Name: identification, histological characterization, and dissection of mouse prostate lobes for in vitro 3d spheroid culture models
Uploaded: 2018-09-18T14:30:00
Description: 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

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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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 ...

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