We thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript. This work was supported by the National Cancer Institute at the National Institutes of Health through grant numbers RO1CA160079, RO1CA138642, UO1CA184966 and VA funded program project number 1P1 BX001604.

Protocols for all procedures involving animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC). Follow officially approved procedures for the care and use of laboratory animals. Intra-prostatic injection requires open-abdominal surgery and animals should be kept in a pathogen-free environment with a designated surgery room where proper surgical aseptic techniques are used during the entire procedure.
1. Preparation of Cells for Implantation
NOTE: Based on research need, any prostate cancer cell line can be used. Cell lines are cultured according to the supplier&#39;s instructions.
<ol>
	<li>For the PC3M-Luc-C6 cell line that stably expresses the firefly luciferase gene, culture cells in Minimum Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS), 1x Non-essential aminoacids, 1x Phleomycin D1 and 1mM sodium Pyruvate. Maintain cells in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37 &#176;C. PC3M-Luc-C6 cells were procured from UCSF core facility.</li>
	<li>Harvest cells by trypsinization. Wash culture plates once with phosphate buffered saline (PBS). Add 2 ml 0.05% Trypsin to a 10 cm dish and incubate for 3-5 min in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37 &#176;C till cells are detached.
	<ol>
		<li>To avoid clumping, do not agitate the cells by hitting or shaking the dish while waiting for cells to detach. Collect the cells in 5 ml of complete media and spin down for 5 minutes at 200 x g. Wash the cell pellet with PBS to remove trypsin.</li>
	</ol>
	</li>
	<li>Enumerate live cells by trypan blue exclusion assay. Mix 10 &#181;l of the cell suspension in PBS with 10 &#181;l of 0.4% (wt/v) trypan blue solution. Load the mixture into a hemocytometer or counting chamber slide and count cells immediately under a microscope or read the chamber slide in a cell counter.
	<ol>
		<li>Prepare a cell suspension containing 2.5 x 105 cells in 10 &#181;l media. Mix the cell suspension with 10 &#956;l basement membrane-like extracellular matrix extract and place the cells on ice. Add luciferin to the cell suspension (1:200 &#181;l; stock 30 mg/ml concentration) before injecting into mice. 
		NOTE: This allows immediate bio-imaging of animals to check the consistency of cell injections among different experimental groups. Inject cells as quickly as possible, preferably within 30 min after trypsinization since cell viability decreases rapidly after detachment.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Surgical Area
<ol>
	<li>Perform surgery in an uncluttered, disinfected area that promotes asepsis.</li>
	<li>Sanitize the counter/lab bench with bleach solution before surgery. 
	NOTE: The use of alcohol is discouraged due to long contact time required to take effect (15 min).</li>
	<li>Use sterile drapes, clean absorbable pads or towels, and replace these materials after each surgical session. Sterilize all instruments prior to use. Preferred methods are a steam autoclave, glass bead sterilizer, ethylene oxide gas, or hydrogen peroxide vapor sterilization.</li>
	<li>Use an aseptically cleaned dissecting microscope to perform the surgical procedure or experienced researchers can perform it without a microscope.</li>
</ol>
3. Implantation of Tumor Cells
<ol>
	<li>Use male 6-8 week old immunocompromised Balb/c or NOD/SCID mice. 
	NOTE: It is more difficult to operate on smaller animals and larger animals tend to have slower kinetics of tumor growth and metastasis.</li>
	<li>Inject pre-surgery pain medication according to the animal facility&#39;s instructions. For example, intra-peritoneal Buprenorphine at a dose of 0.1 mg/kg body weight can be used.</li>
	<li>Anesthetize animals by placing them into an isoflurane chamber with 1-3% isoflurane in oxygen and wait until animals are fully anesthetized. Ensure that there is no toe reflex of muscle tone at this point. Use proper veterinary ophthalmic ointment lubricant to prevent blindness due to xerophthalmia during general anesthesia. 
	NOTE: Anesthetize the animals by the investigator&#39;s preferred method e.g., for Pentobarbital Sodium, 0.05 mg per gram body weight is administered intra-peritoneal or Ketamine/Xylazine solution (concentration: 17.16 mg/ml) at a dose of 65 mg/kg body weight is used subcutaneously. Isoflurane inhalation is a preferred method of anesthetization.&#160;The eye ointment should be gently applied without rubbing against the cornea.</li>
