This work was supported by the grant from National Cancer Institute, R01CA161018 to LK.

All mouse experiments described here were performed according to the guidelines outlined in the institutional IACUC-approved protocols at SUNY Upstate Medical University.
1. The Urogenital System (UGS) Dissection
Note: The schematic is presented in Figure 1.
<ol>
	<li>Euthanize a 3-month-old male C57BL/6 mouse using the CO2 inhalational euthanasia method or another approved technique. 
	Note: Mice between the ages of 3 and 12 months can be used successfully for the experiment. Older mice (&#62; 6 months) will most likely have more fat around the UGS, which will need to be cleared. Identification and separation of the lobes is often difficult in mice younger than 3 months. Other strains can be used for the experiment, as well.</li>
	<li>Place the mouse on its back and secure the legs using pins so that the ventral side of the animal is exposed.</li>
	<li>Spray 70% ethanol on the mouse&#8217;s abdomen and wipe it clean. 
	Note: Shaving the abdomen hair before dissection is not required.</li>
	<li>Lift the skin from the abdomen, along with the muscle layer, with a pair of medium blunt forceps and make an inverted Y-shaped incision on the abdomen using scissors.
	<ol>
		<li>First, make a straight incision with a pair of sharp scissors from just above the penis to the sternum (Figure 2a).</li>
		<li>Cut from the base of the incision towards each toe, up to the thighs (Figure 2b). Fold back the skin on both sides and at the bottom to view the entire abdominal area (Figure&#160;2c-e). 
		Note: The cut size varies depend on the mouse age and size. The size of the initial straight incision may range from 1.5 to 4 cm, depending on the size of the mouse.</li>
	</ol>
	</li>
	<li>Move over the other organs to expose the UGS. Lift and move the brown-yellow intestines (Figure 2f). Pick up the abdominal fat pads (the opaque white spongy tissue) with forceps and move them to the sides (Figure 2g). 
	Note: The UGS comprises of the seminal vesicles, urethra, prostate, ductus deferens (vas deferens), and urinary bladder. It can be identified by the characteristic pair of opaque white semicircular arches (which are the seminal vesicles), with the fluid-filled bladder sac attached to the base. The translucent tissue located right under the seminal vesicles is the prostate.</li>
	<li>Locate the UGS, firmly hold the urinary bladder with blunt forceps, and lift the entire UGS upwards from the mouse abdomen. 
	Note: If the bladder is full, drain it first with a small syringe to provide a better grip with the forceps and reduce the risk of rupturing the bladder.</li>
	<li>Continuing to pull up on the bladder, slide a pair of scissors underneath the bladder and prostate all the way to the spine, and make a cut. Cut through any remaining connections to the abdominal cavity (Figure&#160;2h and 2i). Be careful not to cut too close to the UGS to avoid accidental cutting through any prostate tissue. 
	Note: While making the cut under the UGS, the scissors should be inserted all the way to the back of the mouse, so that the cut snaps the vertebral column as well. This method results in severing nearly all of the connections between the UGS and the abdominal cavity in one snip, reducing the time needed to extract the tissue from the mouse.</li>
	<li>Remove the UGS and place it in 2-6 mL (enough to cover the tissue) of phosphate buffered saline (PBS) or Dulbecco&#39;s modified Eagle medium (DMEM, high glucose) in a 6 cm Petri dish (Figure&#160;2j and 2k) and move it to a dissection microscope (10X magnification). 
	Note: Change the dissecting media as required in the remaining steps.</li>
</ol>
2. Dissecting the Prostate
<ol>
	<li>Clear all the fat from both the dorsal and ventral sides with a pair of fine forceps and microdissection scissors (Figure 3a). Use similar surgical instruments for the rest of the dissection protocol. 
	Note: The fat is often closely entwined with the UGS (can be identified by its white spongy appearance); hence, this step must be performed carefully to avoid accidentally snipping off any prostate tissue. It can take up to 10-15 min to clear all the fat.</li>
	<li>Hold and pull the bladder with the forceps and snip it at the base with the scissors (Figure 3b).</li>
