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. M., Bergan, R. C. <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+murine+model+of+human+prostate+cancer+metastasis.">An orthotopic murine model of human prostate cancer metastasis.</a> J Vis Exp. (79), e50873 (2013).</li><li>Pettaway, C. 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=Selection+of+highly+metastatic+variants+of+different+human+prostatic+carcinomas+using+orthotopic+implantation+in+nude+mice.">Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice.</a> Clin Cancer Res. 2 (9), 1627-1636 (1996).</li><li>Rembrink, K., Romijn, J. C., van der Kwast, T. H., Rubben, H., Schroder, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+implantation+of+human+prostate+cancer+cell+lines:+a+clinically+relevant+animal+model+for+metastatic+prostate+cancer.">Orthotopic implantation of human prostate cancer cell lines: a clinically relevant animal model for metastatic prostate cancer.</a> Prostate. 31 (3), 168-174 (1997).</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=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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clinical+mouse+models+of+human+prostate+cancer+and+their+utility+in+drug+discovery.">Pre-clinical mouse models of human prostate cancer and their utility in drug discovery.</a> Curr Protoc Pharmacol. Chapter 14, Unit 14.15 (2010).</li><li>Johnson, L. 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. 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=Metastatic+model+for+human+prostate+cancer+using+orthotopic+implantation+in+nude+mice.">Metastatic model for human prostate cancer using orthotopic implantation in nude mice.</a> J Natl Cancer Inst. 84 (12), 951-957 (1992).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+metastatic+mouse+models+for+anticancer+drug+discovery+and+evaluation:+a+bridge+to+the+clinic.">Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic.</a> Invest New Drugs. 17 (4), 343-359 (1999).</li><li>Wang, X., An, Z., Geller, J., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-malignancy+orthotopic+nude+mouse+model+of+human+prostate+cancer+LNCaP.">High-malignancy orthotopic nude mouse model of human prostate cancer LNCaP.</a> Prostate. 39 (3), 182-186 (1999).</li><li>An, Z., Wang, X., Geller, J., Moossa, A. R., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+orthotopic+implantation+allows+high+lung+and+lymph+node+metastatic+expression+of+human+prostate+carcinoma+cell+line+PC-3+in+nude+mice.">Surgical orthotopic implantation allows high lung and lymph node metastatic expression of human prostate carcinoma cell line PC-3 in nude mice.</a> Prostate. 34 (3), 169-174 (1998).</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=Reduced+c-Met+expression+by+an+adenovirus+expressing+a+c-Met+ribozyme+inhibits+tumorigenic+growth+and+lymph+node+metastases+of+PC3-LN4+prostate+tumor+cells+in+an+orthotopic+nude+mouse+model.">Reduced c-Met expression by an adenovirus expressing a c-Met ribozyme inhibits tumorigenic growth and lymph node metastases of PC3-LN4 prostate tumor cells in an orthotopic nude mouse model.</a> Clin Cancer Res. 9 (14), 5161-5170 (2003).</li></ol>

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><tbody><tr><td>PC3 prostate cancer cell line&amp;nbsp;</td><td>ATCC</td><td>CRL-1435</td><td></td></tr><tr><td>Minimum Essential Medium (MEM)&amp;nbsp;</td><td>GIBCO,Life Technology</td><td>11095-080</td><td></td></tr><tr><td>PBS</td><td>GIBCO,Life Technology</td><td>10010-023</td><td></td></tr><tr><td>FBS</td><td>GIBCO,Life Technology</td><td>10437-028</td><td></td></tr><tr><td>Zeocin</td><td>Invitrogen,Life Technology</td><td>R250-01</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>GIBCO,Life Technology</td><td>25300-54</td><td></td></tr><tr><td>IVIS&amp;nbsp;</td><td>Xenogen-Caliper</td><td></td><td></td></tr><tr><td>Insulin Syringes (300 &amp;micro;l, 28.5 g)</td><td>Becton Dickinson</td><td>309300</td><td></td></tr><tr><td>Mice</td><td>Charles River Laboratories, Inc</td><td></td><td></td></tr><tr><td>Alcohol Swabs</td><td>MEDEquip Depot</td><td>326895 BD</td><td></td></tr><tr><td>PVP Iodine Prep Pad</td><td>MEDEquip Depot</td><td>C12400PDI</td><td></td></tr><tr><td>Surgical CatGut Chromic Suture</td><td>Demetech</td><td>CC224017F0P</td><td></td></tr><tr><td>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

Research

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

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

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

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  ...

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...