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) 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.
Since the late 1980s, the ability to alter genes by homologous recombination has greatly advanced the study of biological systems1. Inducible, tissue-, or cell-specific promotor systems and site-specific recombinases, such as Cre-loxP, has advanced genetic studies by facilitating control over genetic modifications both temporally and spatially2,3,4. The combination of these genetic strategies has created a wide array of experimental model systems5,6,7.
Genetically engineered mouse models (GEMMs) are an integral tool to assess how individual genes or groups of genes affect mammalian development and disease. In pre-clinical cancer research, GEMMs are the most physiologically-relevant and rigorous method to study cancer development, progression, and treatment8. Our laboratory specializes in generating and characterizing cancer GEMMs.
The most highly diagnosed non-cutaneous cancer among men in the United States is prostate cancer (PCa). The majority of patients with PCa have low-risk disease and high likelihood of survival, but survival rates decline drastically when disease is diagnosed at advanced stages or if targeted hormonal therapy induces progression to aggressive, non-curable PCa subtypes9,10. Our laboratory has developed GEMMs that utilize floxed alleles of one or more tumor suppressor genes. Recombination and loss of tumor suppressor gene expression occurs specifically in the prostate because we have introduced a transgene with Cre recombinase downstream of the probasin promoter activated only in prostate epithelial cells11,12. We have also bred our GEMMs to contain a Cre reporter transgene called mT/mG, which induces Tomato fluorescent protein expression in cells lacking Cre and green fluorescent protein (GFP) expression in cells with Cre13. While the presentation of this method and our representative results show GEMMs we study in our laboratory, this protocol can be used to generate prostate cancer organoids from any mouse model. However, as discussed in detail in our representative results section, we have observed that certain tumor characteristics are optimal for prostate cancer organoid generation.
In the last decade, new methods of culturing cells from tissues of epithelial origin has led to significant advances in our ability to model organ systems in vitro14,15. The term "3D cell culture" has been attributed to the techniques involved in establishing and maintaining organoids, which can be generally defined as structures made up of cells that assemble secondary architecture driven by organ-specific cell lineage characteristics16. These new methods are distinct from classic 2D cell culture in that cells do not require transformation or immortalization for long term growth; thus, 3D cultures of normal cells can be compared to diseased cells. This is particularly valuable in cancer research where normal cell control cultures have typically not been available. In addition, organoids spontaneously form secondary tissue architectures with appropriately differentiated cell types, making them a better model system to understand cancer in vitro than 2D cell lines17. Our laboratory has created 3D organoid lines from tumor issue isolated from our PCa GEMMs to complement our in vivo data and perform experiments which would not be feasible in GEMMs.
In this article, we present written and visual protocols for the complete necropsy of PCa GEMMs, including dissection of distinct mouse prostate lobes and metastatic lesions. We describe and show a step-by-step method for generating organoids from mouse prostate tumors based on a protocol previously published by Drost et al. for deriving organoids from normal mouse prostate epithelial tissue18.
Animal procedures described here were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Resources, Roswell Park Comprehensive Cancer Center, Buffalo, New York.
NOTE: Male mice to be dissected to isolate prostates or prostate tumors for generation of organoids should have at least reached the age of sexual maturity — about 8-10 weeks of age. Specific ages of mice can vary amongst studies. Some factors to consider when choosing age include age-dependent changes in prostate cell populations, age-dependent expression of specific promoter-driven Cre transgenes, and rate of prostate tumor progression in a particular GEMM.
1. Dissection and Imaging of Mouse ProstateTumor and Metastatic Tumors
2. Generation of 3D Organoids from Prostate Tumor Tissue
NOTE: Figure 3 shows a pictorial description of the procedure for generation of tumor organoids.
