A modular and flexible ESC-based mouse model of pancreatic cancer

Abstract

Genetically engineered mouse models (GEMMs) have greatly expanded our knowledge of pancreatic ductal adenocarcinoma (PDAC) and serve as a critical tool to identify and evaluate new treatment strategies. However, the cost and time required to generate conventional pancreatic cancer GEMMs limits their use for investigating novel genetic interactions in tumor development and maintenance. To address this problem, we developed flexible embryonic stem cell (ESC)-based GEMMs that facilitate the rapid generation of genetically defined multiallelic chimeric mice without further strain intercrossing. The ESCs harbor a latent Kras mutant (a nearly ubiquitous feature of pancreatic cancer), a homing cassette, and other genetic elements needed for rapid insertion and conditional expression of tetracycline-controlled transgenes, including fluorescence-coupled shRNAs capable of efficiently silencing gene function by RNAi. This system produces a disease that recapitulates the progression of pancreatic cancer in human patients and enables the study and visualization of the impact of gene perturbation at any stage of pancreas cancer progression. We describe the use of this approach to dissect temporal roles for the tumor suppressor Pten and the oncogene c-Myc in pancreatic cancer development and maintenance.

Keywords: RMCE; embryonic stem cell; kras; mouse model; pancreas; pancreatic cancer.

Figure 1.

Development of ESC-based RMCE-competent mouse models of pancreatic cancer. (A) Technical outline: ESC lines were established from multiallelic transgenic mice and targeted by Flp/Frt-mediated recombination. Chimeric mice were generated from the untargeted ESC line or targeted ESC clones by injection into albino B6 blastocysts. The p48 GEMM-ESC-derived mice are characterized by high ESC contribution as assessed by coat color (agouti). For comparison, mice shown in this figure were all scored >95% coat color chimerism. From left to right, the Pdx GEMM-ESC animals with black coat color were scored 40%, 30%, 15%, 60%, and 10%. (B) Bright-field and fluorescence images of pancreata of mice derived from both GEMM-ESCs expressing mKate2. (C) H&E and mKate immunohistochemistry of pancreata from mice generated by the Pdx1 GEMM-ESCs and the p48 GEMM-ESCs. mKate2 expression was detected in the vast majority of exocrine cells in the p48 GEMM-ESC mice and shows a mosaic expression pattern in the Pdx1 GEMM-ESC animals. Bars, 50 µm.

Figure 2.

Chimeric mice generated with the PDAC GEMM-ESCs display Kras^G12D-induced neoplasia. (A–D) H&E shows PanIN lesions in chimeric mice generated by the Pdx1 GEMM-ESCs. PanINs are mKate2-positive, as opposed to the abundant stroma, and stain-positive for mucins (Alcian blue). Cytokeratin 19 confirmed ductal differentiation. (E–L) PDAC that developed in a 9-mo-old Pdx1 GEMM-ESC mouse. (E–G) Well-differentiated area from K showing mKate2-expressing invasive glandular lesions. Ductal differentiation was confirmed by CK19 immunohistochemistry in G. (H–J) Poorly differentiated area from K in which cells are mKate2-expressing but mostly stain-negative for cytokeratin 19. (L) Liver metastasis found in the same animal and displaying well-differentiated malignant lesions and abundant stromal desmoplasia. (M) PanINs from a mouse with pancreatic Kras^G12D expression, generated by traditional breeding for comparison. Bars, 50 µm if not otherwise specified.

Figure 3.

