Some of the figures in this manuscript were generously provided by the laboratory of Bart O. Williams. The authors are supported by National Cancer Institute grants U54CA143803, CA163124, CA093900, AND CA143055.

This protocol meets and follows the guidelines set by the Johns Hopkins University Institutional Animal Care and Use Committee.
1. Micro-dissecting a Mouse Prostate
<ol>
	<li>Euthanize the mouse by carbon dioxide asphyxiation or an alternative approved method in accordance with institutional animal care and use guidelines.</li>
	<li>Pin the mouse in a supine position to a dissection board by putting a pin through each of the four paws.</li>
	<li>Spray the abdomen with 70% ethanol.</li>
	<li>Hold the skin of the lower abdomen with forceps, and use scissors to cut through the skin and peritoneum approximately 1 cm anterior to the opening of the penis, careful not to cut any organs.</li>
	<li>Cut away the skin and peritoneum from the lower abdomen, up both sides of the abdomen to the rib cage, exposing the peritoneal cavity.</li>
	<li>Identify the bladder and urogenital tract, and expose them by gently moving fat and other organs to the side.</li>
	<li>Using forceps, grip the vas deferens at the base near the urethra, and tear it away.</li>
	<li>Repeat with the opposite vas deferens.</li>
	<li>Using forceps, carefully grip the bladder and pull it up, while simultaneously using scissors to cut through the urethra below the bladder and ventral prostate (approximately 1 cm below the base of the bladder). 
	Note: As the bladder is pulled up, the entire urogenital tract (containing bladder, a section of the urethra, all prostate lobes, and the seminal vesicles) will come with it. There may be some resistance.</li>
	<li>Place the urogenital tract (UGT) into a 60-100 mm Petri dish containing approximately 5-10 ml phosphate buffered saline (PBS).</li>
	<li>Perform the remaining part of the protocol under a dissection microscope. Use two fine forceps, one in each hand. Hold the forceps &#34;like a pencil&#34; and rest the forearms on the bench top so as to steady them.</li>
	<li>Use fine forceps to position the UGT such that the bladder is on top, the urethra is pointed down, and the seminal vesicles are positioned on either side of the bladder.</li>
	<li>Grip the urethra with one forceps and carefully pull away fat with the other forceps without tearing away any prostate tissue. Note: Fat will appear &#34;shiny&#34; relative to the surrounding tissue.</li>
	<li>Once the fat is removed, use forceps to tear the connective tissue between the two ventral lobes of the prostate and separate them.</li>
	<li>To remove one of the ventral lobes, use one forceps to grip a single ventral lobe near the tip, and use the other forceps to grip that lobe at the base as close to the urethra as possible. While still gripping the base of the lobe, use the other forceps to grip the urethra. Pull the lobe away from the urethra with a firm, smooth motion. Make sure no prostate tissue remains attached to the urethra. Place the lobe into the appropriate medium, depending on the next experimental step.
	<ol>
		<li>To fix and paraffin-embed the sample for histological analysis, place the lobe in 10% neutral-buffered formalin (NBF) 8.</li>
		<li>Place the lobe in freezing medium, such as OCT, for cryopreservation and sectioning 8.</li>
		<li>Place the lobe in cold PBS for single cell isolation for culture or flow cytometry 9.</li>
	</ol>
	</li>
	<li>Repeat step 1.15 with the other ventral lobe.</li>
	<li>To remove one of the anterior lobes, use forceps to gently pull the lobe away from the seminal vesicle, making sure not to puncture the seminal vesicle. Once the lobe is independent of the seminal vesicle, remove it in the same fashion as the ventral lobe in step 1.15.</li>
	<li>Repeat step 1.17 with the other anterior lobe.</li>
	<li>Flip the remaining urogenital tract over such that the dorsal prostate is visible.</li>
	<li>To remove one of the dorsolateral lobes, use forceps to tear the connective tissue between the two dorsolateral lobes and also the connective tissue between the lobes and the posterior region of the seminal vesicles. Remove the lobe in a similar fashion as the ventral lobe in step 1.15.</li>
	<li>Repeat step 1.20 with the other dorsolateral lobe.</li>
</ol>
2. Surgical Castration
Note: This is a survival surgery, so asepsis, anesthesia, and pain management are important to the ethical and successful completion of this protocol. Follow all institutional animal care and use guidelines. Use an external heat source, such as a recirculating water blanket, during the surgical procedure to prevent hypothermia. &#160;Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Autoclave all surgical instruments before use in surgery.
<ol>
	<li>On a clean, sterile surface anesthetize the mouse using appropriate measures (e.g. 2% inhaled isoflurane) and position the mouse in a supine position. Note: Alternative anesthesia methods, such as ketamine, may also be used, depending on the approved animal protocol.</li>
	<li>Check the mouse&#39;s reflexes by pinching the toes. If the mouse does not react, proceed to the next step. If it does react, wait 1-3 min and check again. Shave the surgical area with an electric razor.</li>
	<li>Aseptically prepare the abdomen of the mouse using alternating scrubs of 70% ethanol and iodine or procedures recommended by the institutional veterinary staff or IACUC.</li>