	<li>Place the remaining tissue ventral side up. Hold one ductus deferens with forceps, trace it to the base with scissors, and snip it (Figure 3c), then repeat on the other side. Remove the ductus deferens, now leaving behind the prostate (translucent and verminous), seminal vesicles (opaque and white, semicircular), and urethra (pink-red and opaque tube) (Figure&#160;3d and 3e).</li>
	<li>Insert forceps in between the inner arch of the seminal vesicles and prostate tissue anterior lobes. Pry apart the seminal vesicles and prostate and snip any connective tissue as needed (Figure 3f). Trace the seminal vesicles to their base at the urethra and remove them (Figure&#160;3g and 3h). Be careful not to puncture them.</li>
	<li>Proceed to dissect out the individual prostate lobes.</li>
</ol>
3. Gross Anatomy of the Prostate and Individual Lobe Microdissection (Figures 3i-n, Figure 4)
<ol>
	<li>Flip the tissue with a pair of blunt forceps so that the dorsal side faces up, showing the dorsal lobes, which will resemble a butterfly&#8217;s wings.</li>
	<li>Collect the dorsal lobes by holding the lobe with forceps and snipping at the base with scissors (Figure 3j).</li>
	<li>Flip over the remaining tissue to the ventral side.</li>
	<li>Collect the lateral lobes, which are small and usually wrap the urethra on the side, which are wedged between the anterior, ventral, and dorsal lobes (Figure 3k).</li>
	<li>Next, collect the ventral lobes, which are larger than the lateral lobes and lie on the urethra ventrally (Figure 3l).</li>
	<li>Last, harvest the anterior lobes, the largest of the four, by cutting and discarding the urethra (Figure 3m).</li>
	<li>Process the tissue pieces according to experimental needs. Proceed to section 4 for histology, or to section 5 for the spheroid culture.</li>
</ol>
4. Lobe Identification and Morphology from Hematoxylin and Eosin-Stained Slides
<ol>
	<li>Fix the prostate tissue and embed it in paraffin. Proceed with staining the slides with hematoxylin and eosin (H&#38;E) to view and identify differences in prostate lobes based on histology, using the characteristics outlined by Oliveira et al.12 (Figure 5).</li>
</ol>
5. Processing the Tissue for 3D Culture10
Note: This is outlined in Figure 6.
<ol>
	<li>Proceed with culturing the whole prostate or individual lobes as per the experimental needs. Transfer the prostate tissue to a 10 cm dish containing 2-3 mL of DMEM. With a scalpel, mince the prostate tubules as finely and evenly as possible under the dissection microscope.</li>
	<li>Continue with the remaining steps under a tissue culture hood, under sterile conditions.</li>
	<li>Transfer the tissue pieces along with the DMEM to a 15 mL tube and increase the volume to 9 mL with DMEM. Add 1 mL of 10x collagenase stock solution to the DMEM-tissue mixture and vortex to mix. 
	Note: Collagenase stock solution is 10 mg/mL in Roswell Park Memorial Institute (RPMI) medium, and the final concentration in the 15 mL tube is 1 mg/mL.</li>
	<li>Place the tube on a gyratory shaker or tube rotator for 2 h at 37 &#176;C, for the collagenase to degrade the extracellular matrix.</li>
	<li>Centrifuge the tube at 400 x g for 5 min at room temperature.</li>
	<li>Discard the supernatant and resuspend in 2 mL of warm 0.05% trypsin-EDTA to cleave the cell-cell and cell-matrix adhesions. Transfer the tube to 37 &#176;C for 5 min. 
	Note: If the tissue chunks are too big to mix well, cut the pipette tip with a razor blade to make a wider bore.</li>
	<li>Break up any tissue clumps by pipetting with a P1000 pipette 8-10 times, then repeating with a P200 pipette.</li>
	<li>Neutralize the trypsin with 3 mL of complete DMEM. Add 500 U of DNAase I and mix well.</li>
	<li>Pass through a 5 mL syringe 5-10 times with an 18 G needle. Then pass through 5 times with a 20 G needle.</li>
	<li>Repeat steps 4-8 once more if the solution still contains big tissue chunks.</li>
	<li>Filter the cell suspension through a 40 &#956;m filter placed on a 50 mL tube. Rinse the 15 mL tube with 5 mL of DMEM and pass through the same filter. Repeat the rinse.</li>
	<li>Discard the filter and centrifuge the tube containing the flow-through at 400 x g for 5 min at room temperature.</li>
</ol>
6. Plating and Culturing the Cells
<ol>
