<p>PDF file - 248K, Supplementary Figure 1. (A) Simplified schematic of mucin type O-glycans analyzed in this report. Mucin-type O-linked glycans are initiated by the addition of an N-acetyl galactose sugar residue (the Tn epitope), which can be extended into the T antigen or core 3 structures, or Tn can be sialylated creating terminal STn that cannot be further extended. T antigen can be extended into core 2 structures that lead to the Lewis Blood Group antigens - LeX, SLeX, SLeC and sialyl Lewis A (the CA19-9 antigen, a widely utilized biomarker of adenocarcinoma progression). B & C - Representative immunohistochemical results for comparison of expression of mucin core proteins, glycans, and glycopeptides taken at 200x magnification. (B) Serial sections of primary tumor from autopsy patient 3 stained for indicated antigens. (C) Serial sections of liver metastasis from autopsy patient 20 for same antigens seen in primary tumor.</p>
<p>PDF file - 320K, Supplementary Figure 2. Representative immunohistochemical results for comparison of cancer fieldeffects in autopsy and resection tissue samples from the same patients. Two different autopsy patients who underwent surgical resection are presented. Serial sections stained for the antigens indicated are shown in the resection tissue samples alongside the autopsy samples of their primary tumors. 200x magnification.</p>
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.
Abstract There were an estimated 43,140 new cases and 36,800 deaths due to pancreatic adenocarcinoma (PA) for 2010, ranking PA as the third leading cause of cancer related death. Virtually all long term survivors are diagnosed early but these survivors only account for 7% of those diagnosed highlighting the need for an early diagnostic test. CA19-9, the only current clinical serum-based assay used to monitor PA, detects an oligosaccharide blood group antigen sialyl Lewisa (SLeA; Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAc) that can be present as an O-linked oligosaccharide on glycoproteins and is expressed at relatively low levels on normal epithelial cells. Unfortunately, approximately 15% of the population cannot synthesize SLeA due to a lack of expression of the necessary enzyme to add the final fucose residue thus lowering the sensitivity of the assay. We investigated the expression of other O-linked glycans expessed in PA – sialyl Tn (STn; NeuAcα2-6GalNAc), Tn (GalNAc), T (Galβ1-3GalNAc) and sialyl LewisC (SLeC; Neu5Acα2-3Galβ1-3GlcNAc) – and the aberrant expression of mucin core proteins. In an effort to establish an antigenic signature for PA, we have performed immunohistochemical analysis on primary tumor and liver metastatic tumor samples from 28 autopsy patients diagnosed with PA to analyze their mucin and glycosylation expression. STn and Tn were expressed in the majority of patients in both the primary and liver metastatic tumors and SLeC expression mirrored that of SLeA albeit at lower levels. In those patients lacking SLeA expression SLeC could be detected indicating that the CA19-9 test could be enhanced when combined with these glycans. MUC1, MUC4 and MUC6 were detected in ≫70% of the primary tumor samples analyzed. We also investigated the potential of mucin glycopeptides (STn/Tn on MUC1, STn/Tn on MUC4, and ST/T on MUC1) in providing a unique signature for PA. We found that both STn/Tn on MUC1 and STn/Tn on MUC4 were expressed in >90% of the primary tumor samples analyzed while ST/T on MUC1 was predominantly expressed in normal controls. Differences were also observed in the mucin and glycan signatures between primary and liver metastatic tumors. For example, MUC2 and MUC5B were expressed in the liver metastases while MUC17 was restricted to primary tumor. Finally, we analyzed the expression of MUC1, MUC4, STn/Tn on MUC1, STn/Tn on MUC4, STn and Tn in 4 pancreatitis samples. These antigens were observed in 75-100% of these cases indicating that these particular antigens may by induced by the early inflammatory process leading towards a pro-tumorigenic microenvironment. We conclude that glycopeptide structures comprise an expression pattern unique to PA with potential as biomarkers of early disease and warrant further investigation. Supported by grants U01CA111294 and U01CA128437 from the NCI Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 895. doi:10.1158/1538-7445.AM2011-895
<p>PDF file - 248K, Supplementary Figure 1. (A) Simplified schematic of mucin type O-glycans analyzed in this report. Mucin-type O-linked glycans are initiated by the addition of an N-acetyl galactose sugar residue (the Tn epitope), which can be extended into the T antigen or core 3 structures, or Tn can be sialylated creating terminal STn that cannot be further extended. T antigen can be extended into core 2 structures that lead to the Lewis Blood Group antigens - LeX, SLeX, SLeC and sialyl Lewis A (the CA19-9 antigen, a widely utilized biomarker of adenocarcinoma progression). B & C - Representative immunohistochemical results for comparison of expression of mucin core proteins, glycans, and glycopeptides taken at 200x magnification. (B) Serial sections of primary tumor from autopsy patient 3 stained for indicated antigens. (C) Serial sections of liver metastasis from autopsy patient 20 for same antigens seen in primary tumor.</p>
Abstract We describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of breast cancer, using immunocompetent pigs. Primary mammary epithelial cells were isolated from the porcine gland. Primary mammary cells were immortalized with hTERT, and then transformed cell lines were generated from these immortalized cells with oncogenic KRAS and dominant negative p53. The transformed cell lines outperformed the primary cells in terms proliferation, population doubling time, soft agar growth, 2D migration, and Matrigel invasion. Three transformed cell lines were selected based on in vitro performance, and were able to grow tumors when injected subcutaneously in nude mice, with undifferentiated morphology. Tumorigenic porcine mammary cell lines were generated in this report.
