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

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.

. Responses to the NIH Preclinical Research Guidelines. 16 Table S2. Primers and other short sequences. 19 Table S3. Antibody information. 20 Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.

Introduction 5 Methods: Ethical Statement
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Methods 6 Methods: Study Design
For each experiment, give brief details of the study design including: (a) The number of experimental and control groups.
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Methods: Experimental Procedures
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Methods 8
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Discussion: Funding
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Acknowledgements NA = not applicable. 1. Isolate ducts from mass tissue removing as much of the acinar and islet cell tissue as possible in a sterile hood. 2. Mince up the tissue with a razor blade into 1mm 3 pieces and add 10 mg/mL of collagenase I plus 100 ug/mL DNase I. 3. Incubate cells at 37°C for 30 min and do mechanical dissociation with a 10 mL pipette every 10 min. 4. Centrifuge at 4°C (500 g x 5 min) to pellet cells and decant supernatant. 5. Resuspend in 10 mL of red blood cell lysis buffer ( and incubate for 10 min at room temp, then add 10 mL of media (DMEM + 5%FBS) and spin cells down again. 6. Take the cell pellet and resuspend with media and pass through a 70 µm nylon sieve (Corning™ Sterile Cell Strainers, Thermo Fisher Scientific, cat. no. 07-201-431). 7. Centrifuge cells again at 500 g x 5 min and decant supernatant. 8. Resuspend cells in 3 mL whole media and plate 1 mL in each well of a porcine gelatin coated 6 well plate. 9. After 4 hours take the media off the well and put in new well in 6 well plate to help remove fibroblast contamination. 10. Incubate cells overnight and the next day wash the wells with PBS and add fresh media (half DMEM, half ECM). 11. After 4 days epithelial cobblestone growth will start to appear and the epithelial cells will start to expand. 12. The well with the least amount of contaminating cells is used for further studies. 13. If fibroblasts are present the wells are trypsinized for 2-3 min and then washed with PBS x 2 to remove the fibroblasts. Remaining epithelial cells will expand from this and can then be plated in a 10 cm dish for expansion. 14. When a 10 cm dish is confluent the epithelial cells will be plated back into a 2-6 well plates for immunocytochemistry and cellular transformation.

A B
A B *** SV40T band exposed with IR Dye 800 CW. First 4 bands are samples used for the paper.

A B
Pan-Keratin exposed with IR Dye 800CW. First 4 samples are samples used for the paper. This blot was stripped and re-probed for pan-keratin.

A B
Mutant Kras G12D exposed with IR Dye 680RD. First 4 samples are samples used for the paper. Actin is also showing up in this exposure.  (B) E-cadherin exposed with IR Dye 680RD. (C) p53 exposed with IR Dye 800CW. (D) b-actin exposed with exposed with IR Dye 680RD. (E) p21 and b-actin exposed with IR Dye 680RD (p21 was ran on a different gel but the same samples were used).     A wild type Minnesota minipig (female, 79 kg) underwent a ventral vertical midline incision under general anesthesia. The posterior surface of the duodenal lobe of the pancreas was exposed by medial rotation of the C-loop of the duodenum (A). A titanium marking clip was place on the duodenal lobe of the pancreas at its margin with the duodenum (white arrow in A). Transformed PDECs (SV40T LVGK CL2) were trypsinized and suspended in porcine collagen (1.6 mg/mL; physiologic pH) at a final concentration of 4 x 10 7 cell/mL. Four aliquots of cells (250 µL/aliquot, or 10M cell/aliquot) were injected into the duodenal lobe of the pancreas (p) with an 18-gauge needle (yellow arrowhead in A), all clustered within a centimeter of the marking clip (total of 40M cells injected). There was no back leakage of injectate. The surgical incision was closed and the animal recovered uneventfully. The subject was receiving cyclosporine A (10 mg/kg orally twice per day), begun one week prior to implantation, and continued until necropsy.
The subject was observed for 8 weeks, and exhibited no abnormal signs or behavior. At necropsy performed at 8 weeks post-implantation (B), there was no gross abnormality (neither visible or palpable) at the site of injection (p = pancreas; d = duodenum; arrow = marking clip). The pancreatic segment of interest (containing the clip; arrow in C) was resected, and immediately underwent thin slicing in the fresh state for formalin fixation and histology.

Histology
(D) H&E. On a few slices, there were microscopically-evident abnormalities. In the section shown there is a circumscribed abnormality (yellow arrows) ~800 µm in diameter. It is surrounded by cords of immune cells which by morphology appear to be activated T-lymphocytes (t). The circumscribed abnormality appears to be a collection of metaplastic ductal cells, whose nuclear morphology does not have the neoplastic characteristics of the tumor xenografts shown in Fig. 3. Surrounding patches of normal-looking pancreatic acini (a) also are shown.

Magnification of the region of interest (dashed rectangle) is shown in panel H. (E)
Immunohistochemical negative control. All reagents except primary antibody. (F) SV40T immunohistochemistry. There is minimal signal present (not nearly to the extent seen with the murine xenografts in Fig. 4). (G) KRAS G12D immunohistochemistry. The circumscribed abnormality is positive for mutant KRAS.

Replicates
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Biological Variables
Sex, age, weight, and underlying health conditions, are often critical factors affecting health or disease. In particular, sex is a biological variable that is frequently ignored in animal study designs and analyses, leading to an incomplete understanding of potential sexbased differences in basic biological function, disease processes and treatment response.
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Key biological and/or chemical resources include, but are not limited to, cell lines, specialty chemicals, antibodies and other biologics.
Briefly describe methods to ensure the identity and validity of key biological and/or chemical resources used in the proposed studies. These resources may or may not be generated with NIH funds and: • May differ from laboratory to laboratory or over time; • May have qualities and/or qualifications that could influence the research data; • Are integral to the proposed research.