Pten is required for tumor maintenance, and its restoration leads to rapid tumor regression and a significant survival benefit. (A) On dox versus off-dox (14 d) comparison of pancreatic tumors from two shPten mice. H&E shows area of focal invasive pancreatic cancer in the top panel. Pten immunohistochemistry confirmed Pten depletion in the on-dox mouse within the Pdx1-Cre recombined neoplastic lesions and restoration of Pten expression in the off-dox mouse. Immunofluorescence analysis showed persistent mKate2 expression and dox-dependent GFP expression. The only remaining pancreatic cells with strong EGFP expression in the off-dox tumor were morphologically normal acinar cells, likely due to a low turnover rate and retention of EGFP protein. (B) Rapid tumor regression upon restoration of Pten expression in mice taken off dox food as assessed by three-dimensional ultrasound imaging (each line represents one individual mouse). (C) KM survival of shPten mice randomized into on-dox (n = 4) and off-dox (n = 4) treatment arms after detection of an 800- to 1100-mm³ pancreatic tumor. (D) Four weeks to 6 wk after dox deprivation, pancreata still harbor mKate2-positive neoplastic lesions.

Figure 4.

c-Myc plays a major role in Kras^G12D-induced neoplasia. (A) H&E and immunofluorescence for GFP and carboxypeptidase A1 (CPA) from pancreata of p48 GEMM-ESC mice expressing shRNAs directed against Renilla, Kras, and c-Myc after 4 wk on dox feed, starting on postnatal day 11. (B) Immunoblot confirming knockdown of c-Myc in sorted GFP- and mKate2-positive adult pancreatic cells from dox-treated mice. (C) Knockdown of Kras and c-Myc leads to retention of intact acinar tissue, as assessed by CPA-positive over total pancreatic area. (D) Decreased pancreas/body weight ratio in shMyc and shKras mice. Bars, 100 µm.

Figure 5.

The tandem cTGM targeting vector allows for combined inhibition of two independent genes. (A) Schematic of the tandem cTGM targeting vector. Depletion of shp53/shRpa3-expressing relative to shp53/shRenilla-expressing Pdx1 ESC clones functionally confirmed inhibition of Rpa3 by shRpa3 in the second position. Pdx1 ESCs were treated with adenoviral Cre recombinase to activate the latent rtTA₃ within the CAGS-LSL-RIK and were subsequently treated with dox-containing growth medium. (B) KM survival curve of chimeric mice generated with the Pdx1 GEMM-ESCs and expressing shp53/shRenilla (n = 16) or shp53/shRpa3 (n = 8). Mice received dox-containing feed starting on postnatal day 5. shp53/shRenilla mice succumbed to pancreatic cancer with a median latency of 60 d, while expression of shRpa3 efficiently inhibited pancreatic tumor development. (C,D) At 5–6 wk of age, pancreata of shp53/shRenilla and shp53/shRpa3 mice expressed mKate2 and GFP as a surrogate for tandem cTGM expression. (E) A tumor from a 9-wk-old shp53/shRenilla mouse expressed mKate2 and GFP. (F) A pancreas from a 19-wk-old shp53/shRpa3 mouse depicts almost complete depletion of mKate2- and GFP-expressing cells from the pancreas. Histopathology from mostly poorly differentiated pancreatic adenocarcinomas that developed in shp53/shRenilla mice are shown in E and F. Histopathology from mostly poorly differentiated pancreatic adenocarcinomas that developed in shp53/shRenilla mice are shown in G and H. Pdx1-Cre recombined cells can be identified by mKate2 and GFP expression. Bars, 50 µm.

Figure 6.

Knockdown of c-Myc impairs pancreatic cancer development. (A) Immunoblot demonstrating that the efficiency of target gene knockdown is equally potent upon introduction of single or tandem shRNAs into the CHC locus. (B) c-Myc depletion by shp53/shMyc-1 and shp53/shMyc-2 leads to a significant survival benefit and strongly impairs pancreatic cancer development (P = 0.0006; P < 0.0001, respectively; shp53/shRen, n = 11; shp53/shMyc-1, n = 5; shp53/shMyc-2, n = 10). Mice expressing shp53/shMyc-1 and shp53/shMyc-2 do not show a significant difference in survival (P = 0.1988). (C) Tumors developing in shp53/shMyc-1-expressing mice are positive for mKate2 and GFP and are of poorly differentiated histology (comparable with shp53/shRen tumors). Bars, 50 µm.