This research is funded by the AACR Henry Shepard Bladder Cancer Research Grant (09-60-30-LI).

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco.
1. Cell Preparation
<ol>
 <li> The human bladder cancer cell line KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) is grown in RPMI-1640 media supplemented with 10% fetal bovine serum and 50 &mu;g/ml gentamicin. </li>
 <li> Cultures are maintained at 37 &deg;C, 5% CO2 with media changes every two to three days. </li>
 <li> Cells were harvested by trypsin digestion and counted using a hemocytometer. A single cell suspension of 2 x 106 cells in 50 &mu;l was prepared in phosphate buffered saline and kept on ice. </li>
</ol>
2. Orthotopic Bladder Tumor Model
<ol>
 <li> Female nude (nu/nu) mice 8-10 weeks old (Simonsen Laboratory, Gilory, CA) were used. </li>
 <li> Place a sterile drape over a heating pad to serve as a surgical platform. Sterile lubricating jelly, 24-guage catheters with stylet needles, silver nitrate solution, and sterile water are prepared. </li>
 <li> General anesthetic is induced in the mice with inhaled isoflurane (3% for induction, 1-2% for maintenance) and maintained via nosecone. Sterile ophthalmic ointment is applied to the animal's eyes, and a heat pad is used to maintain body heat. </li>
 <li> Place the mouse in supine position.</li>
 <li> Gently grasp the animal's vulva with forceps for stabilization. </li>
 <li> Using a 24-guage intravenous catheter with the needle stylet removed, the mouse urethra is catheterized. Ample lubrication should be used. With the mouse supine, the urethra is located immediately posterior to the vulvar folds and anterior to the vagina. A 45-degree angle is initially taken in order to cannulate the urethra and pass beneath the pubic bone. A more shallow angle is then taken to pass through the urethra into the bladder. Gentle pelvic pressure to straighten the urethra can often help. Care must be taken to avoid pushing too hard against resistance, as this can lead to urethral or bladder perforation. Urine seen within the catheter confirms its location within the bladder lumen. </li>
 <li> Aspirate urine from the catheter and bladder lumen using the stylet needle. The stylet needle is used for all subsequent aspirations and injections, leaving the catheter in place. This allows for injection of accurate volumes by eliminating the dead-space within the catheter, and it makes repeated catheterizations unnecessary.</li>
 <li> To impair the glycosaminoglycan layer of the bladder urothelium, 10 &mu;L of 0.5 M silver nitrate is injected and allowed to dwell for 10 seconds. This may form a white precipitate with remaining urine. </li>
 <li> Aspirate and discard the precipitate and bladder contents using a stylet needle. </li>
 <li> Wash the bladder by injecting 100 &mu;L sterile water. Aspirate and discard the water. Repeat wash with sterile water 3 times for a total of 4 washes. </li>
 <li> Place a 4-0 silk tie around the urethral meatus to occlude the urethra in preparation for catheter removal. </li>
 <li> Inject previously prepared 2 x 106 KU-7-luc2-GFP cells in 50 &mu;L using the stylet needle. </li>
 <li> Simultaneously remove the catheter and stylet needle, leaving the urethra occluded by the silk tie. </li>
 <li> The mouse will be maintained under general anesthetic for 2 hours before the silk tie is removed and the animal is awakened. </li>
</ol>
3. Ultrasound 
<ol>
 <li> General anesthetic is induced with vaporized isoflurane and maintained via nose cone. </li>
 <li> A VisualSonics Vevo 770 In Vivo High-Resolution Micro-Imaging System (VisualSonics Inc, Toronto, Ontario, Canada) with the RMV-704 40 MHz probe was used.</li>
 <li> Place the mouse in supine position on the ultrasound platform. </li>
 <li> Secure the animal's hind legs with tape to avoid excess movement during manipulation of the ultrasound probe. </li>
 <li> Apply ultrasound gel to the mouse pelvis. </li>
 <li> Lower a RMV-704 (40 MHz) ultrasound probe onto the mouse pelvis to obtain transverse or longitudinal images. </li>
 <li> If tumor is found, images can be saved for measurement of tumor dimensions. </li>
</ol>
4. Bioluminescent Imaging
<ol>
 <li> Perform intraperitoneal injection of 200 &mu;L (150 mg/kg) luciferin substrate. </li>
 <li> Induce general anesthetic with vaporized isoflurane and maintain via nose cone. </li>
 <li> Place mice in supine position within the bioluminescence imager. </li>
 <li> Five minutes after luciferin injection, measure the bioluminescence from the animal in photons per second. </li>
</ol>
5. saRNA Preparation
<ol>
 <li> Synthesize RNA oligonucleotides at a scale of 50-100 &mu;mol. </li>
 <li> Anneal complementary single-stranded oligonucleotides into duplex saRNAs. </li>
 <li> Lipid-nanoparticle (LNP)-saRNA are prepared with the ionizable lipid 1,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), disteroylphosphatidyl choline, cholesterol, and PEG&#x2013;DMG using a spontaneous vesicle formation formulation procedure as previously described.1 </li>
</ol>
6. Intravesical Administration of Formulated saRNA
<ol>
 <li> After establishing the orthotopic bladder tumor model, mice are treated with intravesical small activating RNA (saRNA) formulated in lipid nanoparticles (LNPs) (Table 1). </li>
 <li> General anesthetic is established and maintained with vaporized isoflurane, and sterile ophthalmic lubricant is applied.</li>
 <li> Place the animal in supine position. </li>
 <li> Perform urethral catheterization as previously described. </li>
 <li> Place a 4-0 silk tie around the urethra and catheter. </li>
 <li> Aspirate all urine from the catheter and bladder using an empty syringe and stylet needle. </li>
 <li> Inject LNP-saRNA intravesically for a total dose of 50 &mu;L (3 mg/kg). Simultaneously remove the catheter and stylet needle, leaving the urethra occluded by the silk tie.</li>
 <li> The mouse will remain anesthetized with the urethra occluded for 2 hours. </li>
 <li> Treatment with intravesical LNP-saRNA was begun on Day 4 after tumor implantation. Mice were treated serially, every 3 days, for a total of 14 treatments.</li>
</ol>
7. Representative Results
Bladder tumors stemming from KU-7-luc2-GFP cells can be monitored by luciferase bioluminescent imaging (Figure 1A and C) and bladder ultrasound (Figure 1B and D). Tumors are often detectable within 3-5 days after cell inoculation. We found successful bladder tumor implantation in over 90% of mice. As treatment with intravesical dsRNA ensues, the progression of tumor can be monitored with these modalities. After the animal is sacrificed, tumor growth can also be confirmed using GFP fluorescence. In general, mean bioluminescence correlated with mean ultrasound dimensions for bladder tumors, as seen visually in Figures 1C and 1D. However, after animals were sacrificed and tumor was confirmed by GFP, we found that bioluminescence correlated more closely with the presence of viable tumor than did ultrasound measurements. This was especially apparent among mice treated with saRNA (as opposed to controls) because residual scar tissue in treated mice was often visualized by ultrasound but could not be differentiated from live tumor. 
<img src="/files/ftp_upload/4207/4207fig1.jpg" alt="Figure 1" /> 
Figure 1. Tumor growth kinetics of bladder tumors. (A) Bioluminescence imaging measures the luciferase activity within bladder tumors to confirm their location and growth. (B) In vivo bladder ultrasound using a 40MHz probe produces tumor images whose dimensions can be accurately measured. (C) Graphs illustrating the tumor's progressive increase in bioluminescence and ultrasound dimensions. Values represent mean (+/- standard deviation) measurements from 6 animals. <a href="https://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <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 international. 100, 1377-1384 (2007).</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), e2523 (2011).</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.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <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> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

