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 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 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.
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.
2. Dissecting the Prostate
3. Gross Anatomy of the Prostate and Individual Lobe Microdissection (Figures 3i-n, Figure 4)
4. Lobe Identification and Morphology from Hematoxylin and Eosin-Stained Slides
5. Processing the Tissue for 3D Culture10
Note: This is outlined in Figure 6.
6. Plating and Culturing the Cells
7. Harvesting the Spheres
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.
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 4d) show the different lobes outlined for identification. Lobes can be separated from mice as young as 3 months.
The different prostate lobes differ significantly in histology (Figure 5). All mouse prostate lobes are composed of multiple gland profiles composed of a lumen surrounded by secretory epithelial cells; however, the shape of the lobes, organization of the cells, and nature of the secretion vary from lobe to lobe. The identifying characteristics from H&E-stained mouse prostates have been efficiently outlined by Oliveira et al.12, briefly outlined here. Anterior lobes have moderate-to-large acini with frequent infoldings and strongly eosinophilic secretion. Dorsal lobes appear like anterior with eosinophilic secretion but have much smaller acini and less infoldings. Lateral lobes have small to large acini, with characteristic flat luminal borders and eosinophilic secretion. Ventral lobes are structurally like lateral lobes, also with flat luminal borders, but have a unique pale non-eosinophilic luminal secretion.
Mouse prostate cells can be cultured as spheroids in the basement membrane ECM and exhibit epithelial-like characteristics. This is outlined as a schematic in Figure 6. Cells were isolated from the mouse prostate as described above and cultured in the basement membrane ECM. The cells start growing into organoids in as early as 4 days of culture. Figure 7 (top) shows representative fields from 8-day-old cultures showing the organoids in the basement membrane ECM. Under the above-mentioned culture conditions, most of the cells grow into solid spheres, with a fraction having a partial or full lumen inside. These organoids usually have an even spherical morphology. Organoids were harvested and immunostained as described in Colicino et al.13. Figure 7 (bottom panel) shows an organoid without a lumen (left) and with a lumen (right), stained with phalloidin (F-actin marker, green) and DAPI (nucleus, blue). β-catenin is a cell-cell adhesion marker strongly expressed at cell-cell interfaces in epithelial cells. Figure 8 demonstrates strong β-catenin staining (green) at the cell-cell junctions in spheroids that co-localize with F-actin (red). The spheroids also express cytokeratin 5, cytokeratin 8, p63, and androgen receptor proteins, which provide evidence that the spheroids are indeed derived from cells originating in the prostate10.
Figure 1: Schematic flowchart demonstrating dissection of the mouse urogenital system and isolation of the prostate. Flow chart for the protocol steps described in sections 1-2. Please click here to view a larger version of this figure.
Figure 2: Dissection of the mouse UGS. Step-by-step images from the dissection of the UGS from a 3-month-old mouse: (a and b) making the incisions, (c-e) pulling back the skin to expose the abdominal area, (f) shifting the intestines, (g) moving the abdominal fat pads, (h and i) extracting the UGS from the abdominal cavity, (j) the extracted UGS, (k) the extracted UGS with the different organs and tissues marked as follows: SV (yellow) = seminal vesicles; P (red) = prostate; F (purple) = fat; V (light blue) = vas deferens; and B (green) = bladder. Please click here to view a larger version of this figure.
Figure 3: Dissection of the mouse prostate. Step-by-step images for the dissection of the mouse prostate from the UGS: (a) removing fat, (b) removing the urinary bladder, (c) removing the vas deferens, (d) ventral view of the prostate with the urethra, (e) dorsal view of the prostate with the urethra, (f-h) removing the seminal vesicles, (i) ventral view of the prostate, (j) dissecting a dorsal lobe, (k) dissecting a lateral lobe, (l) dissecting a ventral lobe, (m) dissecting an anterior lobe, and (n) the dissected prostate lobes: anterior = AP, dorsal = DP, lateral = LP and ventral =VP. Please click here to view a larger version of this figure.