Abstract Human mucin 7 (MUC7) is the smallest of the mucin core proteins at 39 kD and its fully glycosylated form ranges from 125-250 kD. This secreted, non-gel forming mucin is normally expressed by the mucousal and serosal salivary glands and sub-mucosal glands in the lower respiratory tract. Recently MUC7 has been observed to be expressed in such diseases as allergic-asthma, COPD, cystic fibrosis and bladder cancer. The tandem repeat domain consists of 5-6 repeats of a 23 amino acid sequence, which contains 3 SEA domains which may serve as sites of auto-cleavage. Our immunohistochemical studies of pancreatic adenocarcionoma revealed that MUC7 was expressed in over 50% of primary tumors, liver metastases, and lymph node metastases. MUC7 was found to be primarily expressed by the immune cells in the lymph node metastases rather than tumor cells. In contrast, MUC7 was not expressed by infiltrating immune cells in primary tumors and liver metastases, but it was present in the tumors themselves. Further characterization of the immune cells expressing MUC7 in the lymph nodes (by means of immunofluorescence and flow cytometry) revealed that the majority were macrophages and myeloid-derived suppressor cells together with a small population of dendritic cells. MUC7 has been posited to play a role in inflammation. This function is likely due to a unique histatin-like domain allowing it to bind numerous microbes to promote their clearance.MUC7 has been shown to be upregulated by IL-1β, IL-4, IL-13, TNF-α, LPS, and EGF in inflammed airways, and its minimal proximal promoter contains binding sites for NF-κB, AP1 and STAT transcription factors; all of which are involved in promoting inflammation. We found that MUC7 expression was up-regulated in the pancreatic cancer cell line Hs766T and the breast cancer cell line BT474 upon incubation with Th1 cytokines IFN-γ, IL-1β and TNF-α and the Th2 cytokines IL-4, IL-10 and IL-13. MUC7 over-expression was achieved in a time-dependent manner when incubated with the Th1 cytokines; however immediate and sustained expression was observed when cells were treated with the Th2 cytokines. Global gene expression by microarray was evaluated on samples of primary and metastatic pancreatic adenocarcinoma that expressed or did not express MUC7 to identify candidate genes affected by MUC7 expression. The microarray data revealed that a number of genes involved in tissue remodeling and inflammation were differentially regulated by MUC7 expressing tumors. Together, the data from the analyses presented here led us to propose that MUC7 has a role in establishing a pro-tumorigenic microenvironment through enhancing the Th2 inflammatory response and aiding in tissue remodeling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 296. doi:1538-7445.AM2012-296
Abstract Background . A large animal model of pancreatic cancer would permit development of diagnostic and interventional technologies not possible in murine models, and also would provide a more biologically-relevant platform for penultimate testing of novel therapies, prior to human testing. Here, we describe our initial studies in the development of an autochthonous, genetically-defined, large animal model of pancreatic cancer, using immunocompetent pigs. Methods . Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs; epithelial origin was confirmed with immunohistochemistry. Three transformed cell lines subsequently were generated from these primary cells using expression of oncogenic KRAS and dominant negative p53, with/without knockdown of p16 and SMAD4. We tested these cell lines using in vitro and in vivo assays of transformation and tumorigenesis. Results . The transformed cell lines outperformed the primary cells in terms proliferation, population doubling time, soft agar growth, 2D migration, and Matrigel invasion, with the greatest differences observed when all four genes (KRAS, p53, p16, and SMAD4) were targeted. All three transformed cell lines grew tumors when injected subcutaneously in nude mice, demonstrating undifferentiated morphology, mild desmoplasia, and staining for both epithelial and mesenchymal markers. Injection into the pancreas of nude mice resulted in distant metastases, particularly when all four genes were targeted. Conclusions . Tumorigenic porcine pancreatic cell lines were generated. Inclusion of four genetic “hits” (KRAS, p53, p16, and SMAD4) appeared to produce the best results in our in vitro and in vivo assays. The next step will be to perform autologous or syngeneic implantation of these cell lines into the pancreas of immunocompetent pigs. We believe that the resultant large animal model of pancreatic cancer could supplement existing murine models, thus improving preclinical research on diagnostic, interventional, and therapeutic technologies.