L.C.L. is a named inventor on pending patent applications related to saRNA which have been filed by the University of California San Francisco and licensed to Alnylam Pharmaceuticals.

We present a protocol for the establishment of a mouse orthotopic xenograft bladder tumor model followed by intravesical treatment of the tumors with LNP formulated saRNA that targets the promoter of p21CIP1/WAF1 (p21) for transcriptional activation.2, 3 The resultant tumors can be confirmed and monitored with bioluminescent imaging and bladder ultrasound. Orthotopic models may be superior to intraperitoneal, subcutaneous, or intravenous models by providing a milieu of urine and urothelium that mor...

<table border="1">
<tbody>
 <tr>
 <td> Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>KU-7-luc-GFP cells</td>
 <td>Caliper</td>
 <td>128091</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> thymic 			nude mice ( nu/nu) </td>
 <td>Simonsen Laboratories</td>
 <td>6-7 weeks old</td>
 <td>Sim:(NCr) 			nu/nu fisol</td>
 </tr>
 <tr>
 <td>Ultrasound unit</td>
 <td>Visualsonics</td>
 <td>Vevo 770</td>
 <td>RMV-704 probe</td>
 </tr>
 <tr>
 <td>Bioluminescence unit</td>
 <td>Caliper</td>
 <td>IVIS Spectrum</td>
 <td></td>
 </tr>
 <tr>
 <td>Exel safelet catheter</td>
 <td>Fisher</td>
 <td>14-841-21</td>
 <td>24G X &frac34;"</td>
 </tr>
 <tr>
 <td>Silver nitrate</td>
 <td>Sigma</td>
 <td>S8157</td>
 <td></td>
 </tr>
 <tr>
 <td>Hamilton syringe</td>
 <td>Hamilton company</td>
 <td>80301</td>
 <td></td>
 </tr>
 <tr>
 <td>LNP-saRNA</td>
 <td>Alnylam Pharmaceuticals</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>
Table 1. Specific reagents and equipment.

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.

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

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

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 ...

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14.6K Views.  University of California, San Francisco. Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

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

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 ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

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 ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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.

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 ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

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.

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

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 ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...