	<li>Using a sterile scalpel, make a 1 cm vertical incision through the skin in the midline of the lower abdomen, approximately 1.5 cm anterior to the penis.</li>
	<li>Make a small incision (&#60;1 cm) through the peritoneum, careful not to cut any organs.</li>
	<li>Using sterile forceps, grip the cut edge of the peritoneum and lift it up gently so as to be able to see the peritoneal cavity beneath.</li>
	<li>Using another sterile forceps, reach into the peritoneal cavity and grip either the left or right testicular fat pad, lateral to the bladder. Pull the fat pad through the opening in the peritoneum and skin until the testis comes out as well. Be careful not to injure any other tissues or organs during this step.</li>
	<li>Using a cautery pen, cut through the fat pad that is holding the testis. Cut slowly through the testicular artery with the cautery pen Note: If the testicular artery is cut too fast, there may be too much bleeding and the mouse may have to be euthanized.</li>
	<li>Once the fat pad and artery are cut, remove the testis and fat pad.</li>
	<li>Repeat steps 2.6-2.9 with the other testis.&#160;Note: An alternative method for removal of the testes is to surgically ligate the testicular artery, followed by cutting the vessel with scissors.</li>
	<li>Suture the peritoneum closed with absorbable suture.</li>
	<li>Staple the skin closed with surgical wound clips.</li>
	<li>Administer an analgesic to manage pain, and place the mouse in a clean cage on a cage warmer for 5-10 min. Monitor the mouse for signs of pain or bleeding.</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.">Cancer statistics.</a> CA Cancer J Clin. 65 (1), 5-29 (2015).</li><li>Roehrborn, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+benign+prostatic+hyperplasia.">Pathology of benign prostatic hyperplasia.</a> Int J Impot Res. 20 (suppl 3), 11-18 (2008).</li><li>Valkenburg, K. C., Williams, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+prostate+cancer.">Mouse models of prostate cancer.</a> Prostate Cancer. , 895238 (2011).</li><li>Shappell, S. B., 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=Prostate+pathology+of+genetically+engineered+mice:+definitions+and+classification.+The+consensus+report+from+the+Bar+Harbor+meeting+of+the+Mouse+Models+of+Human+Cancer+Consortium+Prostate+Pathology+Committee.">Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee.</a> Cancer Res. 64 (6), 2270-2305 (2004).</li><li>Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery+in+prostate+cancer+mouse+models.">Drug discovery in prostate cancer mouse models.</a> Expert Opin Drug Discov. , 1-14 (2015).</li><li>El-Alfy, M., Pelletier, G., Hermo, L. S., Labrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+features+of+the+basal+cells+of+human+prostate+epithelium.">Unique features of the basal cells of human prostate epithelium.</a> Microsc Res Tech. 51 (5), 436-446 (2000).</li><li>Prins, G. S., Huang, L., Birch, L., Pu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+estrogens+in+normal+and+abnormal+development+of+the+prostate+gland.">The role of estrogens in normal and abnormal development of the prostate gland.</a> Ann N Y Acad Sci. 1089, 1-13 (2006).</li><li>Valkenburg, K. 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=Activation+of+Wnt/beta-catenin+signaling+in+a+subpopulation+of+murine+prostate+luminal+epithelial+cells+induces+high+grade+prostate+intraepithelial+neoplasia.">Activation of Wnt/beta-catenin signaling in a subpopulation of murine prostate luminal epithelial cells induces high grade prostate intraepithelial neoplasia.</a> Prostate. 74 (15), 1506-1520 (2014).</li><li>Lukacs, R. U., Goldstein, A. S., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation+and+characterization+of+adult+murine+prostate+stem+cells.">Isolation, cultivation and characterization of adult murine prostate stem cells.</a> Nat Protoc. 5 (4), 702-713 (2010).</li><li>Michiel Sedelaar, J. P., Dalrymple, S. S., Isaacs, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+men--warning:+intact+versus+castrated+adult+male+mice+as+xenograft+hosts+are+equivalent+to+hypogonadal+versus+abiraterone+treated+aging+human+males,+respectively.">Of mice and men--warning: intact versus castrated adult male mice as xenograft hosts are equivalent to hypogonadal versus abiraterone treated aging human males, respectively.</a> Prostate. 73 (12), 1316-1325 (2013).</li><li>Wang, X., 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+luminal+epithelial+stem+cell+that+is+a+cell+of+origin+for+prostate+cancer.">A luminal epithelial stem cell that is a cell of origin for prostate cancer.</a> Nature. 461 (7263), 495-500 (2009).</li><li>Pascal, L. E., 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=5alpha-Reductase+inhibition+coupled+with+short+off+cycles+increases+survival+in+the+LNCaP+xenograft+prostate+tumor+model+on+intermittent+androgen+deprivation+therapy.">5alpha-Reductase inhibition coupled with short off cycles increases survival in the LNCaP xenograft prostate tumor model on intermittent androgen deprivation therapy.</a> J Urol. 193 (4), 1388-1393 (2015).</li><li>Shen, M. M., Abate-Shen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+prostate+cancer:+new+prospects+for+old+challenges.">Molecular genetics of prostate cancer: new prospects for old challenges.</a> Genes Dev. 24 (18), 1967-2000 (2010).</li></ol>