	<li>Resuspend the pellet in 0.5 mL of complete Prostate Epithelial Cell Growth Medium (PrEGM). Count the cell density using a cell counter or hemocytometer and dilute the cells to 5 x 105 cells/mL in complete PrEGM. 
	Note: The yield from a 3-month-old mouse whole prostate can range from 3 x 105-106 cells. Hence, it is important to initially resuspend the pellet in no more than 0.5 mL of PrEGM (as mentioned above) so that the desired cell density can be achieved, even with low yield.</li>
	<li>Mix the required volume of cells with the basement membrane ECM in a 2:3 volume ratio (60% basement membrane ECM and 40% cell suspension) and plate in a multi-well plate or chamber slide as per the experimental needs. 
	Note: For immunostaining, plate on glass-bottom plates or chamber slides, covering the entire well or middle of the well. Plate around the rim of the well if counting the resulting organoids, or plate as several droplets all over the well if plating higher volumes. Be mindful to not spread it too thick or too thin. Plate at least 100 &#181;L of matrix-cell mixture per 1 cm2 of plating area; for example, in a chamber slide with a 2 cm2-well surface area, 200 &#181;L of matrix-cell mixture containing 5 x 104 cells should be plated.</li>
	<li>Transfer the plate/slide to a 37 &#176;C incubator with 5% CO2 for 30 min for the basement membrane ECM to solidify. Then, add pre-warmed complete PrEGM to cover the well, taking care not to disturb the basement membrane ECM plug.</li>
	<li>Remove half of the media and add fresh media every 2-3 days. Grow spheroids for 5-10 days.</li>
	<li>Proceed with harvesting (step 7), immunostaining (step 8), and/or imaging for downstream applications.</li>
</ol>
7. Harvesting the Spheres
<ol>
	<li>To harvest spheres, aspirate the media carefully without disturbing the basement membrane ECM gel plug and add 1 mL of 1 mg/mL dispase solution in PrEGM per 100 &#181;L of basement membrane ECM. 
	Note: It is easier to harvest spheroids if they are plated in the middle of the well or the rim (instead of covering the whole well), to ensure that an adequate amount of the dispase solution can be added to the well.</li>
	<li>Scrape the bottom of the plate with a cell scraper to lift off the basement membrane ECM gel into the dispase-PrEGM solution.</li>
	<li>Pipette the entire solution up and down once with a wide bore 1000 &#181;L pipette tip or 5 mL pipette to break up the gel plug into smaller pieces. 
	Note: Cut the tip of a 1000 &#181;L pipette tip with a razor blade to make a wide bore tip.</li>
	<li>Incubate in a 37 &#176;C incubator with 5% CO2 for 1-2 h until the basement membrane ECM has completely dissolved. 
	Note: This can be ensured by visual inspection of the well bottom while tilting the plate to confirm that no more chunks of gel remain.</li>
	<li>Collect the cell suspension in a 15 mL conical tube.</li>
	<li>Centrifuge spheres at 250 x g for 5 min at room temperature and continue with downstream applications.</li>
</ol>
8. Immunostaining of the Spheres13
Note: After spheroids have grown for the desired amount of time, the basement membrane ECM can be dissolved and spheres can be stained as described in Colicino et al.13.
<ol>
	<li>Briefly, rinse the cultures with PBS and add 4% paraformaldehyde (PFA) directly to the basement membrane ECM gel plug. Incubate for 30-90 min at room temperature with slow shaking, until the basement membrane ECM is fully dissolved, replacing fresh PFA every 30 min. 
	Note: The PFA will dissolve the gel plug and fix the spheroids during this process.</li>
	<li>Next, wash with PBS 3 times and add 50 mM ammonium chloride for 10 min to quench any autofluorescence from the PFA. Repeat for 3 washes with PBS.</li>
	<li>Permeabilize with 0.1% non-ionic detergent for 5 min and block with PBSAT (PBS, 1% BSA, 0.5% Triton X-100) for 30 min.</li>
	<li>Incubate with a primary antibody in PBSAT overnight at 4 &#176;C, followed by 3 washes with PBSAT and a secondary antibody in PBSAT for 2 h at room temperature.</li>
	<li>Repeat the washes with PBSAT and stain with DAPI according to the manufacturer&#8217;s protocol, if desired. Wash 3 or more times with PBS.</li>
	<li>Add PBS with 200 mM 1,4-diazabicyclo[2.2.2]octane (DABCO) antifade reagent and proceed to imaging.</li>
</ol>