	<li>Remove the animal from the isoflurane chamber and place into a nose cone apparatus with continuous flow of 1-2% isoflurane in oxygen to ensure that the animal is under full anesthesia before proceeding.
	<ol>
		<li>Remove hair from mice by shaving or use a hair removing cream before beginning the procedure. 
		NOTE: It would be preferable to place the mouse on a sterile heating pad during surgery.</li>
	</ol>
	</li>
	<li>Place mouse in a supine position. Clean the lower abdomen with 10% w/w povidone-iodine solution followed by 70% ethanol swabs.</li>
	<li>With a pair of fine forceps, lift an area of skin 2 mm above the preputial gland, about 1-2 cm above the penis sheath, and about 2-3 cm below the bottom of the rib cage.</li>
	<li>Make a midline incision 1cm in length, first through the skin with a scalpel and then through the muscle layer with a scissor (Figure 1).</li>
	<li>Locate the bladder in the body cavity. It is a yellow-light brown spherical organ, located directly under the incision (Figure 1).</li>
	<li>With a pair of fine forceps grip the bladder and lift upward then downward out of the body cavity towards the penis sheath. This will expose the two seminal vesicles which are a pair of white saclike organs and clearly distinct.</li>
	<li>With a cotton swab in each hand, externalize the seminal vesicles, one by one, and pull them out of the body cavity and lay them face down on the outer surface of the abdomen with the bladder in the middle (Figure 2).</li>
	<li>Using the cotton swabs, gently tilt back the seminal vesicles at the point of insertion near the bladder neck, towards the penis sheath, so that the two dorsal prostate lobes are clearly visible. Use wet cotton swabs to avoid tissue damage (Figure 3).</li>
	<li>Agitate the cell suspension with a micropipette before loading in the syringe.</li>
	<li>While placing the seminal vesicles in position with a cotton swab, insert the syringe needle into the dorsal prostatic lobe under the microscope (Figure 4). Slowly inject 20 &#181;l of cell suspension until a bulla formation is identified. A bulging bulla indicates that the injection is correct.</li>
	<li>While retracting the needle, press lightly on the injection site with a cotton swab and hold for a few seconds to prevent leakage.</li>
	<li>Carefully lift the seminal vesicles with cotton swabs and insert them back in to the body cavity one by one followed by the bladder. Avoid &#39;twirling&#39; the internal organs while performing this procedure.</li>
	<li>After placing the organs back into the body cavity, suture the muscle layer first in an interrupted pattern with absorbable 4-0 chromic catgut sutures followed by the skin closure with non-absorbable 4-0 nylon surgical suture. The skin can also be pulled together and closed with surgical clamps to close the incision completely. 
	NOTE: Mice have a habit of scratching and biting at their wound, which may lead to re-opening of the wound, hence use of tissue glue is also recommended along with sutures. Image animals immediately to assure that there is equal bioluminescent intensity among all experimental groups.</li>
	<li>Return the animals to clean cages and keep them under a warming lamp or heating pad. Monitor the animals constantly until they completely recover from anesthesia and maintain sternal recumbency.</li>
</ol>
4. Monitoring of Animals
<ol>
	<li>Monitor the animals regularly until the end of the experiment, according to institutional protocols. If using surgical metal clips, remove after one to two weeks. Duration of the experiment depends on the specific research need. 
	NOTE: This experiment was conducted for 21 days to examine the successful establishment of the implanted cancer cells in the mouse prostate.</li>
	<li>Administer pain medication based on the animal facility&#39;s instructions. For example, intra-peritoneal Buprenorphine at a dose of 0.1 mg/kg body weight can be used at the time of the procedure with a second dose after 6 hr and additional doses every 8-12 hr as needed.</li>
	<li>Monitor animal weight, food consumption, skin color and texture, activity and frequency of urination and defecation. Euthanize animals immediately if there is a significant loss of body weight greater than 15%.
	<ol>
		<li>For euthanasia, deliver CO2 from a pressurized tank into an un-crowded cage at a flow rate to displace 10-30% of the chamber or cage volume/minute, allowing CO2 to enter the chamber slowly so that unconsciousness and complete narcotization occur prior to death.</li>
