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...

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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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Mouse surgical instruments (Mouse Dissecting kit)</td>
			<td style="width:260px;">World Precision Instruments</td>
			<td style="width:196px;">MOUSEKIT</td>
			<td style="width:620px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Dissection microscope</td>
			<td style="width:260px;"></td>
			<td style="width:196px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">RPMI medium</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td style="width:196px;">11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Dissection medium (DMEM + 10%FBS)</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Fetal Bovine Serum</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td>10438018</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:415px;">PBS (Phosphate buffered saline)</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td style="width:196px;">10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Collagenase</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td style="width:196px;">17018029</td>
			<td>Make 10x stock (10mg/ml) in RPMI, filter sterilize, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td style="width:196px;">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">DNase I</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td>10104159001&#160;ROCHE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Syringes and Needles</td>
			<td style="width:260px;">Fisher Scientific</td>
			<td style="width:196px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Fisherbran&#160;Sterile Cell Strainers, 40&#956;m</td>
			<td style="width:260px;">Fisher Scientific</td>
			<td>22-363-547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrEGM BulletKit&#160;</td>
			<td style="width:260px;">Lonza</td>
			<td>CC-3166</td>
			<td>Add all componenets, aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Matrigel membrane matrix</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Dispase II powder</td>
			<td style="width:260px;">Thermofisher Scientific</td>
			<td style="width:196px;">17105041</td>
			<td>Make 10x stock (10mg/ml) in PrEGM, filter sterilize, aliquot and store at -20 &#176;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&#160;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&#160;4c and...

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 ...

