Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

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.  

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