KRASG12D and TP53R167H Cooperate to Induce Pancreatic Ductal Adenocarcinoma in Sus scrofa Pigs

Although survival has improved in recent years, the prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite substantial differences in anatomy, physiology, genetics, and metabolism, the overwhelming majority of preclinical testing relies on transgenic mice. Hence, while mice have allowed for tremendous advances in cancer biology, they have been a poor predictor of drug performance/toxicity in the clinic. Given the greater similarity of sus scrofa pigs to humans, we engineered transgenic sus scrofa expressing a LSL-KRASG12D-TP53R167H cassette. By applying Adeno-Cre to pancreatic duct cells in vitro, cells self-immortalized and established tumors in immunocompromised mice. When Adeno-Cre was administered to the main pancreatic duct in vivo, pigs developed extensive PDAC at the injection site hallmarked by excessive proliferation and desmoplastic stroma. This serves as the first large animal model of pancreatic carcinogenesis, and may allow for insight into new avenues of translational research not before possible in rodents.


Intra-Pancreatic Injection of Adeno-Cre in LSL-KRAS G12D -TP53 R167H Pigs Established Predominantly Metastastic Metastatic Leiomyosarcoma
LSL-KRAS G12D -TP53 R167H pigs (N=3) were anesthetized, and 4x10 9 Adeno-Cre injected directly into the gland parenchyma ( Figure S3A). Approximately two weeks following the surgery, the pigs began showing signs of pancreatic insufficiency including vomiting though there were no signs of jaundice/bile duct obstruction. Animals were euthanized 16 days following Adeno-Cre infection.
Gross necropsy revealed several abnormalities (summarized in Table S1) in 3/3 pigs, though these were particularly severe in one animal. This included 10-15 ml of a serosanguinous fluid in the abdominal cavity and 20-30ml of similar fluid in the thoracic cavity (data not shown). We also observed large abnormal masses surrounding the pancreatic duct and tapering toward the duodenum, which were continuous with the duodenal tunica muscularis. These masses were firmly adherent to the serosal surface of the duodenum, as well as multi-focally adherent to the omentum. On cut surface, the tumor appeared homogenous, firm and displayed extensive fibrosis. These masses were expanding into the pancreatic lobules, which were pale with noticeable hemorrhage and friable pale regions attributed to saponified fat ( Figure S3A).
The squamous region of the stomach contained multiple, small erosions, and the omentum was thickened by numerous demarcated plaques ( Figure S3A). Similar multifocal to coalescing plaques were observed on the splenic and hepatic capsules, with lesions being more pronounced on the lateral right lobe of the liver. Additionally, the abdominal surface of the diaphragm was granular and red, with plaques restricted only on the capsule and not invading the parenchyma ( Figure S3B). Finally, blood was collected for complete blood count (CBC) and blood chemistry, both of which showed little to no abnormality (data not shown).
To exclude the possibility that these masses were spontaneous or an indirect effect of the virus, the primary tumor was subject to RT-PCR, confirming the presence of the TP53 R167H transcript ( Figure S3C,D). Primary tumors were next sectioned, and staining for RAS G12D and TP53 R167H affirmed their expression in malignant tissues and strongly localized with PCNA, a surrogate marker of cell proliferation ( Figure S3E). However, consistent with our initial assessment, these malignant tissues were morphologically distinct from known pancreatic neoplasms, instead showed mesenchymal characteristics consistent with leimyosarcoma ( Figure S3E). Furthermore, the peripancreatic lymph node was markedly enlarged in these animals (approximately 6-8 x the normal size) and lacked a distinct cortex/medulla, showing clear signs of metastasis consistent with the described sarcoma ( Figure S3E). The peri-gastric and hepatic hilar lymph nodes were also enlarged, though they displayed distinct cortices and medullas (data not shown). The diagnosis of leiomyosarcoma was confirmed via immunohistochemistry. The malignant tissue was positive for mutant KRAS G12D and had strong expression of the KRAS effector pERK as well as proliferation surrogate PCNA ( Figure S3F). This was not observed in the adjacent normal tissue, which had low expression of both pERK and PCNA ( Figure S3F). Similarly, the infiltrating sarcoma was negative for the epithelial marker E-Cadherin, and highly positive for mesenchymal markers α-Smooth Muscle Actin (αSMA) and Vimentin, and displayed little of the fibrosis classically associated with PDAC ( Figure S3F).

