Generation of tumorigenic porcine pancreatic ductal epithelial cells: toward a large animal model of pancreatic cancer

Abstract

23Background. A large animal model of pancreatic cancer would permit development of 24

Porcine Models of Pancreatic Cancer

“…In contrast to the autochthonous mechanism of tumor induction that the OCM provides, an orthotopic method of tumor induction involves seeding of tumorigenic cells into the pancreas, preferably into an immunocompetent host. In pursuit of this model type, primary cultures of porcine pancreatic ductal epithelial cells (PDECs) were established from explants of normal pancreatic tissue; IHC for cytokeratin-19 in early-passage strains were consistent with epithelial origin of the cultured cells ( 77 ). Strains of PDECs subsequently were infected with a lentiviral vector containing GFP, TP53 R167H , and KRAS G12D (LV-GKP; generated using porcine sequences), producing clones with demonstrable expression of mutant p53 and KRAS; refer to Table 2 ( 77 ).…”

“…In pursuit of this model type, primary cultures of porcine pancreatic ductal epithelial cells (PDECs) were established from explants of normal pancreatic tissue; IHC for cytokeratin-19 in early-passage strains were consistent with epithelial origin of the cultured cells ( 77 ). Strains of PDECs subsequently were infected with a lentiviral vector containing GFP, TP53 R167H , and KRAS G12D (LV-GKP; generated using porcine sequences), producing clones with demonstrable expression of mutant p53 and KRAS; refer to Table 2 ( 77 ). Initial in vitro tumorigenic assays of these clones (denoted as PGKP, for PDECs transformed with LV-GKP) demonstrated increases in migration and soft agar colony formation relative to primary PDECs ( 77 ).…”

“…Strains of PDECs subsequently were infected with a lentiviral vector containing GFP, TP53 R167H , and KRAS G12D (LV-GKP; generated using porcine sequences), producing clones with demonstrable expression of mutant p53 and KRAS; refer to Table 2 ( 77 ). Initial in vitro tumorigenic assays of these clones (denoted as PGKP, for PDECs transformed with LV-GKP) demonstrated increases in migration and soft agar colony formation relative to primary PDECs ( 77 ). To further increase the transformed phenotype of the PGKP cells, RNAi of SMAD4 and CDKN2A were added using additional LV vectors, with ~70–90% knockdown ( 77 ).…”

“…Initial in vitro tumorigenic assays of these clones (denoted as PGKP, for PDECs transformed with LV-GKP) demonstrated increases in migration and soft agar colony formation relative to primary PDECs ( 77 ). To further increase the transformed phenotype of the PGKP cells, RNAi of SMAD4 and CDKN2A were added using additional LV vectors, with ~70–90% knockdown ( 77 ). Relative to primary cells, these secondary clones (PKGPS and PGKPSC) also displayed increased proliferation, soft agar colony formation, invasion, and migration, i.e., evidence of in vitro “tumorigenicity” ( 77 ), with perhaps enhanced capabilities compared to the primary clone (PGKP cells).…”

“…To further increase the transformed phenotype of the PGKP cells, RNAi of SMAD4 and CDKN2A were added using additional LV vectors, with ~70–90% knockdown ( 77 ). Relative to primary cells, these secondary clones (PKGPS and PGKPSC) also displayed increased proliferation, soft agar colony formation, invasion, and migration, i.e., evidence of in vitro “tumorigenicity” ( 77 ), with perhaps enhanced capabilities compared to the primary clone (PGKP cells). The three types of transformed PDECs (summarized in Table 2 ) were then implanted subcutaneously in nude mice; all three cell lines formed tumors and demonstrated equivalent in vivo tumorigenicity ( 77 ).…”