<ol>
	<li>Marks, C. L. <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+Human+Cancers+Consortium+(MMHCC)+from+the+NCI.">Mouse Models of Human Cancers Consortium (MMHCC) from the NCI.</a> Cancer Research. 65 (9 Supplement), 242-243 (2005).</li><li>Marks, C. <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+Human+Cancers+Consortium+(MMHCC)+from+the+NCI.">Mouse Models of Human Cancers Consortium (MMHCC) from the NCI.</a> Disease Models &amp; Mechanisms. 2 (3-4), 111 (2009).</li><li>Day, C. P., Merlino, G., Van Dyke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+mouse+cancer+models:+a+maze+of+opportunities+and+challenges.">Preclinical mouse cancer models: a maze of opportunities and challenges.</a> Cell. 163 (1), 39-53 (2015).</li><li>Society, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Facts and Figures. , (2018).</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 Research. 64 (6), 2270-2305 (2004).</li><li>Berquin, I. M., Min, Y., Wu, R., Wu, H., Chen, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+signature+of+the+mouse+prostate.">Expression signature of the mouse prostate.</a> Journal of Biological Chemistry. 280 (43), 36442-36451 (2005).</li><li>Wu, 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=Generation+of+a+prostate+epithelial+cell-specific+Cre+transgenic+mouse+model+for+tissue-specific+gene+ablation.">Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation.</a> Mechanisms of Development. 101 (1-2), 61-69 (2001).</li><li>Xin, L., Lukacs, R. U., 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=Self-renewal+and+multilineage+differentiation+in+vitro+from+murine+prostate+stem+cells.">Self-renewal and multilineage differentiation in vitro from murine prostate stem cells.</a> Stem Cells. 25 (11), 2760-2769 (2007).</li><li>Hofner, T., 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=Defined+conditions+for+the+isolation+and+expansion+of+basal+prostate+progenitor+cells+of+mouse+and+human+origin.">Defined conditions for the isolation and expansion of basal prostate progenitor cells of mouse and human origin.</a> Stem Cell Reports. 4 (3), 503-518 (2015).</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> Nature Protocols. 5 (4), 702-713 (2010).</li><li>Clarke, M. F., Fuller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+and+cancer:+two+faces+of+eve.">Stem cells and cancer: two faces of eve.</a> Cell. 124 (6), 1111-1115 (2006).</li><li>Oliveira, D. S., 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=The+mouse+prostate:+a+basic+anatomical+and+histological+guideline.">The mouse prostate: a basic anatomical and histological guideline.</a> Bosnian Journal of Basic Medical Sciences. 16 (1), 8-13 (2016).</li><li>Colicino, E. G., 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=Gravin+regulates+centrosome+function+through+PLK1.">Gravin regulates centrosome function through PLK1.</a> Molecular Biology of the Cell. 29 (5), 532-541 (2018).</li><li>Ittmann, 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=Animal+models+of+human+prostate+cancer:+the+consensus+report+of+the+New+York+meeting+of+the+Mouse+Models+of+Human+Cancers+Consortium+Prostate+Pathology+Committee.">Animal models of human prostate cancer: the consensus report of the New York meeting of the Mouse Models of Human Cancers Consortium Prostate Pathology Committee.</a> Cancer Research. 73 (9), 2718-2736 (2013).</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. 2011, 895238 (2011).</li><li>Xiong, 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=Disruption+of+Abi1/Hssh3bp1+expression+induces+prostatic+intraepithelial+neoplasia+in+the+conditional+Abi1/Hssh3bp1+KO+mice.">Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice.</a> Oncogenesis. 1, e26 (2012).</li><li>Liao, C. P., Adisetiyo, H., Liang, M., Roy-Burman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+enhance+the+gland-forming+capability+of+prostate+cancer+stem+cells.">Cancer-associated fibroblasts enhance the gland-forming capability of prostate cancer stem cells.</a> Cancer Research. 70 (18), 7294-7303 (2010).</li></ol>