Representative necropsy images of a mouse with a large fluid-filled primary prostate tumor in the anterior prostate region are shown in Figure 2A. In contrast, Figure 2B, shows representative necropsy images of a mouse with a large solid primary prostate tumor for which individual prostate regions are indistinguishable. Fluorescent dissection images show the same solid prostate tumor from Figure 2B expressing GFP, indicating that the tumors cells express Cre (Figure 2C). Tissue that does not express probasin, such as the bladder, express Tomato and thus does not express Cre (Figure 2C). The liver and lungs from the mouse from Figure 2B have metastatic tumors expressing GFP, showing that they originated from the primary prostate tumor, and are surrounded by normal tissue that expresses Tomato (Figure 2C). Finally, the pelvic lymph node from this mouse expresses GFP and not Tomato, indicating that this metastatic tumor has overtaken this organ and no normal tissue remains (Figure 2C).
We show images in Figure 4 of organoids we have generated from a solid prostate tumor. At Day 1, small organoids are forming, as seen in the representative phase contrast images. Fluorescent images on Day 1 show that both Tomato and GFP expressing cells are present in the tumor organoid culture (arrows). However, by Day 7 when prostate tumor organoids have fully formed, these organoids are expressing GFP and not Tomato. These data suggest that these organoids have originated from tumor cells that were expressing Cre and not from normal epithelial cells. These tumor organoids continue to be only GFP-positive as we expand our culture to passage 1 and 2.
In Figure 5, we show images of organoids we have generated from a fluid-filled prostate tumor. On Day 1, small organoids are forming, and fluorescent images show that both Tomato- and GFP-expressing cells are present in the organoid culture — similar to our observation at Day 1 for organoids generated from a solid prostate tumor (Figure 4). However, organoids from a fluid-filled prostate tumor express either GFP or Tomato at Day 7 — indicating that organoids have formed from cells that do not express Cre. This pattern continues at passage 1 and passage 2, where the culture has both Tomato- and GFP-expressing organoids. Further analysis of these organoids is severely limited because the line is a mixture of normal epithelial organoids and tumor organoids. We believe that fluid-filled prostate tumors are suboptimal in generating tumor organoids simply because there is a greater percentage of normal prostate epithelial cells. Since both normal prostate epithelial cells and prostate cancer cells form organoids, the lines generated from fluid-filled prostate tumors are a mixture of normal and cancer organoids. We obtain pure tumor organoid lines from fluid-filled prostate tumors by flow sorting for GFP-positive cells and generating organoids from that population of cells. Solid prostate tumors are primarily comprised of tumor cells, therefore organoids generated from these tumors are a more pure population of cancer organoids without prior sorting for GFP.
Figure 1: Our recommended dissection order for prostate cancer (PCa) genetically engineered mouse models (GEMMs) and anatomy of the mouse prostate. (A) The order we recommend in our protocol for dissecting the major organs from a PCa GEMM. 1. Urogenital region. 2. Pelvic lymph nodes. 3. Spleen. 4. Liver. 5. Kidneys. 6. Lungs. 7. Tibia and Femur. (B) Map of the mouse urogenital region and prostate anatomy. Fluorescent dissection images of a 12 week old mouse expressing probasin-Cre and the mT/mG Cre reporter transgene. Bladder (BL), seminal vesicles (SV), anterior prostate (AP), ventral prostate (VP), lateral prostate (LP), dorsal prostate (DP), and proximal prostate (PP). Please click here to view a larger version of this figure.
Figure 2: Representative dissection images of prostate cancer (PCa) genetically engineered mouse models (GEMMs). (A) The abdominal cavity prior to removal of the urogenital region and the urogenital region with a fluid-filled prostate tumor. (B) The abdominal cavity prior to removal of the urogenital region and the urogenital region with a solid prostate tumor. (C) Representative Tomato and GFP fluorescent images of a solid prostate tumor, liver, lung, and pelvic lymph node from a PCa GEMM that develops metastatic lesions. Scale bar = 5 mm. Bladder (BL), anterior prostate (AP), and dorsal prostate (DP). Please click here to view a larger version of this figure.
Figure 3: Flow chart of the protocol for generating prostate tumor organoids. After dissecting the prostate tumor, mince the tissue into 1 mm pieces. Digest the tumor pieces in collagenase, collect the cells, and digest in trypsin to obtain a single cell suspension. After counting cells, resuspend in volume of matrix required for a 1.0 x 106 cell/mL cell concentration. Plate domes in dish using a drop-wise method. Please click here to view a larger version of this figure.
