</td><td></td><td></td></tr><tr><td>IVIS Lumina III</td><td>Caliper Life Science</td><td></td><td></td></tr></tbody></table>

minimally invasive establishment of murine orthotopic bladder xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

Intramural injection of three different tumor cell lines (UM-UC1 luc, UM-UC3 luc and UM-UC13 luc) was performed in 50 animals under ultrasound-guidance on three consecutive days. The inoculation was performed efficiently (mean time 5.7 min/animal) and was not associated with any intra- or post-interventional complications.
Monitoring of tumor growth was performed by ultrasound imaging and bioluminescence. On day #3 a tumor could be detected by ultrasound in the anterior bladder of all 50 animals [Figure&#160;4 I]. 98% of mice showed constant tumor growth during the follow-up period [Figures&#160;4 and&#160;5]. Following the inoculation of UM-UC3 luc, one mouse developed intraperitoneal tumor dissemination and the tumor involuted after day #7 in a second animal [Table&#160;1]. This was the first group of mice inoculated with this new technique.
The mice were sacrificed on day #24, #28, and #37 after inoculation of UM-UC3 luc, UM-UC1 luc and UM-UC13 luc, respectively. Xenograft tumors were harvested and examined on hematoxylin and eosin (H&#38;E) sections. All tumors were muscle invasive and some infiltrated into the perivesical fat, but no invasion into adjacent organs was observed [Figure&#160;6 I]. 60% of mice bearing UM-UC13 luc tumors and 20% of mice bearing UM-UC3 luc tumors developed retroperitoneal lymph node metastases which were confirmed by H&#38;E staining [Figure&#160;6 II].
<img alt="Figure 1" fo:content-width="4in" fo:src="/files/ftp_upload/51123/51123fig1highres.jpg" src="/files/ftp_upload/51123/51123fig1.jpg" /> 
Figure 1.&#160;Image and schematic illustration of the experimental setup. The mouse is mounted on the heated operation table (I) and held under anaesthesia (II) with 3% isoflurane/oxygen mixture. The lower limbs are fixed with a rubber band (III). After approaching the ultrasound scanhead (IV) to the skin (longitudinal alignment with a cranial angle of 45-70&#176;) the bladder (V) is visualized on the ultrasound screen. A syringe with a 30 G needle (VI) is guided to the skin in an angle of 30-45&#176; (80-90&#176; relative to the longitudinal axis of the ultrasound scanhead).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51123/51123fig2highres.jpg" src="/files/ftp_upload/51123/51123fig2.jpg" /> 
Figure 2.&#160;Immobilization of the bladder. Dimensions and illustration to construct the bladder stabilization strap (I) The strap is attached to the lower abdomen and immobilizes the bladder (II). Thus an evasion of the bladder during intramural injection is avoided.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51123/51123fig3highres.jpg" src="/files/ftp_upload/51123/51123fig3.jpg" /> 
Figure 3.&#160;Intramural inoculation of tumor cells. Visualization of the bladder on the ultrasound screen (I). Perforation of the skin and abdominal wall muscles (II). Needle insertion into the bladder wall without penetration of the mucosa (III). PBS (50 &#181;l) between the muscular layer and the mucosa after slow injection (IV). Tumor cells suspended in Matrigel in the intramural artificially created space (V, VI).
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51123/51123fig4highres.jpg" src="/files/ftp_upload/51123/51123fig4.jpg" /> 
Figure 4.&#160;Follow-up by ultrasound. Continuous follow-up by ultrasound showed significant increase in tumor volume (I: day #3, II: day #7, III: day #13).
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51123/51123fig5highres.jpg" src="/files/ftp_upload/51123/51123fig5.jpg" /> 
Figure 5.&#160;Follow-up by bioluminescence. Continuous follow-up by bioluminescence showed constant increase in luminescence over the study period.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51123/51123fig6highres.jpg" src="/files/ftp_upload/51123/51123fig6.jpg" /> 
Figure 6.&#160;Histology of xenograft tumor and lymph node metastasis. In toto H&#38;E section of a representative xenograft tumor demonstrating invasive growth into the muscle without invasion into adjacent organs (I). 60% of mice bearing UM-UC13 luc tumors and 20% of mice bearing UM-UC3 luc tumors presented retroperitoneal lymph node metastases (II).
<table border="0" cellpadding="0" cellspacing="0" width="320">
	<tbody>
		<tr>
			<td>Inoculated cell line</td>
			<td></td>
			<td>UM-UC1 luc</td>
			<td>UM-UC3 luc</td>
			<td>UM-UC13 luc</td>
		</tr>
		<tr>
			<td>Number of mice</td>
			<td></td>
			<td>20</td>
			<td>15</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Volume injected, &#181;L</td>
			<td></td>
			<td>40</td>
			<td>50</td>
			<td>50</td>
		</tr>
		<tr>
			<td>Cell count, absolute</td>
			<td></td>
			<td>3.6 x 105</td>
			<td>6 x 105</td>
			<td>5.5 x 105</td>
		</tr>
		<tr>
			<td>Time per animal, min&#160;</td>
			<td></td>
			<td>3.4 (&#177;1.6)</td>
			<td>7.7 (&#177;3.7)</td>
			<td>6.8 (&#177;2.9)</td>
		</tr>
		<tr>
			<td>Tumor incidence</td>
			<td></td>
			<td>49 (98%)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>20 (100%)</td>
			<td>14 (93%)</td>
			<td>15 (100%)</td>
		</tr>
		<tr>
			<td>Lymph node metastasis</td>
			<td></td>
			<td>0</td>
			<td>3 (20%)</td>
			<td>9 (60%)</td>
		</tr>
		<tr>
			<td>Follow up (days)</td>
			<td></td>
			<td>28</td>
			<td>22 [before treatment]</td>
			<td>28 [before treatment]</td>
		</tr>
		<tr>
			<td>Tumor volume (&#956;L)</td>
			<td>day 4</td>
			<td>11.6 (&#177;1.3)</td>
			<td>12.5 (&#177;1.7)</td>
			<td>14.4 (&#177;1.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>end of</td>
			<td>394.6 (&#177;72.4)</td>
			<td>288.7 (&#177;66.1)</td>
			<td>78.3 (&#177;13.4)</td>
		</tr>
		<tr>
			<td>follow-up</td>
		</tr>
		<tr>
			<td>Tumor luminescence (Photons / sec)</td>
			<td>day 4</td>
			<td>4.6 x 108</td>
			<td>2.0 x 108</td>
			<td>5.8 x 108</td>
		</tr>
		<tr>
			<td>(&#177;9.4 x 107)</td>
			<td>(&#177;3.7 x 107)</td>
			<td>(&#177;1.3 x 108)</td>
		</tr>
		<tr>
			<td></td>
			<td>end of</td>
			<td>1.9 x 1010</td>
			<td>1.4 x 1010</td>
			<td>1.5 x 1010</td>
		</tr>
		<tr>
			<td>&#160;follow-up</td>
			<td>(&#177;4.0 x 109)</td>
			<td>(&#177;2.3 x 109)</td>
			<td>(&#177;1.9 x 109)</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Ultrasound-guided tumor cell injection - procedure and results.