The authors declare that they have no competing financial interests.

This paper outlines the methods for dissection of the mouse prostate and identification of individual lobes. Also described is the protocol for culturing mouse prostate cells in a 3D culture for in vitro analysis.
A critical step in the dissection protocol is (1) harvesting the entire UGS out of the mouse and separating the individual organs under a dissection microscope. The prostate tissue is very small and surrounded by the rest of the UGS; thus, it is practically impossible to har...

The scientific community has been attempting to elucidate the complex mechanism of human cancer development for decades. Whereas identification of potential key players and drug targets begins with patient cells and tissue studies, the translational application of such findings often requires the use of pre-clinical animal models. The use of genetically engineered mice models (GEMMs) to model human cancers has steadily risen since the establishment of the Mouse Models of Human Cancers Consortium (NCI-MMHCC), a committee which sought to describe and unify characteristics of mouse cancer models for scientists worldwide1,2. Mouse models fulfill the need for mechanistic studies in pre-clinical studies of most types of cancer, for understanding the development, progression, response to treatments, and acquired resistance3. 
Prostate cancer is the most commonly occurring cancer in men, affecting over 160,000 men every year4. Aggressive forms of the disease claim tens of thousands of lives every year. However, the mechanism of disease progression is still poorly understood. This results in a serious lack of effective treatment options for advanced and metastatic prostate cancer, as evidenced by the high mortality rate in advanced prostate cancer patients4. Hence, there is a growing need for pre-clinical models to study prostate cancer. However, owing to the inherent differences between the mouse and human prostate, modeling of prostate cancer in GEMMs did not gain popularity until the Bar Harbor Classification system was introduced in 2004, which outlined histopathological changes in the mouse prostate upon genetic manipulation, identification of neoplastic changes, and their relation to stages of cancer progression in humans5. One important characteristic of the mouse prostate that must be taken into consideration while studying any prostate GEMM model is the presence of four distinct pairs of lobes: anterior, lateral, ventral and dorsal. The lobes present significant differences in the histopathology and gene expression pattern6. Probasin protein expression pattern can vary between lobes in young post-puberty mice7, which must be considered since Cre-based GEMM models are mostly designed using a probasin-based promotor called Pb-Cre47. The resulting spatial and temporal differences in Cre expression often lead to differences in tumor initiation and progression timelines as well as differences in neoplastic changes between the lobes. Hence, it is important to account for such differences while studying tumor development in the prostate GEMMs, and the individual lobes may need to be evaluated separately to achieve reproducible results. The first part of this article describes the proper methods to dissect a mouse prostate, identify and separate each lobe, and recognize the histological differences between the lobes.
While the analysis of tumor growth and histopathology can provide valuable insights into the tumor development, they do not provide much information about molecular mechanisms. To study the mechanism of tumor development and progression, it is often useful to analyze the tumor cells in vitro. Several methods have been suggested over the years that involve cultures of these cells, including suspension cultures, 3D cultures8 and recently, regular 2D cultures9. Whereas most of these methods result in good cell survival and proliferation rates, the 3D cultures provide an environment that is closest to physiological conditions. In 3D or spheroid cultures grown in a basement membrane extracellular matrix (ECM), the fully differentiated luminal cells usually have very low survival rate; however, the basal and intermediate cells (mostly stem cells) are able to propagate and produce cell clusters called spheroids10. This makes it suitable for a cancer study since epithelial cancers are believed to originate from stem cells (popularly known as cancer stem cells)11. The second part of this protocol describes a method for culturing the mouse prostate cells in 3D cultures. The resulting spheres can be used for several types of downstream analyses, including the study of organoid morphology and behavior by live cell imaging, immunofluorescence staining for different proteins, and the study of responses to chemotherapeutic treatments.
Overall, the goal of this protocol is to outline optimal methods for using mouse models in prostate cancer by describing the anatomy and dissection techniques of the mouse prostate and the processing of the tissue for spheroid cultures and in vitro analysis.

