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

Abstract

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.

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).

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).

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 × 10⁶ cell/mL cell concentration. Plate domes in dish using a drop-wise method.

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.

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.