Figure 4: Representative images from generation of mouse prostate tumor organoids from a solid prostate tumor. Representative phase contrast, Tomato, and GFP fluorescent images from Day 1, Day 7, Passage 1, and Passage 2 of organoids generated from a solid mouse prostate tumor. Scale bar = 100 µm. Arrows indicate individual cells in fluorescent images. Please click here to view a larger version of this figure.
Figure 5: Representative images from generation of mouse prostate tumor organoids from a fluid-filled tumor. Representative phase contrast, Tomato, and GFP fluorescent images from Day 1, Day 7, Passage 1, and Passage 2 of organoids generated from a fluid-filled mouse prostate tumor. Scale bar = 100 µm. Arrows indicate individual cells in fluorescent images. Please click here to view a larger version of this figure.
Critical steps within the protocol for prostate tumor dissection and organoid generation
Removal of non-prostate tissue and fine dissection of the mouse prostate tumor is crucial for the optimal generation of cancer organoids since both non-prostate epithelial cells and normal prostate epithelial cells will generate organoids. For solid prostate tumors specifically, it is crucial to isolate areas of viable tumor to remove contamination with necrotic tissue that would reduce the number of viable cells. During organoid generation, tissue digestion with collagenase should be diligently monitored, as prolonged exposure to collagenase will limit cell viability. With organoids derived from cancer GEMMs, it is crucial to fully genotype each line to ensure that all transgenes and modified alleles that were engineered in the mouse are present in the organoids. Repetition of genotyping after prolonged passaging is also necessary to ensure that genetic modifications are maintained.
Modifications and troubleshooting of prostate tumor dissection and organoid generation
We have observed mouse to mouse variability in prostate tumor characteristics, even amongst animals with the same genotype. Therefore, specific modifications to the prostate dissection protocol described here may be necessary for each mouse. In addition, adaptability is necessary when dissecting metastatic tumors since it is difficult to predict the severity of these lesions prior to starting the dissection.
On a few occasions, we have observed excess contamination of our cell pellet with what appears to be connective tissue, even after digestion with both collagenase and trypsin. When this occurs, we resuspend the pellet in at least 2 mL of AdDMEM F12(+++) and use a 40 µm cell strainer to remove the connective tissue. Since there is lot-to-lot variability in the solidification rate of the matrix, increasing or decreasing the time for dome solidification may be necessary prior to application of organoid media.
Limitations in using GEMMs
While GEMMs are the most rigorous method for pre-clinical cancer studies, this approach requires significant time, expense, and training. In addition, mouse to mouse variability can, as in the study of humans, complicate interpretation of data.
Limitations in using 3D cell culture
Compared to 2D cell culture, generating and maintaining organoid lines require increased time and cost. For instance, our tumor organoid lines are passaged every 2-3 weeks, while cell lines can be passaged every 2-3 days. This slower growth rate of organoids increases the time required to complete experiments considerably. Organoid culture media contains several specialized growth factors and reagents, which can be costly depending on source, thus generating and maintaining organoids is more expensive than traditional 2D cell lines. Finally, our laboratory and others have observed lot to lot differences in matrix and other reagents – creating a challenge for maintaining consistency in organoid growth for long term experiments.
Significance in using 3D cell culture with respect to existing/alternative methods
Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived xenograft models. Cell growth in 2D requires transformation/immortalization — thus both in vitro and xenograft studies using 2D cultures typically do not have unaltered normal cell lines to serve as non-cancer controls. The last decade of research in 3D organoid culture of normal epithelial-derived tissues has now allowed for the growth of non-cancerous epithelial tissues that can be used to compare to analogous organoids derived from cancer tissue. Cancer organoids can also be used to establish xenografts to further understand tumor development. In addition, non-cancer organoids can be used to generate control xenografts — which was not possible before 3D cell culture methods were developed16.