Figure 4: Anatomy of the mouse prostate lobes. Representative images from 3-month-old mouse UGS after removal of the fat, bladder, and vas deferens. Images show prostate lobes with seminal vesicles and urethra, with dorsal (a and c) and ventral (b and d) views. Top panels (a and b) show untouched images, while bottom panels (c and d) show the lobes outlined in blue (dorsal), red (anterior), yellow (ventral), and green (lateral). Please click here to view a larger version of this figure.
Figure 5: The different prostate lobes vary significantly in histology. Here is an H&E-stained whole prostate image showing the seminal vesicles, urethra, and the four prostate lobes. Lobes are outlined in orange (anterior), blue (dorsal), green (lateral), and black (ventral). Inset: representative images from each lobe, taken under a 10x objective. Scale bar represents 100 µm. Please click here to view a larger version of this figure.
Figure 6: Schematic flowchart demonstrating digestion of the prostates and subsequent spheroid cultures. Flow chart for the protocol steps described in sections 5 and 6. Please click here to view a larger version of this figure.
Figure 7: The prostate cells grow into even spherical spheroids with or without a lumen. Prostate tissue harvested from a wild-type 3-month-old mouse was cultured as per the protocol. Cultures were imaged on a plate imager under a 4X objective, at 8 days post-culture, showing the formation of spheroids (top). The spheroids were harvested and stained as described in the protocol with a fluorescent dye-conjugated phalloidin for F-actin (green) and DAPI for nuclei (blue), then imaged on a spinning disk confocal microscope under a 40X water objective (bottom). The spheroid on the right shows evidence of a lumen. Scale bars represent 50 μm. Please click here to view a larger version of this figure.
Figure 8: The mouse prostate organoids show junctional localization of β-catenin. Spheroids harvested from 6-day-old 3D cultures were stained, using a primary antibody against β-catenin and a fluorescent dye-conjugated secondary antibody, and imaged on a spinning disk confocal microscope under a 40X water objective. Representative images show immunostaining for β-catenin (green, left), F-actin (red, middle), and DAPI (blue). The right panel shows all 3 channels merged. Scale bar represents 20 μm. Please click here to view a larger version of this figure.
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 harvest the organ directly from the body cavity while procuring all four pairs of lobes intact. Hence, it is important to harvest the entire UGS from the mouse and then proceed to extract the prostate. It is also critical in this protocol to (2) perform the steps in a timely fashion but remain meticulous. Time is of the essence during dissection in order to minimize tissue degradation. The specific technique described here for extracting the UGS from the mouse is faster compared to alternatives and is highly recommended. The entire dissection and lobe isolation procedure usually takes 35-40 min, which can be lowered to 25 min with more practice. The most challenging part of the dissection is cleaning the fat surrounding the UGS, which takes up a large fraction of total dissection time. The fat must be meticulously removed to make sure none of it remains with the prostate, but it is important to be extremely careful not to accidentally lose any prostate tissue in the process. Another critical step is (3) to not remove the urethra before separation of the prostate lobes. The urethra essentially forms the base on which all four pairs of prostate lobes are arranged. It is easiest to identify the lobes if they are still attached to the urethra, based on their spatial distribution with respect to the urethra.
A critical step in the spheroid culture protocol is (1) mincing the tissue well. Mincing the prostate tissue with a scalpel is a tedious step, especially since the prostate tissue has a sticky nature. However, mincing the tissue as small as possible is essential to obtain maximum yield. Another essential step is (2) pipetting after trypsinization and passing through syringes of the sizes specified post-neutralization. Both these steps must be performed correctly and repeated, if required, to achieve optimum cell yield. Finally, it is important (3) not to plate the basement membrane ECM too thick or too thin. If the basement membrane ECM is plated too thin (i.e., less than 100 µL/cm2), cells will not grow into proper organoids in the middle of the well, where the gel will be the thinnest. Plating it too thick (i.e., more than 250 µL/cm2) will result in the basement membrane ECM not solidifying properly and sliding off during media changes. Plating in the middle of the well will help to achieve the most even gel thickness.
The protocol described can be slightly modified according to experimental needs. The technique for the extraction of the UGS from the abdominal cavity requires a firm grip on the urinary bladder with forceps. In case the bladder is too full, it is important to drain the bladder with a 1 mL syringe and thin (26G or similar) needle, to ensure a proper grip before starting to extract the tissue. The tissue should be kept moist with PBS or DMEM throughout the dissection process. Using DMEM is preferred if the tissue is going to be used for spheroid cultures. The harvested cell suspension can be sorted by flow cytometry to isolate sub-populations of cells before plating, if desired, as described by Lukacs et al.10.
