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
<p class="j...

<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 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 ...

The overall goal of this procedure is to establish a mouse orthotopic prostate cancer xenograft model that provides a useful approach to studying the molecular events of primary tumor development in the early events of the metastatic cascade. This model can help answer key questions regarding the spastic interactions between genetically and molecularly altered tumor cells in the micro-environment of the growing tumor. It can also help in the pre-clinical evaluation of new therapeutic approaches against the human prostate cancer.The main advantage of this technique over the spontaneous metastatic models are that it promotes the study of the molecular events of primary tumor development and also the recapitulation of the initial phase of metastasis prior to entry and embolism of tumor cells into the circulation. Orthotopic model of prostate cancer is one of the best pre-clinical models to understand how prostate cancer cells metastasize to different organs. Most prostate cancer patients die of metastasis, and therefore this model is very important to investigate the molecular mechanisms of metastasis.Using this model we can design better strategies for the treatment of metastasis by using neural compounds, those are specific to metastatic pathways. Demonstrating this procedure will be Melissa Colden, a grad student and Dr.Varahram Shahryari, a research associate from our laboratory. Before beginning the procedure, wash the tumor cell culture of interest with PBS and incubate the cells with 2 milliliters of 05 percent trypsin for 3 to 5 minutes in the cell culture incubator.When the cells have detached, stop the reaction with 5 milliliters of complete medium and count the cells by trypan blue exclusion. Dilute the cells to a 2.5 times 10 to the 5 cells per 10 microliters of medium concentration, and mix the cell suspension with 10 microliters of basement membrane-like extracellular matrix extract. Hold the cells on ice, then place an anesthetized six to eight week old immunocompromised male experimental mouse in the supine position on a sterile heating pad and confirm a lack of response to toe pinch.Apply ointment to the animals eyes and remove the hair from the lower abdomen. Disinfect the surgical area with 10 percent povidone iodine scrub, followed by 70 percent ethanol about 1 to 2 centimeters above the penis sheath, and about 2 to 3 centimeters below the bottom of the rib cage. Use a pair of fine forceps to lift 2 millimeters of skin above the preputial gland.Using a scalpel, make a 1 centimeter mid-line incision through the skin, followed by a 1 centimeter incision through the muscle layer. Locate the yellow, light brown spherical bladder directly below the incision. Then use the fine forceps to grip the bladder and to lift the organ up and out of the body cavity toward the penis sheath to expose the seminal vesicles.With a wet cotton swab in each hand, carefully externalize the vesicles one at a time, and lay them top down on the outer surface of the abdomen with the bladder in the middle. Next, use the cotton swabs to 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. Now use a micro-pipette to re-suspend the cells in a single-cell suspension and label them with luciferin.Aspirate 25 microliters of the cells into a 0.5 cc syringe equipped with a 28.5 gauge needle and transfer the mouse under a dissecting microscope. Then, insert the syringe needle into a dorsal prostatic lobe and slowly inject 20 microliters of the cell suspension until a bulla formation is observed. When all of the cells have been administered, retract the needle, pressing lightly on the injection site with a cotton swab for a few seconds to prevent leakage.Then use the swabs to carefully return the vesicles to the body cavity one at a time, followed by the bladder. After placing the organs back into the body cavity, suture the muscle layer first in an interrupted pattern with absorbable 4-0 chromic cat gut sutures, followed by the skin closure with non-absorbable 4-0 nylon surgical sutures. The skin can also be pulled together and closed with surgical clamps to close the incision completely.Then, immediately image the animal, and return the mouse to a clean cage under a warming lamp with monitoring until it is fully recovered. Following the orthotopic implantation of luciferin-labeled prostate cancer cells into the posterior prostatic lobe, as just demonstrated, weekly imaging using live animal bioluminescence imaging reveals a successful colonization of the prostate lobes by the tumor cells. Indeed, an increase in the bioluminescence over time is indicative of the enlargement of the primary tumor over the course of the experiment, confirming the effectiveness of this technique in non-invasive, radiographic, fluorescent or luminescent imaging for tumor growth and metastatic lesion monitoring.While attempting this procedure, it is important to remember that intra-prostatic injection requires open abdominal surgery. So, it should be performed in a pathogen-free environment using proper aseptic surgical techniques. Working with human cancer cells can be extremely hazardous and precautions, such as wearing personal protective equipment, and working within biosafety level two containment should always be taken while performing this procedure.This model paved the way for the transplant of histologically intact tumor fragments to examine the effects of potential drug therapies on tumor growth or original lymph node metastasis. After watching this video, you should have a good understanding of how to implant cancer cells into the dorsal prostatic lobes for the establishment of an orthotopic human prostate cancer xenograft mouse model.

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13.9K visualizações.  VA Medical Center and UCSF. The overall goal of this procedure is to establish a mouse orthotopic prostate cancer xenograft model that provides a useful approach to studying the molecular events of primary tumor development in the early events of the metastatic cascade. This model can help answer key questions regarding the spastic interactions between genetically and molecularly altered tumor cells in the micro-environment of the growing tumor. It can also help in the pre-clinical evaluation of new therapeutic approach...