Significance in using 3D cell culture in prostate cancer research
In recent studies, organoids have been used to recapitulate GEMM prostate tumor characteristics. Dardenne et al. show that organoids generated using prostate tumors from GEMMs that simultaneously lack the tumor suppressor Pten and overexpress the MYCN oncogene had greater growth potential than organoids generated using prostates from control GEMMs. In addition, both sequencing and immunohistochemistry showed that tumor organoids recapitulated the expression profiles of prostate tumors both lacking Pten and overexpressing MYCN21. Blattner et al. show that simultaneous prostate overexpression of an oncogenic mutant of Speckle Type BTB/POZ Protein (SPOP) and deletion of Pten increases the rate of tumorigenesis in GEMMs. When prostate organoids were generated to overexpress mutant SPOP, their proliferation was increased compared to control prostate organoids and lineage marker expression recapitulated original prostate tumors22. Together, these studies demonstrate that organoids are an optimal model for further study of prostate tumor characteristics in GEMMS.
Organoid culture has also been used as a tool to assess individual subpopulations of prostate tumor cells. Using GEMM tumors that lack Pten and both Pten and Trp53 tumor suppressors in prostate epithelial cells, Agarwal et al. fractionated cells into basal and luminal progenitors, propagated these subpopulations as organoids, and further characterized their specific phenotypes23. Thereby using 3D cell culture, it is possible to characterize subpopulations of tumor cells which may be limited in abundance within prostate tumors themselves.
As described above, 3D cell culture techniques permit the growth of normal epithelial cells. Thereby, prostate organoids generated from GEMMs lacking a Cre driver provide a unique model for real time monitoring of tumorigenesis by induction of Cre recombinase in vitro. Indeed, Dardenne et al. assessed how NMYC overexpression affects growth potential in the context of Pten loss over time by ectopically expressing ERT2-Cre and treating with tamoxifen21. Additionally, the effect of NMYC overexpression on androgen receptor (AR), the major target of therapy for prostate cancer, was assessed after induction of Cre recombinase in organoids generated from GEMMs21. The same inducible Cre system was used by Blattner et al. in prostate organoids to measure how overexpression of mutant SPOP affects prostate cancer cell proliferation and AR expression22. Notably, experiments inducing Cre expression in vitro have a built-in non-cancer control with vehicle-treated organoids.
Specific limitations in using 3D cell culture in prostate cancer research
While organoid growth of normal epithelial cells is an advantage of using 3D cell culture techniques, capacity to grow normal organoids has also presented a challenge in prostate cancer research studies. As shown in our representative results section, we have observed outgrowth of normal prostate organoids in lines generated from prostate tumors which are less aggressive (Figure 5). One way to address this phenomenon is to generate organoids from GEMMs expressing a Cre reporter transgene, such as mT/mG. Fluorescent microscopy can be used to assess the relative ratio of normal to tumor organoids by observing expression of Tomato and GFP. In addition, GFP expression can be used to flow sort organoid cells to generate pure prostate tumor organoid lines. Agarwal et al. show a sorting method for separation of normal epithelial cells and cancer cells from GEMM prostate tumors without a Cre reporter. They show that epithelial cell adhesion molecular (EpCAM)-positive cells from prostate tumors did not separate into subpopulations when sorted using either CD24 or Sca-1 cell surface markers23 — thus, these markers could be employed to exclude normal prostate epithelial cells from GEMM prostate tumors prior to organoid generation. Our laboratory and others have observed that the conditions under which prostate cancer cells form organoids appear to either select for or promote lineage specific gene expression programs characteristic of prostate basal epithelial cells. This is a significant challenge because prostate tumors in both mice and humans are primarily luminal in nature, expressing AR, CK8, and other luminal markers, and rarely express basal lineage markers such as p63 or CK5. While this phenomenon has yet to be published in detail, immunohistochemistry analysis shows that AR is decreased in Ptenf/+ organoids compared to Ptenf/+ prostates21. The outgrowth of basal epithelial cells in prostate cancer organoids calls into question whether these lines are truly an accurate pre-clinical model of prostate cancer.