		<li>Maintain CO2 flow for at least one minute after respiratory arrest and leave animals in the chamber for a sufficient time so that death occurs prior to performing a physical method.</li>
		<li>Perform decapitation, cervical dislocation or any other IACUC approved physical method after euthanizing the animals chemically. 
		NOTE: Significantly reduced body weight often indicates a lethargic condition. Tumor bearing animals should be in good health except for the presence of tumors until the end of the experiment. Euthanasia should be consistent with the AVMA Guidelines on Euthanasia and must be listed in the approved IACUC protocol.</li>
	</ol>
	</li>
</ol>
5. Non-invasive Bio-imaging of Animals
<ol>
	<li>Monitor the animals weekly using a non-invasive imaging technique to track colonization of cancer cells, tumor growth and any distant metastasis. 
	NOTE: Imaging modalities such as GFP-imaging, Luciferase imaging, X-rays or 3D micro-computed tomography (uCT) etc. can be used based upon the specific research need12-15.</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2015.">Cancer statistics, 2015.</a> CA Cancer J Clin. 65 (1), 5-29 (2015).</li><li>Andrieu, 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=Heat+shock+protein+27+confers+resistance+to+androgen+ablation+and+chemotherapy+in+prostate+cancer+cells+through+eIF4E.">Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E.</a> Oncogene. 29 (13), 1883-1896 (2010).</li><li>Fusi, 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=Treatment+options+in+hormone-refractory+metastatic+prostate+carcinoma.">Treatment options in hormone-refractory metastatic prostate carcinoma.</a> Tumori. 90 (6), 535-546 (2004).</li><li>Hughes, C., Murphy, A., Martin, C., Sheils, O., O'Leary, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathology+of+prostate+cancer.">Molecular pathology of prostate cancer.</a> J Clin Pathol. 58 (7), 673-684 (2005).</li><li>Pavese, J., Ogden, I. 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J., 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=Blockade+of+epidermal+growth+factor+receptor+signaling+in+tumor+cells+and+tumor-associated+endothelial+cells+for+therapy+of+androgen-independent+human+prostate+cancer+growing+in+the+bone+of+nude+mice.">Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice.</a> Clin Cancer Res. 9 (3), 1200-1210 (2003).</li><li>Kim, S. J., 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=Targeting+platelet-derived+growth+factor+receptor+on+endothelial+cells+of+multidrug-resistant+prostate+cancer.">Targeting platelet-derived growth factor receptor on endothelial cells of multidrug-resistant prostate cancer.</a> J Natl Cancer Inst. 98 (11), 783-793 (2006).</li><li>Park, S. I., 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=Targeting+SRC+family+kinases+inhibits+growth+and+lymph+node+metastases+of+prostate+cancer+in+an+orthotopic+nude+mouse+model.">Targeting SRC family kinases inhibits growth and lymph node metastases of prostate cancer in an orthotopic nude mouse model.</a> Cancer Res. 68 (9), 3323-3333 (2008).</li><li>Zhang, J., 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=AFAP-110+is+overexpressed+in+prostate+cancer+and+contributes+to+tumorigenic+growth+by+regulating+focal+contacts.">AFAP-110 is overexpressed in prostate cancer and contributes to tumorigenic growth by regulating focal contacts.</a> J Clin Invest. 117 (10), 2962-2973 (2007).</li><li>Park, S. I., Kim, S. J., McCauley, L. K., Gallick, G. 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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=Longitudinal+live+animal+micro-CT+allows+for+quantitative+analysis+of+tumor-induced+bone+destruction.">Longitudinal live animal micro-CT allows for quantitative analysis of tumor-induced bone destruction.</a> Bone. 48 (1), 141-151 (2011).</li><li>Steinbauer, M., 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=GFP-transfected+tumor+cells+are+useful+in+examining+early+metastasis+in+vivo,+but+immune+reaction+precludes+long-term+tumor+development+studies+in+immunocompetent+mice.">GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice.</a> Clin Exp Metastasis. 20 (2), 135-141 (2003).</li><li>Yang, M., 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+fluorescent+orthotopic+bone+metastasis+model+of+human+prostate+cancer.">A fluorescent orthotopic bone metastasis model of human prostate cancer.</a> Cancer Res. 59 (4), 781-786 (1999).</li><li>Stephenson, R. 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The authors declare that they have no competing financial interests.