The authors declare that they have no competing financial interests.

Prostate micro-dissection allows for lobe-specific experimentation and analysis of the mouse prostate (Figure 1). In genetically engineered mouse models, phenotypes may be seen in one lobe that is not seen in others. Also, for histological analysis, this protocol assures that the maximum amount of pure prostate tissue can be sectioned and stained, without other urogenital tract tissues present in the section. Finally, for single-cell experiments, this protocol allows for isolation of almost pure prostate cells. Epithelial cell enrichment kits can further purify prostate cells from stromal cells. Limitations to micro-dissection include the use of a dissection microscope, and the learning curve necessary to use fine forceps while looking through the dissection microscope. Alternatively, prostate tissue may be sectioned for histology as part of the UGT, making micro-dissection unnecessary. However, the downside to sectioning the entire UGT is that other tissues will be present in the section, making it not as clean. In addition, for generating single prostate cell populations, the lobes must be separated from the UGT to avoid contamination.
Surgical castration is a fundamental surgery for many prostate research labs and is essential for the study of the effects of testosterone on prostate development and disease. This protocol is quick (typical time of surgery for an experienced user is about 5 min) and removes almost 100% of testosterone from circulation (Figure 3). Of note, testosterone levels in mice differ significantly, and may vary by as much as 30-fold from mouse to mouse 10. Castration removes this variation. Following castration, researchers may decide to add testosterone back in to regenerate the prostate (Figures 3-4). This can be done by subcutaneously implanting a source of testosterone, such as an osmotic pump, a testosterone pellet, or a semi-permeable silastic tube containing testosterone powder 8. Depending on the amount of testosterone added, this procedure can provide physiological to superphysiological testosterone levels in the circulation (Figure 3). This protocol can also be used to study prostate cell lineage and stem cell biology 11. Another option is chemical castration, or androgen deprivation therapy, via treatment of mice with compounds that inhibit the production of testosterone, such as dutasteride 12. This also mimics a common treatment of prostate cancer patients, who are rarely surgically castrated. However, surgical castration provides a reliable, permanent method for removal of virtually all testosterone. It is important to consider that the adrenal glands also synthesize androgen species, such as androstenedione and dehydroepiandrosterone, which can be converted into testosterone, though in much smaller amounts, relative to the testes 13. In some cases, the adrenal glands can also be removed surgically, though this procedure is more difficult.
Overall, these two protocols are useful for a myriad of research into prostate diseases, as well as hormonal studies. These protocols are a reliable and consistent way to analyze prostate biology in hormonally normal, castrated, or hormonally regenerated conditions.