While prostate cancer organoids have been documented to model the tumor from which they are derived better than traditional 2D culture, there is potential for organoids to undergo genetic changes in culture, especially after several passages. Currently, we are not aware of any published studies that have documented spontaneous genetic mutations, genetic gains or losses, or epigenetic changes that are common after prolonged passaging of prostate cancer organoids. To limit variability as a result genetic or epigenetic changes that may occur due to prolonged passaging, experiments should be performed in early organoids from early passages (<10) as often as possible.
Future applications of 3D cell culture
While it is impossible to predict all future applications that will be developed using 3D cell culture in cancer research, there are several avenues which appear to have the most potential. As with 2D cell lines, carrying out in vitro genetic modification is relatively straightforward in organoids. Modifying specific genes in either normal or cancer organoids opens up many possibilities in the study of the mechanisms governing tumorigenesis, cancer progression, and treatment — especially when genetically-modified organoids are used to generate organoid xenografts. Genetic modification of organoids is greatly advantageous when GEMMs do not exist for a specific gene or establishing a new GEMM is outside the scope of a particular study.
Cancer organoid culture also has many potential applications for clinical research. A library of relevant tumor subtypes within each organ system from both patients and animal models could be used to quickly assess efficacy of a new drug or new combination of existing drugs. As 3D cell culture becomes mainstream and increases in efficiency, generating patient-derived organoids for the purpose of personalized medicine has the potential to help tailor treatment for each cancer patient by testing all available drugs and combinations of drugs using his or her individual organoid line16.
The authors have nothing to disclose.
The authors would like to thank the Calvin Kuo Laboratory at Stanford University for providing HEK293 cells stably transfected with either HA-mouse Noggin-Fc or HA-mouse Rspo1-Fc. We would also like to thank Dr. Dean Tang for allowing us access the fluorescent dissection microscope in his laboratory. This work was supported by CA179907 to D.W.G. from the National Cancer Institute. Shared resources at Roswell Park Comprehensive Cancer Center were supported by National Institutes of Health Cancer Center Support Grant CA016056.
0.25 % Trypsin+2.21 mM EDTA | Sigma | 25-053 | |
1 1/4 in, 23 gauge, disposable syringe needles | Becton Dickinson | Z192430 | |
10 % neutral buffered formalin | Sigma | HT501128 | |
32 % paraformaldehyde | Electron Microscopy Services | 15714 | |
A83-01 | MedChemExpress | HY-10432 | |
Advanced DMEM/F12+++ | Gibco | 12634 | |
Analytical balance | Mettler Toledo | 30216623 | |
B27 (50X) | Gibco | 17504044 | |
Collagenase II | Gibco | 17101015 | |
Dissecting Board | Thermo-Fisher | 36-1 | |
EHS Sarcoma matrix, Pathclear Lot#19814A10 | Manufactured by Trevigen | Requistitioned from the National Cancer Institute at the Frederick National Laboratory | Holder of grants from the National Cancer Institute can request matrix |
HEPES (1M) | Sigma | 25-060 | |
human recombinant Epidermal growth factor (EGF) | PeproTech | AF-100-15 | |
L-glutamine (200 mM) | Sigma | 25-005 | |
N-Acetyl-L-Cysteine | Sigma | A9165 | |
Penicillin-Streptomycin | Sigma | P4333 | |
Precision balance | Mettler Toledo | 30216561 | |
Scalpel #23 | World Precision Instruments | 504176 | |
Scalpel Handle #7, 16 cm | World Precision Instruments | 500238 | |
Single-edge carbon razor blade | Fisherbrand | 12-640 | |
Stainless steel dissecting scissors, 10 cm, straight | World Precision Instruments | 14393 | |
Stainless steel Iris forceps, 10 cm, curved tip, serrated | World Precision Instruments | 15915 | |
Stainless steel Nugent utility forceps, straight tip, serrated | World Precision Instruments | 504489 | |
Y-276632 (Rock Inhibitor) | APExBIO | A3008 |