Watch this Scientific Journal Video about Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts at JoVE.com

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

minimally-invasive-establishment-murine-orthotopic-bladder

Research

JoVE Journal

Medicine

15.8K Views.  University of British Columbia. The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Video: Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

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.

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 ...

Cancer Research

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

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.

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

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Patient-derived Tumor Xenografting: A Technique to Generate Experimental Mouse Model for Evaluating Urothelial Cell Carcinoma

Source: Moret, R. et al. <a href="https://www.jove.com/video/59223" target="_blank">Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.</a> J. Vis. Exp. (2019)
This video describes the procedure to generate an experimental xenograft murine model, which can be used for studying urothelial cell carcinoma progression.

Source: Moret, R. et al. Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and ...

JoVE Encyclopedia of Experiments

Urinary Tract Cancer

In vivo studies

Bladder Tumor Organoid Model: A Method for Orthotopic Transplantation of Bladder Organoids into Recipient Murine Bladder

To prepare the bladder tumor organoids for orthotopic transplantation, add 500 microliters of collagenase/dispase to the organoid medium in a 24-well plate. Pipette the basement membrane matrix and the medium up and down. Then, incubate the plate for 20 minutes at 37 degrees Celsius.
After the incubation, collect the cells into a 15-milliliter tube and add 5 milliliters of pre-warmed DMEM. Then, centrifuge the tube at 400 x g for 3 minutes at 4 degrees Celsius and aspirate the supernatant. Resuspend the pellet with 1 milliliter of DMEM and transfer the solution to a 90-millimeter Petri dish.
Under a microscope, pick up 10 to 100 tumor organoids with a P200 pipette and collect them into a microtube on ice. Centrifuge the microtube and carefully remove the supernatant. Then, maintain the pellet on ice until the mice are ready for surgery. Prior to submucosal bladder wall transplantation, keep the syringes, pipette tips, and basement membrane matrix on ice until ready to use.
After the mouse has been anesthetized, lay it in a supine position and maintain anesthesia by mask inhalation of 2% vaporized isoflurane. Apply povidone-iodine with a sterile gauze and wipe it down with 70% ethanol. Make a small transverse incision in the skin and muscular wall of the lower midline abdomen with sterile surgical scissors. Expose the bladder from the abdominal cavity and support it with a saline-soaked cotton swab.
Resuspend the organoid pellets in 80 microliters of organoid medium containing 50% high concentration basement membrane matrix and inject the suspension into the anterior aspect of the bladder dome using the insulin syringe. Close the incision with a 4-0 nylon suture and disinfect the surgical site with povidone-iodine and 70% ethanol. Allow the mouse to recover under an infrared irradiator for 10 to 15 minutes. Then, monitor the mouse again until it regains consciousness.

EoE Sub Articles

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

Once a female mouse has been anesthetized, provide hydration by an intraperitoneal injection of Hartmann&#39;s solution at 0.1 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 temperature. 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.
Next, load a 1-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 1-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-milliliter 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. Position it 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.