The prostate is the most common site of cancer in men in the US. Nearly 220,000 men each year will be diagnosed with prostate cancer, and approximately 27,000 men will succumb to their disease 1. Men have a 1 in 7 lifetime risk of a prostate cancer diagnosis in the US 1. Benign prostate hyperplasia (BPH), an age-related noncancerous enlargement of the prostate, is also a widespread condition, affecting over 80% of men over 80 2. As such, the prostate is the focus of considerable research.
Mouse models have been used widely to study diseases of the prostate 3. Overall, the mouse prostate is an excellent representative of the human gland 4, but there are similarities and differences between mouse and human prostate anatomy and physiology 5. In both species, the prostate is located at the base of the bladder, surrounding the urethra. The human prostate gland is a single lobe, split into four zones: central, transition, peripheral, and anterior fibromuscular stroma. In contrast, the mouse prostate consists of three paired lobes at different positions around the urethra: anterior, ventral, and dorsolateral. On a cellular level, the main difference is that the basal cell to luminal cell ratio in human is about 1:1, but it is 1:4 in mice 6. The murine stroma is also different in mice relative to humans &#8211; humans have more extensive smooth muscle, while the muscle layer is much thinner in murine prostate. The proper identification, dissection, and handling of the mouse prostate are essential to mouse prostate research.
Hormone levels drastically affect the development and homeostasis of the prostate gland in both humans and mice. While estrogen seems to play a role in prostate development 7, the most important hormone in the prostate is testosterone. Testosterone is essential for glandular development and maintenance. Following either physical or chemical castration, approximately 90% of the luminal cells in the adult prostate apoptose, and the gland shrinks. If testosterone is reintroduced to an individual under castration conditions, the prostate is able to regenerate itself to full capacity. Testosterone also seems to drive prostate cancer, which is why androgen deprivation therapy is a commonly used therapeutic strategy. However, many prostate cancers become resistant to androgen deprivation. In addition, men undergoing androgen deprivation therapy for treatment of prostate cancer undergo cycles of castrated and regenerated conditions. In the mouse, surgical castration, along with hormonal regeneration of the prostate by reintroducing testosterone, is an important tool with which to study castration resistance and the effects of testosterone cycling on the prostate.
In this article, we will discuss and demonstrate the proper techniques by which to locate and micro-dissect a mouse prostate, as well as to surgically castrate a mouse.

<table><tbody><tr><td>Surgical tools</td><td>Roboz</td><td>Various</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F8775</td><td></td></tr><tr><td>OCT medium</td><td>VWR</td><td>25608-930</td><td>Must be placed on dry ice to freeze, and stored at -80 &amp;deg;C</td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010</td><td></td></tr><tr><td>Isoflurane</td><td>Johns Hopkins</td><td></td><td>Also available from vendors online</td></tr><tr><td>Cautery Pen</td><td>Medline</td><td>ESCT002</td><td></td></tr><tr><td>Silk suture</td><td>AD Surgical</td><td>M-S330R19</td><td></td></tr><tr><td>Surgical Wound Clips</td><td>Roboz</td><td>RS-9265</td><td></td></tr></tbody></table>

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.

All six prostate lobes were removed from a mouse via prostate micro-dissection (Figure 1). The complete urogenital tract (UGT) is composed of all prostate lobes, the bladder, seminal vesicles, and the urethra (Figure 1a). The vas deferens attaches to the urethra, but is unnecessary for prostate micro-dissection, and can thus be detached before removal of the UGT by cutting the urethra (Figure 1b-1c). The urogenital tract will remain together once removed from the abdomen (Figure 1d). At this point, the UGT remains covered in fatty tissue, which needs to be removed to gain access to the prostate lobes (Figure 1e). All six lobes (ventral, anterior, and dorsolateral pairs) can be seen surrounding the urethra at the base of the bladder. Pure prostate tissue can then be removed following the protocol above (Figure 1f). These lobes may be placed in the appropriate medium for further analysis.
Castration is the removal of the testes, which are the primary producers of testosterone. Testosterone is present in the mouse until castration occurs, at which point the prostate regresses (Figure 2a). Hormonal regeneration via reintroduction of testosterone can follow castration (Figure 2b). We performed castration, followed by two cycles of hormonal regeneration, and testosterone levels were monitored in mouse serum by ELISA (Figure 3). Under castration conditions, testosterone levels virtually disappear. Alternatively, tamoxifen (a selective estrogen receptor modulator) shows no significant difference to the hormonally normal condition. After castration, testosterone was re-introduced to the mouse, causing testosterone levels to spike dramatically (Figure 3). We then micro-dissected the prostate lobes of the mice and imaged them as individual anterior lobes (Figure 4a). We also formalin-fixed, paraffin-embedded, and sectioned the tissue. Figure 4b shows hematoxylin and eosin stained anterior prostate tissue under normal, castrated, and regenerated conditions. Castrated prostate lobes shrink to approximate one-tenth the size of a normal prostate, and that hormonal regeneration restores the prostate to its original size.
<img alt="Figure 1" src="/files/ftp_upload/53984/53984fig1.jpg" /> 
Figure 1: Gross Histology of the Prostate Micro-dissection Protocol. (a) The urogenital tract (UGT) in its natural state, exposed in the murine abdomen. The dotted lines encompass the various parts of the UGT, including the seminal vesicles (SV), prostate lobes (anterior prostate (AP) and ventral prostate (VP) are visible here), bladder (B), and urethra (located beneath the bladder). The vas deferens (VD) is also visible. (b) The vas deferens have been removed. The arrow points to the region from which the vas deferens has been removed. (c) The UGT has been removed from the abdomen by cutting the urethra below the ventral prostate lobes. (d) The UGT has been placed in PBS and is seen beneath a dissection microscope. (e) Fat has been removed from the UGT, allowing for the visibility of all six lobes of the prostate. The arrows point to the two anterior lobes, the ventral lobes, and the dorsolateral lobes. (f) All six individual prostate lobes have been removed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53984/53984fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53984/53984fig2.jpg" /> 
Figure 2: Timeline for Castration and Hormonal Regeneration. (a) Testosterone is present at physiological levels until castration, when testosterone disappears and regression of the prostate begins. (b) Castration removes testosterone, until testosterone is reintroduced, causing hormonal regeneration of the prostate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53984/53984fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53984/53984fig3.jpg" /> 
Figure 3: Testosterone Levels in Various Hormonal Conditions. Testosterone ELISA under tamoxifen treated, hormonally normal, castrated, and hormonally regenerated conditions. Regenerated x 2 refers to implantation and removal of synthetic testosterone, followed by implantation of a second round of synthetic testosterone. Error bars represent standard deviation. This figure has been modified from 5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53984/53984fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53984/53984fig4.jpg" /> 
Figure 4: Prostate Appearance under Castration and Regeneration Conditions. (a) Gross appearance of anterior prostate lobes under castrated, hormonally normal, and hormonally regenerated conditions. Regenerated x 2 refers to implantation and removal of synthetic testosterone, followed by implantation of a second round of synthetic testosterone. (b) Formalin-fixed, paraffin-embedded anterior prostate lobes sectioned and stained with hematoxylin and eosin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53984/53984fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Prostate Micro-dissection and Surgical Castration at JoVE.com