Adeno-Cre Injected LSL-KRAS G12D -TP53 R167H Pigs Present with PanIN Disease with Several Hallmark Features of Pancreatic Tumorigenesis
While leimyosarcoma was the predominant histotype observed and accounted for all masses observed upon necropsy, histological analysis of the pancreas revealed several large, luminal masses as well as pronounced abnormalities to the ductal tree. In each animal, there were several lesions resembling a combination of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasms (PanINs) ( Figure S4A). While the majority of structures were well differentiated, they exhibited heterochromatic nuclei and there were isolated regions of metaplastic/neoplastic growth. These lesions were accompanied by extensive leukocyte infiltration and fibrosis, as well as the occasional intra-pancreatic hemorrhage in areas with invading leimyosarcoma ( Figure S4A).
To affirm the PanIN lineage, tissues were stained for immunohistochemistry. Unlike the observed leiomyosarcoma, these lesions were highly positive for E-Cadherin indicating epithelial origin. Additionally, these lesions also exhibited strong PCNA staining suggesting increased cell proliferation and consistent with a PanIN phenotype. Additionally, these lesions were surrounded by a dense, desmoplastic stroma that stained positive for Masson's Trichrome as well as mesencyhmal markers Vimentin and αSMA, though the lesions themselves were negative for both Vimentin and αSMA ( Figure S4B). Tissues were next stained for the duct marker CK19 and acinar cell marker Pancreatic Amylase.
Consistent with previous observations, the invading sarcoma was negative for both makers. However, several of these lesions were dual positive for both CK19 and Pancreatic Amylase, confirming acinar-to-ductal metaplasia, an early event in pancreatic carcinogenesis ( Figure S4C). Similarly, CK19-positive lesions displayed strong PCNA staining indicative of cell proliferation, and were surrounded by a αSMA-rich tumor stroma ( Figure S4C). Figure S1. Comparative histology between human, mouse, and pig pancreas.

SUPPLEMENTARY FIGURE LEGENDS
Human, mouse, and pig pancreata were sectioned and H&E stained to assess structural similarities in (A) pancreatic acini, (B) pancreatic ductal system, and (C) neuroendocrine Islets of Langerhans.

Figure S9. Intraductal delivery of Ad-Cre increases ERK activation and cell proliferation
Tumors from Adeno-Cre injected LSL-KRAS G12D -TP53 R167H pigs were sectioned and stained for pERK or PCNA. Cells positive for either (A) pERK or (B) PCNA were quanitified per 40X field by two blinded investigators. Counts were averaged and displayed as mean ± SEM (*P < 0.05).

Table S1
Pancreas Leimyosarcoma with: severe necrotizing and lymphoplasmacytic pancreatitis with fibrosis, acinar degeneration, acinar-to-ductal metaplasia, severe necrotizing peripancreatic steatitis, isolated endocrine/exocrine cell necrosis, and fibrosis/inflammaton associated with the main pancreatic duct. Tumor is mostly centered and surrounds the common arm of pancreatic duct and pancreas.

Small Intestine
Neoplastic cells similar to those described previously are present in the duodenum.

Liver
Secondary tumors consistent with that described previously. Portal triads are infiltrated by small numbers of lymphocytes and plasma cells. Neoplastic cells similar to those described previously are present on the capsule and are invading the underlying subcapsular parenchyma.

Bile Duct
Neoplastic cells similar to those described previously are present in the periductal tissue.

Lymph Nodes
Peripancreatic lymph node 6-8x normal size and lacking a distinct cortex and medulla and showing clear signs of metastasis. Perigastric and hepatic hilar lymph nodes also enlarged with no evidence of metastases.

Spleen
Multifocal to coalescing plaques were observed on the splenic capsule consistent with metastases.

Omentum
Neoplastic cells similar to those described previously are present.

Colon
No Significant Lesion (NSL), moderate lymphocytes/plasma cells and rare eosinophils present within the lamina propria.

Lung
NSL, rare peribronchial aggregates of lymphocytes and plasma cells.

Kidneys
Cortex and medulla are infiltrated by small aggregates of lymphocytes and plasma cells. Thickened glomerular mesangium.

Diaphragm
Granular and red with focal lesions consistent with the previously descried neoplasms.

Bladder NSL
Stomach NSL, multiple, small erosions. Figure S1. Comparative histology between human, mouse, and pig pancreas Human, mouse, and pig pancreata were sectioned and H&E stained to assess structural similarities in

Pancreas
Leimyosarcoma with: severe necrotizing and lymphoplasmacytic pancreatitis with fibrosis, acinar degeneration, acinar-to-ductal metaplasia, severe necrotizing peripancreatic steatitis, isolated endocrine/exocrine cell necrosis, and fibrosis/inflammaton associated with the main pancreatic duct. Tumor is mostly centered and surrounds the common arm of pancreatic duct and pancreas.

Small Intestine
Neoplastic cells similar to those described previously are present in the duodenum.

Liver
Secondary tumors consistent with that described previously. Portal triads are infiltrated by small numbers of lymphocytes and plasma cells. Neoplastic cells similar to those described previously are present on the capsule and are invading the underlying subcapsular parenchyma.

Bile Duct
Neoplastic cells similar to those described previously are present in the periductal tissue.

Lymph Nodes
Peripancreatic lymph node 6-8x normal size and lacking a distinct cortex and medulla and showing clear signs of metastasis. Perigastric and hepatic hilar lymph nodes also enlarged with no evidence of metastases.

Spleen
Multifocal to coalescing plaques were observed on the splenic capsule consistent with metastases.

Omentum
Neoplastic cells similar to those described previously are present.

Colon
No Significant Lesion (NSL), moderate lymphocytes/plasma cells and rare eosinophils present within the lamina propria.

Lung
NSL, rare peribronchial aggregates of lymphocytes and plasma cells.

Kidneys
Cortex and medulla are infiltrated by small aggregates of lymphocytes and plasma cells. Thickened glomerular mesangium.

Diaphragm
Granular and red with focal lesions consistent with the previously descried neoplasms.