This method can help in the start of genetically-engineered mouse models of prostate cancer and investigation of prostate cancer mechanism through in vivo and in vitro analysis. The main advantage of this technique is that it allows for a complete analysis of the prostate cancer mouse model, all the way from tissue dissection to cell isolation and culturing. After exposing the urogenital system, or UGS, firmly grasp the urinary bladder with medium blunt forceps, and lift the entire system up from the mouse abdomen.Slide a pair of scissors under the bladder and prostate all the way to the spine to make an incision through the spinal cord, and cut through any remaining connections to the abdominal cavity to allow removal of the entire UGS. Transfer the UGS to a six-centimeter Petri dish containing two to six milliliters of PBS under a dissecting microscope. And use a pair of fine forceps and microdissection scissors to carefully clear all of the fat from both the dorsal and ventral sides of the tissue system without snipping any prostate tissue.When all of the fat has been removed, pull the bladder with the forceps and excise the bladder at its base. Place the remaining tissue ventral side up and holding one duct ends with the forceps, follow the vessel to its base with scissors and excise the vessel at its base. Remove the same vessel on the other side and insert the forceps between the seminal vesicles and prostate to allow snipping of any adjoining connective tissue as necessary.Then trace the seminal vesicles to their base at the urethra and remove the vessels without puncturing them. When both vesicles have been removed, place the tissue dorsal side up so the dorsal lobes which resemble a butterfly's wings are visible. Holding each dorsal lobe with forceps, cut the tissue at its base with scissors to collect the dorsal lobes and turn the tissue to the ventral side.Collect the lateral lobes, which are small and usually wrap the urethra on the side and are wedged between the anterior, ventral, and dorsal lobes in the same manner. Collect the ventral lobes, which are larger than the lateral lobes and lie on the urethra ventrally. Then cut and discard the urethra to harvest the anterior lobes.To process the prostate tissue for 3D culture, transfer the lobes to a 10-centimeter dish containing two to three milliliters of DMEM, and use a scalpel to mince the prostate tubules as finely and evenly as possible. Transfer the tissue fragments to a sterile tissue culture hood and add the tissue solution to a new 15-milliliter tube. Bring the volume in the tube up to nine milliliters with fresh medium, and add one milliliter of TenX collagenase stock solution to the tissue mixture.After vortexing, incubate the tube with shaking for two hours at 37 degrees Celsius to degrade the extracellular matrix. At the end of the incubation, collect the tissue by centrifugation and re-suspend the pellet in two milliliter of warm 05%strips in EDTA. After five minutes of 37 degrees Celsius to facilitate cleaving of the cell-to-cell and cell-to-matrix adhesions, use a P1000 pipehead with a wide board tip to triturate the tissue eight to 10 times to break up any clumps.Repeat the dissociation with a P200 pipehead tip. Then neutralize the trypsin with three milliliters of complete medium. Mix 500 units of DNASE1 into the tissue slurry and pass the solution five to 10 times through a five milliliter syringe equipped with an 18-gauge needle, followed by five times through a 20-gauge needle.When no more large tissue pieces are visible, filter the suspension through a 40-micrometer filter into a 50-milliliter conical tube, and collect the cells by centrifugation. For cell plating and culture, re-suspend the pellet in 0.5 milliliters of complete prostate epithelial cell growth medium, and allude the cells to a five times 10 to the fifth cells per milliliter of prostate epithelial cell growth medium concentration. Mix the cells with basement membrane extracellular matrix at a two to three volume of solution ratio, and plate the cell suspension in the appropriate experimental cell culture container.Then place the cells in a cell culture incubator for 30 minutes. When the extracellular matrix has solidified, cover the culture with pre-warmed complete prostate epithelial cell growth medium, taking care not to disturb the basement membrane extracellular matrix plug. Then return the cells to the cell culture incubator for five to 10 days, refreshing half of the medium every two to three days.To harvest spheres, aspirate the medium carefully without disturbing the basement membrane extracellular matrix gel plug, and add one milliliter of Disbase solution for 100 microliters of basement membrane extracellular matrix. Using a cell scraper, scrape along the bottom of the plate to lift the basement membrane extracellular gels from the bottom of the plate into the supernatant, and pipe up the entire solution one time to break up the plug into smaller pieces. Incubate the extra cellular matrix pieces in the cell culture incubator for one to two hours until the basement membrane extracellular matrix has completely dissolved.Then transfer the cell suspension to a 15-milliliter tube. And collect the cells by centrifugation for further downstream manipulation. The mouse prostate is composed of four pairs of lobes located dorsally and ventrally to the seminal vesicles and urethra.The prostate lobes are composed of multiple gland profiles, each comprised of a lumen, surrounded by secretory epithelial cells. The shapes of the lobes, the organization of the cells and the nature of the secretion vary from lobe to lobe. The isolated prostate cells begin growing into organoids as early as four days after basement membrane extracellular matrix culture.Over the next one to six days, most of the cells grow into solid spheres with a fraction of the spheres exhibiting a partial or full lumen. The spheroids also demonstrate a strong beta catenin staining at the cell-to-cell junctions that co-localize with F-actin staining. While attempting this procedure, it's very important to follow meticulously the microdissection steps.For example, isolate the prostate lobes while they're still attached to the urethra, so you know which lobe is which based on where they are with respect to the urethra. Following the dissection procedure, the isolated lobes can be used for downstream analysis, such as RMA sequencing, Western blotting or immunostaining. The primary 3D culture technique allows you to gain further insights into the prostate cancer mechanism, because you can monitor life with spheroids for morphology and behavior changes and identify any neoplastic changes in the same.In summary, this prostate dissection and culture methods can be incorporating to various downstream applications to provide valuable information for investigating the mechanism of prostate cancer using genetically engineered mouse models.

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Cancer Research

19.3K visualizações.  SUNY Upstate Medical University. Este método pode ajudar no início de modelos de camundongos geneticamente modificados de câncer de próstata e investigação do mecanismo de câncer de próstata através de análise in vivo e in vitro. A principal vantagem dessa técnica é que ela permite uma análise completa do modelo de camundongos cancerosos de próstata, desde a dissecção tecidual até o isolamento celular e a cultura. Depois de expor o sistema urogenital, ou UGS, segure firmemente a bexiga urinária com fórceps...