Histological characteristics of the different prostate lobes have been outlined in this protocol, and one should be able to identify the lobes in healthy prostate tissue by following these guidelines. However, there are certain limitations of the process. In cases of hyperplasia or aggressive tumors, the epithelial morphology is disrupted, which can make it significantly harder to distinguish between lobes based on the H&E staining. In such instances, separation of lobes (as outlined in this protocol) before embedding and staining is necessary if lobe-specific information is needed. Even in healthy tissue, it is often necessary to analyze lobes separately since they present wide variability in anatomy and histology and have differential expression signatures6. However, the translational relevance of separating lobes in mouse prostate may be debated, since the human prostate is not divided into lobes. In spite of this, the mouse remains the best model to study PCa in vivo, and several mouse models have been developed14,15. The 3D culture method especially is a key technique that can be used to investigate mechanisms of the disease. However, one caveat of the spheroid culture method arises from the differential expression pattern of probasin, which is the commonly used promoter for Cre recombinase in prostate cancer mouse models. The probasin promoter is predominantly activated in luminal and intermediate cells but not in basal cells7. The culture conditions described here promote spheroid formation predominantly from basal and intermediate cells, but not from luminal cells10. Hence, the resulting cultures actually produce a mixture of Cre-expressing and non-Cre-expressing spheroids (i.e., a mixture of control and knock-out spheroids). As a result, during analysis, it is important to immunostain for the protein of interest to identify knock-out organoids and draw conclusions on organoid behavior based on gene knock-outs.
The significance of these methods lies in the multi-faceted applications of using GEMMs in prostate cancer studies. The dissection method described has detailed steps which will enable researchers to extract prostates faster and more effectively. The sequence of the dissection process is designed to ensure that individual lobes can be identified and extracted, even by those with minimal experience in prostate dissections. The culture method outlined can be used to further analyze GEMM models of prostate cancer. The culture conditions should allow for growth and survival of both control and tumor cells. In our experience, we have successfully used GEMM prostates that exhibit mPIN16. Mouse prostates with higher grade tumors have also been used for spheroid culture analysis17. The control mouse spheroids exhibit an even spherical morphology; however, spheroids derived from prostate tumor cells may demonstrate differences in morphology and behavior due to their neoplastic potential. Hence, studying organoid behavior in vitro will provide an extended look into the characteristics of these cells, and possibly observations of spheroid behavior in real-time through live-cell imaging.
In conclusion, the prostate dissection and culture methods described in this protocol can be incorporated into various types of studies involving GEMMs to continue providing information on prostate cancer in all downstream applications.
The authors have nothing to disclose.
This work was supported by the grant from National Cancer Institute, R01CA161018 to LK.
Mouse surgical instruments (Mouse Dissecting kit) | World Precision Instruments | MOUSEKIT | |
Dissection microscope | |||
RPMI medium | Thermofisher Scientific | 11875093 | |
Dissection medium (DMEM + 10%FBS) | Thermofisher Scientific | 11965-084 | |
Fetal Bovine Serum | Thermofisher Scientific | 10438018 | |
PBS (Phosphate buffered saline) | Thermofisher Scientific | 10010031 | |
Collagenase | Thermofisher Scientific | 17018029 | Make 10x stock (10mg/ml) in RPMI, filter sterilize, aliquot and store at -20 °C |
Trypsin-EDTA (0.05%) | Thermofisher Scientific | 25300054 | |
DNase I | Sigma-Aldrich | 10104159001 ROCHE | |
Syringes and Needles | Fisher Scientific | ||
Fisherbran Sterile Cell Strainers, 40μm | Fisher Scientific | 22-363-547 | |
PrEGM BulletKit | Lonza | CC-3166 | Add all componenets, aliquot and store at -20 °C. |
Matrigel membrane matrix | Thermofisher Scientific | CB-40234 | |
Dispase II powder | Thermofisher Scientific | 17105041 | Make 10x stock (10mg/ml) in PrEGM, filter sterilize, aliquot and store at -20 °C |