This manuscript describes a detailed procedure for establishing a human orthotopic prostate cancer xenograft mouse model. This model was established by direct implantation of the human prostate cancer cell line PC3M-Luc-C6 into the dorsal prostatic lobes of immunocompromised mice. Tumors were allowed to develop over the course of the experiment. Tumor growth was monitored weekly by a non-invasive bioluminescence imaging system during the experiment.
The most important factor in establishing xe...

Prostate cancer is the second most prevalent cause of cancer deaths (9%) among males in the United States, next to cancer of the lung and bronchus (28%)1. According to recent data, it is estimated that 220, 800 newly diagnosed prostate cancer cases and 27, 540 deaths will occur in 20151. The five year relative survival rate of early stage prostate cancer is &#62;99% while that of advanced metastatic disease is only 28%1. A major challenge for treatment of advanced metastatic disease is the lack of understanding of molecular mechanisms underlying the propensity of this disease to metastasize to other organs, particularly to the bone, which is a frequent site for prostate cancer. Hence, there is a clear need to study the molecular makeup of these prostate tumors in order to develop effective therapeutic regimens against progression to advanced metastatic disease2,3.
Prostate tumors exhibit high biological heterogeneity without a well-defined pathway to progression. Metastases often occur with no prior indication of tumor invasiveness4. This clinical heterogeneity is attributed to the molecular diversity of prostate cancer. Understanding the molecular makeup of these lethal tumors is the key to design better diagnostic and therapeutic strategies for this disease. Consequently, prostate cancer research is currently focused on understanding and preventing metastasis.
Pre-clinical in vivo mouse models offer a variety of options to understand the molecular mechanisms of prostate cancer progression to advanced metastatic disease. In addition, these models are important for preclinical evaluations of new therapeutic strategies against this disease. The most commonly used animal models include transgenic mouse models, tail-vein injection, intra-cardiac implantation and human orthotopic mouse models. Transgenic studies are time consuming and correlation of prostate cancer development in mice with that of humans have shown variability11. In spontaneous metastatic mouse models, cells are injected directly into the circulation and though, they have rapid turnaround time, they cannot be used to study the primary tumor or the initial steps in the metastatic cascade5. Orthotopic xenograft models have the limitation of developing bone metastatic lesions, the common site of prostate cancer metastasis. Nonetheless, the human orthotopic prostate cancer xenograft mouse model is well characterized and widely used to study the molecular events of primary tumor development, cross-talk between tumor and organ microenvironment, initial phase of the metastatic disease and use of experimental drugs for therapeutic intervention6,7,8-11.

<table border="0" cellpadding="0" cellspacing="0" width="732">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PC3 prostate cancer cell line&#160;</td>
			<td style="width:188px;">ATCC</td>
			<td style="width:111px;">CRL-1435</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Medium (MEM)&#160;</td>
			<td>GIBCO,Life Technology</td>
			<td>11095-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>GIBCO,Life Technology</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO,Life Technology</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeocin</td>
			<td>Invitrogen,Life Technology</td>
			<td>R250-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin&#160;</td>
			<td>GIBCO,Life Technology</td>
			<td>25300-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS&#160;</td>
			<td>Xenogen-Caliper</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin Syringes (300ul, 28.5g)</td>
			<td>Becton Dickinson</td>
			<td>309300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mice</td>
			<td>Charles River Laboratories, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol Swabs</td>
			<td>MEDEquip Depot</td>
			<td>326895 BD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVP Iodine Prep Pad</td>
			<td>MEDEquip Depot</td>
			<td>C12400PDI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical CatGut Chromic Suture</td>
			<td>Demetech</td>
			<td>CC224017F0P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>Corning</td>
			<td>354248</td>
			<td></td>
		</tr>
	</tbody>
</table>

pre-clinical orthotopic murine model of human prostate cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Following orthotopic implantation of PC3M-Luc-C6 cells into the posterior prostatic lobe, mice were weekly imaged by using a live animal bioluminescence imaging system to monitor the colonization of cells and tumor growth over the course of experiment (Figure 5A-B). Quantification of the bioluminescent signal indicated that PC3M-Luc-C6 cells successfully colonized the prostate lobes. Increased bioluminescence is indicative of increased primary tumor growt...

Watch this Scientific Journal Video about Pre-clinical Orthotopic Murine Model of Human Prostate Cancer at JoVE.com

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

pre-clinical-orthotopic-murine-model-of-human-prostate-cancer

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JoVE Journal

Medicine

14.1K Views.  VA Medical Center and UCSF. Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Video: Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

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 ...

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 ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

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 ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and 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  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.