<table><tbody><tr><td>Mouse surgical instruments (Mouse Dissecting kit)</td><td>World Precision Instruments</td><td>MOUSEKIT</td><td></td></tr><tr><td>Dissection microscope</td><td></td><td></td><td></td></tr><tr><td>RPMI medium</td><td>Thermofisher Scientific</td><td>11875093</td><td></td></tr><tr><td>Dissection medium (DMEM + 10%FBS)</td><td>Thermofisher Scientific</td><td>11965-084</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>10438018</td><td></td></tr><tr><td>PBS (Phosphate buffered saline)</td><td>Thermofisher Scientific</td><td>10010031</td><td></td></tr><tr><td>Collagenase</td><td>Thermofisher Scientific</td><td>17018029</td><td>Make 10x stock (10mg/ml) in RPMI, filter sterilize, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Thermofisher Scientific</td><td>25300054</td><td></td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>10104159001&amp;nbsp;ROCHE</td><td></td></tr><tr><td>Syringes and Needles</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Fisherbran&amp;nbsp;Sterile Cell Strainers, 40&amp;mu;m</td><td>Fisher Scientific</td><td>22-363-547</td><td></td></tr><tr><td>PrEGM BulletKit&amp;nbsp;</td><td>Lonza</td><td>CC-3166</td><td>Add all componenets, aliquot and store at -20 &amp;deg;C.</td></tr><tr><td>Matrigel membrane matrix</td><td>Thermofisher Scientific</td><td>CB-40234</td><td></td></tr><tr><td>Dispase II powder</td><td>Thermofisher Scientific</td><td>17105041</td><td>Make 10x stock (10mg/ml) in PrEGM, filter sterilize, aliquot and store at -20 &amp;deg;C</td></tr></tbody></table>

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.

The mouse prostate lobes can be identified and dissected using their locations with respect to the seminal vesicles and urethra. The mouse prostate is composed of 4 pairs of lobes located dorsally and ventrally to the seminal vesicles and urethra. Figure 4a and 4b (top) show the dorsal and ventral views of the intact prostate, along with the seminal vesicles and urethra. The bottom panels (Figure 4c and <strong...

Watch this Scientific Journal Video about Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models at JoVE.com

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

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 ...

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20.0K Views.  SUNY Upstate Medical University. Deze methode kan helpen bij het starten van genetisch gemanipuleerde muismodellen van prostaatkanker en onderzoek van prostaatkanker mechanisme door middel van in vivo en in vitro analyse. Het belangrijkste voordeel van deze techniek is dat het zorgt voor een volledige analyse van de prostaatkanker muis model, helemaal van weefsel dissectie tot celisolatie en culturing. Na het blootstellen van het urogenitale systeem, of UGS, stevig grijpen de urineblaas met medium stompe tangen, en til het h...

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

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 ...

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

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Bioengineering

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 ...

Medicine

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 ...

Extracting Prostate from Mouse Urogenital System: A Technique to Separate Prostate Gland from Other Urinary and Genital System Organs in Male Mouse

Source: Nath, D. et al. <a href="https://www.jove.com/video/58397" target="_blank">Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models.</a> J. Vis. Exp. (2018)
In this video, we describe the procedure for the dissection of the urogenital system or UGS from a male mouse model. This UGS is further dissected ex vivo to isolate the prostate gland and study its role in prostate cancer development.

Source: Nath, D. et al. Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models. J. ...

JoVE Encyclopedia of Experiments

Prostate Cancer

Animal model studies

Prostate Organoid Assay: A Matrix Gel Ring-based Ex Vivo Culture Technique to Study the Differentiation Capacity of Prostate Epithelial Cells

Source: Crowell, P. D. et al. <a href="https://www.jove.com/video/60223" target="_blank">Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the culturing of isolated mouse prostate epithelial cells using a matrix gel-ring based culture technique. This approach can provide complementary information about the differentiation capacity of prostate epithelial cells to form 3D glandular structures in response to genetic or pharmacological manipulation.

Source: Crowell, P. D. et al. Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture. J. Vis. Exp. (2019)
In ...

Assays and techniques

Individual Lobe Extraction from Prostate: A Technique to Harvest Individual Prostate Lobes from Mouse Prostate

Source: Nath, D. et al. <a href="https://www.jove.com/video/58397" target="_blank">Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models.</a> J. Vis. Exp. (2018)
This video describes a technique to harvest individual lobes from the prostate gland of a mouse model. Individual lobes can then be used for histological analysis and cancer research.

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

Samenvatting

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