Porcine pancreatic ductal epithelial cells transformed with KRASG12D and SV40T are tumorigenic

Abstract

We describe our initial studies in the development of an orthotopic, genetically defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.

Figure 1

Isolation and transduction of primary porcine pancreatic ductal epithelial cells. (A) Immunofluorescent staining for Pan-Keratin and cytokeratin 19 (CK-19) in cultured primary epithelial cells. (B) Immunoblot for SV40T, mutant KRAS^G12D, and Pan-Keratin in primary epithelial cells, SV40T immortalized cells, and SV40T immortalized cells transformed with KRAS^G12D (SV40T LV-GK; two lines, CL1 and CL2) Samples are from the same gel/blot with different IR Dye exposures depending on the secondary for the primary antibody. For full blot images see Fig. S3. Black arrow = band of interest; MWM = molecular weight marker. (C) Expression of ZsGreen1 (a green fluorescent protein variant) in primary epithelial cells and SV40T immortalized cells transduced with the LV-GK; phase and inverted fluorescent microscopy of living cells. Measure bars = 400 µm.

Figure 2

In vitro cell transformation assays. Cultured primary epithelial cells from porcine pancreatic duct were compared with SV40T immortalized and transformed (SV40T + KRAS^G12D) cell lines. (A) Cell culture population doubling time (count-based analysis). (B) Proliferation rate using metabolic dye assay, represented as fold change with respect to primary cells (defined as 1). (C) Phase contrast images of soft agar assay (bar = 1000 µm). (D) Plot of soft agar assay. (E–F) Transwell migration assay (absence of BME), phase images and plot. (G–H) Transwell invasion assay (presence of BME), phase images and plot. Each bar or data point in this Figure represents the mean ± SD of triplicate wells; each experiment performed three times on separate days (one representative experiment shown in each panel); ****p < 0.0001; ns = not significant (p ≥ 0.05).

Figure 3

In vivo tumorigenesis assay. (A) Representative xenograft tumor explanted from a nude mouse 42 days after subcutaneous injection of transformed epithelial cells (SV40T LV-GK CL2). (B) Comparison of xenograft tumor volume between primary epithelial cells versus the SV40T immortalized and SV40T LV-GK CL2 cell lines; ****p < 0.0001; ns = not significant (p ≥ 0.05). (C) H&E microscopic image of xenograft tumor. (D) Higher power H&E image; region of interest box shown in panel C. Black arrows = hyperchromatic nuclei; yellow arrows = nuclei with large nucleoli; red arrows = pyknotic nuclei. (E) Higher power H&E image; region of interest box shown in panel D. *Asterisks indicate lumen of gland-like structures lined with ductal-like cells; lumen were filled with mucin in the Alcian blue staining (Fig. S6).

Figure 4

Immunohistochemistry of tumor xenografts from subcutaneous nude mouse assay. (A) Pan-Keratin; section from human PDAC (original tumor, not a xenograft); utilized as a positive control (nuclear counterstain = hematoxylin). Panels B-G are all from xenograft tumor, obtained with implantation of the SV40T LV-GK CL2 cell line. (B) Negative control (all reagents except no primary antibody). Black arrows = ductal-like structures; dashed yellow circles = acinar-like structures. (C) Pan-Keratin. (D) KRAS^G12D (antibody specific to mutant KRAS). (E) SV40T. (F) Ki-67. (G) p21.