Murine Prostate Micro-dissection and Surgical Castration

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 ...

murine-prostate-micro-dissection-and-surgical-castration

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45.3K Views.  Johns Hopkins University. 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.

Video: Murine Prostate Micro-dissection and Surgical Castration

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 ...

Cancer Research

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 ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

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  ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Bioengineering

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.

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 ...

Murine Orchiectomy and Ovariectomy to Reduce Sex Hormone Production

Sex hormone signaling plays a critical role in multiple organ systems as well as in the progression of various diseases, including neurodegenerative disease. The manipulation of sex hormone levels in the murine model system allows for the study of their impact on organs/tissues and within disease progression. Orchiectomy - the surgical removal of the testes - and ovariectomy - the surgical removal of the ovaries - provide a method to deplete the endogenous sex hormones so that the precise hormone levels can be provided through drug or other delivery methods. Here, we provide rapid and minimally invasive methods for both orchiectomy and ovariectomy in the murine model system for the reduction of sex hormones. This protocol details the surgical preparation and excision of the testes through the scrotal sac, and excision of the ovaries via two incisions in the right and left lateral dorsum.

This manuscript describes a consistent way to quickly perform survival rodent orchiectomies and ovariectomies.

Sex hormone signaling plays a critical role in multiple organ systems as well as in the progression of various diseases, including neurodegenerative ...

Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells

Source: Riedel, M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57525" target="_blank">Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration.</a> J. Vis. Exp. (2018)
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.

Modelowanie raka prostaty w genetycznie modyfikowanych modelach mysich: technika zlokalizowanej edycji genów za pośrednictwem CRISPR/Cas9 w komórkach przedniego płata prostaty myszy

Source: Riedel, M. et al. Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration. J. Vis. Exp. (2018)
This video describes a method ...

JoVE Encyclopedia of Experiments

Prostate Cancer

Animal model studies

Intra-prostatic Injection of Cancer Cells: A Technique to Deliver Cancer Cells for Establishing Orthotopic Prostate Cancer Mouse Model

Source: Anker, J. F. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57301" target="_blank">Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the procedure for administering intra-prostatic injection of prostate cancer cells into a mouse model. Orthotopic murine models can be used to study the disease origin and response to treatment therapies in a non-invasive and clinically relevant environment.

Wstrzyknięcie komórek rakowych do gruczołu krokowego: technika dostarczania komórek rakowych w celu ustalenia ortotopowego modelu myszy raka prostaty

Source: Anker, J. F. et al. Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate ...