Normal Pancreata Research Articles

Abstract A057: Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) originates from precursor lesions, the most common of which are known as pancreatic intraepithelial neoplasia (PanIN). PanIN formation and progression is accompanied by accumulation of a precursor microenvironment (PME) composed of desmoplastic stroma that plays an important role in promoting tumor growth and immune evasion. The crosstalk between tumor cells and stromal cells is an area of active investigation. While PanIN and the PME have been extensively studied in mouse models, their nature in humans have remained elusive as there is no clinical indication to sample healthy pancreas. Through a unique collaboration with the Gift of Life Organ and Tissue donation program, we performed transcriptomic analysis on normal pancreata declined for transplant. We noted that epithelial cells in the PanIN and cancer microenvironment were transcriptomically similar; however, their surrounding stromal fibroblasts were markedly distinct. Given that most precursor lesions remain quiescent while some progress to tumor, we sought out to elucidate how the transcriptional programs in epithelial cells influence fibroblast cell fate. To do so, we developed a 3D ex-vivo organoid-fibroblast coculture system using primary organoid and fibroblast cultures successfully derived from Gift of Life pancreata or PDAC patients undergoing surgical resection. Our coculture system retains histological features of heterogeneity observed in vivo. Using single cell RNA-sequencing analysis of matched fibroblast-organoid cocultures we identified factors that were highly expressed in fibroblasts in tumor cocultures, but not in normal cocultures. We are currently interrogating candidate ligand/receptor interactions in our cocultures to determine the mediators of this epithelial-mediated fibroblast polarization. This model will allow us to gain an understanding about how components of the microenvironment restrain or promote malignant transformation and serve as a great tool to study therapies targeting tumor and stromal cells in a patient specific manner. Citation Format: Padma Kadiyala, Ahmed Elhossiny, Jianhua Liu, Alexander Bray, Katelyn Donahue, Arvind Rao, Jiaqi Shi, Yaqing Zhang, Filip Bednar, Timothy Frankel, Eileen Carpenter, Marina Pasca Di Magliano. Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A057.

Abstract A081: Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes

Abstract Pancreatic intraepithelial neoplasia (PanIN) lesions are the microscopic pre-malignant precursor to pancreatic ductal adenocarcinoma (PDAC). Animal models have demonstrated that PanINs arise from acinar cells which have undergone acinar-to-ductal metaplasia (ADM), but the exact molecular mechanisms which govern this transformation remain poorly understood. Novel in vitro models of PanIN genesis are necessary to better characterize pancreatic carcinogenesis and identify clinically actionable biomarkers of early disease. Organoids cultured from normal human pancreata have generated promise in this regard, but the cellular identity and malignant potential of their component cells have not been comprehensively characterized. We performed single-cell RNA sequencing (10X Genomics) on 6 normal pancreatic organoid lines maintained in culture for 3 weeks as well as their parent pancreata obtained from donors with no history of PDAC or other exocrine pancreatic pathology. Integration of whole pancreatic and organoid data using the R package Seurat generated a UMAP with clearly defined “ductal” and “acinar” clusters based on marker gene expression. The majority of organoid cells (47.3-81.5%) colocalized to the “ductal” cluster, and expressed SOX9, KRT19, and PDX1. However, a subset of cells (1.0-33.3%) present in each organoid sample aligned with the “acinar” cluster and expressed PRSS1, PRSS3, REG3A, MUC1, and SPINK1. In accordance with prior reports, organoid cells did not meaningfully express the acinar/endocrine markers PTF1A, CPA1, CHGA, or INS. Further examination of the “acinar” organoid cells revealed heightened expression of CLDN18, TFF1, TFF2, CTSE, CA2, and LYZ, genes associated with PanIN in prior spatial RNA sequencing studies. Other genes significantly enriched in this “acinar” organoid cluster include MUC5AC, a known PanIN marker, OLFM4, a tumor suppressor implicated in intestinal carcinogenesis, and the chemokine CXCL17, which is expressed in cells undergoing ADM. Overall, this data suggests that organoids derived from normal pancreata represent a heterogeneous population of epithelial cells with both acinar and ductal features. Furthermore, these organoids may represent an in vitro model of ADM and/or early PanIN formation, though additional functional mechanistic studies are required for confirmation. Citation Format: Alexander W Bray, Eileen S Carpenter, Timothy L Frankel, Marina Pasca di Magliano. Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A081.

Magnetic Resonance Imaging-Based Assessment of Pancreatic Fat Strongly Correlates With Histology-Based Assessment of Pancreas Composition.

The aim of the study is to assess the relationship between magnetic resonance imaging (MRI)-based estimation of pancreatic fat and histology-based measurement of pancreatic composition. In this retrospective study, MRI was used to noninvasively estimate pancreatic fat content in preoperative images from high-risk individuals and disease controls having normal pancreata. A deep learning algorithm was used to label 11 tissue components at micron resolution in subsequent pancreatectomy histology. A linear model was used to determine correlation between histologic tissue composition and MRI fat estimation. Twenty-seven patients (mean age 64.0 ± 12.0 years [standard deviation], 15 women) were evaluated. The fat content measured by MRI ranged from 0% to 36.9%. Intrapancreatic histologic tissue fat content ranged from 0.8% to 38.3%. MRI pancreatic fat estimation positively correlated with microanatomical composition of fat (r = 0.90, 0.83 to 0.95], P < 0.001); as well as with pancreatic cancer precursor ( r = 0.65, P < 0.001); and collagen ( r = 0.46, P < 0.001) content, and negatively correlated with pancreatic acinar ( r = -0.85, P < 0.001) content. Pancreatic fat content, measurable by MRI, correlates to acinar content, stromal content (fibrosis), and presence of neoplastic precursors of cancer.

OR16-03 Epigenetic Studies Of Pancreatic Neuroendocrine Tumours (PNETs) Reveal Decreased DNA Hydroxymethylation In Non-functioning PNETs But Not Insulinomas

Abstract Disclosure: K.A. English: None. A. Selberherr: None. O.A. Shariq: None. E. O'Neill: None. K.E. Lines: None. R.V. Thakker: None. Background: DNA methylation and hydroxymethylation (5hmC) are epigenetic alterations that can silence or maintain gene transcription, respectively. Pancreatic neuroendocrine tumours (PNETs), which can be hormone-secreting (e.g. insulinomas) or non-functioning (NF-PNETs), have a low mutational burden and are reported to be associated with DNA hypermethylation, but the role of 5hmC in PNETs is unknown. Aim: To determine the patterns of DNA 5hmC in NF-PNETs, insulinomas, and normal pancreatic islets. Methods: NF-PNETs (n=31, mean age 54 ± 13 years (y), 14 males (M), tumour grade (G) G1=15, G2=13, G3=3) and insulinomas (n=21, mean age 50 ± 21y, 9M, G1=17 and G2=4) were collected from patients who did not have a genetic syndrome associated with PNETs together with 14 normal pancreata (mean age 63 ± 12y, 11M). Samples were obtained following ethical approval from the Universities of Oxford and Vienna. DNA 5hmC was assessed by immunohistochemistry (IHC) and MethylationEPIC array (that interrogates 5hmC CpG sites). Multivariate analysis was used to determine variables associated with 5hmC. Transcriptome analysis was undertaken by RNA-Seq. Results: NF-PNETs had reduced 5hmC when compared to insulinomas (p&amp;lt;0.01) and to normal adjacent islets (p&amp;lt;0.001). Thus, 5hmC was ∼3 fold lower in NF-PNETs compared to normal adjacent islets (n=12; p&amp;lt;0.001), whilst 5hmC between insulinomas and normal adjacent islets (n=15) was not significantly different. Multivariate analysis confirmed this finding, with NF-PNETs being associated with lower 5hmC when controlled for patient age, sex and tumour grade (p&amp;lt;0.01). In normal pancreatic tissue 5hmC was ∼2 fold higher in normal islets when compared to exocrine tissue (51 islets assessed in total, range 1-9 islets/sample from 34 fields of 305,866um2 studied; p&amp;lt;0.001). MethylationEPIC array analysis, using DNA from NF-PNETs (n=5), insulinomas (n=4) and normal pancreatic tissue (n=7), revealed NF-PNETs and insulinomas to have a significant decrease in 5hmC compared to normal pancreatic tissue (p&amp;lt;0.001). Furthermore, subtype analysis revealed that NF-PNETs had lower 5hmC compared to insulinomas (0% vs 0.003%, adjusted p&amp;lt;0.08). To determine possible mechanisms underlying these differences in tumour subtype 5hmC, RNA-Seq analysis using NF-PNETs (n=7) and insulinomas (n=6) was performed. This revealed 79 genes to be differentially expressed (adjusted p&amp;lt;0.01), of which 33 were upregulated (including Calcium/Calmodulin Dependent Protein Kinase 2 Gamma (CAMK2G)); and 46 were down-regulated (including Glucagon Like peptide 1 receptor (GLP1R)) in NF-PNETs. Interestingly, CAMK2G and GLP1R are both involved in DNA 5hmC modulation via cAMP. Conclusions: DNA 5hmC, an epigenetic mechanism, is significantly lower in NF-PNETs when compared to normal islets and insulinomas and may involve dysregulation of cAMP-mediated signalling pathways. Presentation: Friday, June 16, 2023

Pancreatic Tissue Dissection to Isolate Viable Single Cells.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Abstract C062: Investigating the role of pancreatic intraepithelial neoplasia in development of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a dismal disease with a 5-year survival rate of 11%. This poor prognosis is due in part to a lack of early disease detection and resistance to current therapies. The most common PDA precursor is pancreatic intraepithelial neoplasia (PanINs); a microscopic lesion that cannot be detected by imaging. Accumulation of a series of genomic mutations in pancreatic exocrine cells leads to formation of PanINs through mechanisms that remain unknown. While PanIN development has been well-studied in genetically engineered mouse models, its onset and pathogenesis in humans has remained elusive due to the inability to study tissue from pathologically normal pancreases. Due to its abundance in digestive enzymes, the pancreas degrades shortly after death and autopsy samples are not suitable for several research applications that require fresh tissue. Through collaboration with the Gift of Life Organ and Tissue donation program, we performed histological and transcriptomic analysis on ten normal pancreata that were refused for transplant. Histological analysis revealed the presence of PanIN lesions in 6 of the 10 donors as early as 25 years of age. To delineate the molecular, cellular, and spatial composition of PanIN lesions to those from acinar and ductal cells we combined single cell RNA sequencing analysis with spatial transcriptomics for the same sample. Annotation of cellular composition of distinct tissue regions demonstrated that PanIN transcriptome closely resembled that of invasive PDA. Using differential expression analysis, we identified factors that were highly expressed in PanINs, but not expressed in normal acini or ducts. We validated the transcript-based findings with immunostaining and RNA in-situ hybridization. We then sought to characterize the microenvironment of the normal pancreas and of PanIN lesions using multiplex immunohistochemical analysis. We observed abundant fibroblasts and macrophages in normal areas of the pancreas as well as surrounding PanIN lesions, while T cells were limited to the latter. Currently, we are characterizing the transcriptome profile of macrophages and fibroblasts. Uncovering the factors that promote the development of sporadic PanINs and their progression to malignancy could aid in designing preventative strategies for high-risk individuals. Citation Format: Padma Kadiyala, Eileen Carpenter, Ahmed Elhossiny, Yaqing Zhang, Sarah Nelson, Fatima Lima, Katelyn Donahue, Wenting Du, Jake McGue, Hannah Watkoske, Holly Attebury, Donnele Daley, Filip Bednar, Arvind Rao, Timothy Frankel, Marina Pasca Di Magliano. Investigating the role of pancreatic intraepithelial neoplasia in development of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C062.

Alterations in the Duodenal Fluid Microbiome of Patients With Pancreatic Cancer

Background & AimsThe tumor microbiome of patients with pancreas ductal adenocarcinoma (PDAC) includes bacteria normally present in the upper gastrointestinal tract. If the predominant source of intratumoral bacteria in patients with PDAC is retrograde migration from the duodenum, duodenal fluid could be a representative biospecimen for determining microbiome profiles of patients with PDAC or at risk of developing PDAC. MethodsWe performed a case-control study comparing bacterial and fungal (16S and 18S rRNA) profiles of secretin-stimulated duodenal fluid collections from 308 patients undergoing duodenal endoscopy including 134 normal pancreas control subjects, 98 patients with pancreatic cyst(s) and 74 patients with PDAC. ResultsAlterations in duodenal fluid microbiomes with diminished alpha diversity were significantly associated with age >70 and proton pump inhibitor use. Patients with PDAC had significantly decreased duodenal microbial alpha diversity compared with age-matched control subjects with normal pancreata and those with pancreatic cyst(s). There was evidence of enrichment of Bifidobacterium genera in the duodenal fluid of patients with PDAC compared with control subjects and those with pancreatic cyst(s). There were also enrichment of duodenal fluid Fusobacteria and Rothia bacteria among patients with PDAC with short-term survival. Duodenal fluid microbiome profiles were not significantly different between control subjects and patients with pancreatic cyst(s). ConclusionPatients with PDAC have alterations in their duodenal fluid microbiome profiles compared with patients with pancreatic cysts and those with normal pancreata. ClinicalTrials.gov, Number: NCT02000089

Abstract A07: Inflammation enables pancreatic acinar cells to overcome resistance to oncogenic Kras by increasing its expression and plasma membrane localization

Abstract Kras mutations are not sufficient to induce precancerous lesions in the pancreas, unlike in other organs such as lungs. Pancreatic intraepithelial neoplasia (PanIN) appear when mutated Kras is associated with pancreatitis. Since acinar cells are thought to be the cell origin of PanIN and pancreatic ductal adenocarcinoma (PDAC), our aim is to understand how acinar cells initially resist to Kras mutations and how pancreatitis enables mutated Kras to promote pancreatic neoplasia. Pancreatitis and associated inflammation were induced by injections of cerulein in tamoxifen-treated Elastase-CreER/LSLKrasG12D mice, in which mutated Kras expression is induced exclusively in acinar cells. RNAseq analysis on FACS-sorted acinar cells revealed that Kras expression and several cancer-associated pathways were rapidly induced in the presence of inflammation. In situ hybridization, RTqPCR, and Western blot (WB) experiments confirmed that Kras and/or KrasG12D mRNA and protein were significantly increased in acinar cells after cerulein treatment. To further explore the control of Kras expression, we generated a novel mouse model in which a citrine gene (a variant of GFP) is fused in frame with the endogenous Kras gene, allowing to detect Kras on tissue sections using a GFP antibody. We demonstrated by WB that the protein expression of Kras, KrasG12D and citrine-Kras fusion was similarly regulated in response to cerulein treatment, suggesting the involvement of the same regulatory elements on all three alleles. The ability of citrine-Kras fusion to bind GTP and translocate to the plasma membrane in acini was not altered compared to wild-type Kras protein. Little or no citrine-Kras was visualized in acinar cells of tamoxifen-treated Elastase-CreER/LSLKrasG12D/citrine-Kras mice. However, a 30-fold increase of citrine-Kras at the cell membrane of neoplastic lesions was detected after cerulein treatment. This indicates that inflammation is required for expression and plasma membrane targeting of Kras and KrasG12D in metaplastic acini. Importantly, protein expression of FT-α/β, Rce1 and ICMT, three enzymes necessary for Kras targeting to the membrane was greatly increased in murine acinar cells during pancreatitis. Basal FT-α/β, Rce1 and ICMT levels were found to be very low in normal mouse and human pancreata, compared to other organs, and their expression increased in primary mouse and human acini grown under conditions inducing the formation of preneoplastic lesions. In addition, computational analysis of a PDAC cohort (n=178) from The Cancer Genome Atlas (TCGA) revealed a positive correlation between expression of KrasG12D and its regulating enzymes. We conclude that Kras and KrasG12D are normally not present in acinar cells in vivo and that inflammation induces a coordinated program driving their expression and translocation to plasma membrane. Our findings provide a first clue to the long-standing observation that in several organs, Kras mutations, alone, are not sufficient to affect tissue homeostasis. Citation Format: Mohamad Nabil Assi, Jean-Nicolas Lodewyckx, Younes Achouri, Claude Gérard, Nicolas Dauguet, Isabelle Houbracken, Yves Heremans, Donatienne Tyteca, Ilse Rooman, Luc Bouwens, Frédéric Lemaigre, Patrick Jacquemin. Inflammation enables pancreatic acinar cells to overcome resistance to oncogenic Kras by increasing its expression and plasma membrane localization [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A07.

Antibody blockade of semaphorin 4D to sensitize pancreatic cancer to immune checkpoint blockade.

26 Background: Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis, and will soon become the second leading cause of cancer mortality. Unfortunately, T-cell directed immunotherapies have failed to demonstrate efficacy in PDAC. These failures may in part be mediated by an immunosuppressive tumor microenvironment (TME). Semaphorin 4D (Sema4D) is a glycoprotein which binds its cognate receptors Plexin B1/B2. Here we present our work in blocking Sema4D in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors and confirmed for disease by ultrasound. Mice were treated with FOLFIRINOX (5-FU, Irinotecan, Oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-Sema4D mAB (bi-weekly). Human and mouse circulating and tumor infiltrating leukocytes were interrogated through flow cytometry (FACS) for immune subset and expression of Sema4D and Plexin receptors. Archived human PDAC tissues were assessed through quantitative immunohistochemistry (IHC) for presence of Sema4D positive infiltrate. Results: Both FACS and IHC analysis of human PDAC specimens confirm the presence and increased prevalence over normal pancreata of Sema4D lymphocytes and Plexin B1/B2 expressing tumor associated macrophages (TAMs). KP2 orthotopically injected mice exhibited longer survival when treated with the triple combination of FOLFIRINOX, ICB, and anti-Sema4D antibody, compared to FOLFIRINOX alone, FOLFIRINOX plus ICB, or FOLFIRINOX plus anti-Sema4D antibody (P &lt; 0.02). Flow cytometric analysis of anti-Sema4D and ICB treated murine tumors show a doubling of penetration by CD 8+ effector T cells within tumors compared to control groups (P = 0.03). A loss in Sema4D fluorescence signal via FACS in tumor-infiltrating CD3+ leukocytes was observed in mice treated with anti-SEMA4D, confirming penetration and target blockade within the TME. Conclusions: Sema4D and Plexin B1/B2 leukocytes penetrate human PDAC tumors, and treatment with Sema4D blocking antibody improved response to ICB in combination with standard of care FOLFIRINOX in preclinical murine studies.

Effects of neoadjuvant FOLFIRINOX and gemcitabine-based chemotherapy on cancer cell survival and death in patients with pancreatic ductal adenocarcinoma.

Background: The progression and response to systemic treatment of cancer is substantially dependent on the balance between cancer cell death (apoptosis and necroptosis) and cancer cell survival (autophagy). Although well characterized in experimental systems, the status of cancer cell survival and cell death in human pancreatic ductal adenocarcinoma (PDAC), especially in response to chemotherapy and different types of chemotherapy is poorly described. Results: The median (95% confidence interval) survival was 31.6 (24.5–44.5) months after FOLFIRINOX versus 15.8 (2.0–20.5) months after gemcitabine-based therapy (p = 0.039). PDAC tissue autophagy was reduced compared to normal pancreata based on reduced BECLIN-1 expression and LC3-Lamp-2 colocalization, whilst necroptosis (RIP-1) was increased. Neoadjuvant therapy was associated with further reduced autophagy based on p62/SQSTM-1 accumulation, and increased necroptosis (RIP3 and pMLKL) and apoptosis (BAX, cleaved CASPASE-9 and CASPASE-3) markers, increased nuclear p65 (NF-κB) and extracellular HMGB1 expression, with greater CD8+ lymphocyte infiltration. Survival was associated with reduced autophagy and increased apoptosis. Necroptosis (RIP-3, pMLKL) and apoptosis (BAX and cleaved CASPASE-9) markers were higher after FOLFIRINOX than gemcitabine-based treatment. Patients and methods: Cancer cell autophagy, apoptosis, and necroptosis marker expression was compared in pancreatic tissue samples from 51 subjects, comprising four groups: (1) surgical resection for PDAC after FOLFIRINOX (n = 11), or (2) after gemcitabine-based (n = 14) neoadjuvant therapy, (3) patients undergoing PDAC resection without prior chemotherapy (n = 13), and (4) normal pancreata from 13 organ donors. Marker expression was undertaken using semi-automated immunofluorescence-FACS-like analysis, defining PDAC cells by CK-7+ expression.

Multiplex immunophenotyping reveals unique characteristics of pancreatic neoplasms

Introduction: The tumor microenvironment of pancreatic adenocarcinoma (PDA) is characterized by a dense stroma and infiltration with primarily immunosuppressive cells. While much is known about the immune cellular changes occurring during tumorigenesis in murine models of PDA, data in humans is lacking. We sought to identify difference in the immune microenvironment of neoplastic and pre-neoplastic lesions using multiplex immunohistochemistry. Method: Operative pancreatic tissue was collected from 289 patients undergoing pancreatectomy at the University of Michigan. Slides were prepared from formalin-fixed, paraffin embedded tissue which were subjected to serial rounds of primary and secondary antibody staining followed by tyramide signal amplification. Result: Analysis was performed on tissue from patients with normal pancreata (n=42), mucinous cystic neoplasms (MCN;n=22), intraductal papillary mucinous neoplasms (IPMN;n=80), pancreatic intraepithelial neoplasia (PanIN;n=37) and PDA (n=108). IPMNs and normal pancreata had significantly more epithelial cells when compared to MCN and PDA. In IPMNs, these cells were more frequently positive for the checkpoint inhibitor PD-L1 (mean = 16.4%; p<0.0001). Mucinous lesions (MCN and IPMN) had significantly more antigen presenting cells than other lesion types (mean = 4.6% and 3.2%, respectively; p=0.0048). While there were no differences in relative infiltration of cytotoxic T lymphocytes, PDA had a far greater overall and relative number of regulatory T lymphocytes (Treg) (mean = 17%; p<0.0001). The accumulation of Tregs paralleled progression of neoplasia with increasing infiltration in normal pancreata, PanIN and PDA (1%, 3%, 17%, respectively; p<0.0001). Conclusion: A better understanding of the immune microenvironment of PDA is needed for the development of novel immunotherapeutic approaches. These data demonstrate an accumulation of Tregs as tumors progress from preinvasive to invasive cancer, suggesting a potential avenue for intervention.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice

Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n= 36) and poorly differentiated (n= 23) PDAC samples by immunohistochemistry. KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

Saa3 is a key mediator of the protumorigenic properties of cancer-associated fibroblasts in pancreatic tumors.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of abundant desmoplastic stroma primarily composed of cancer-associated fibroblasts (CAFs). It is generally accepted that CAFs stimulate tumor progression and might be implicated in drug resistance and immunosuppression. Here, we have compared the transcriptional profile of PDGFRα+ CAFs isolated from genetically engineered mouse PDAC tumors with that of normal pancreatic fibroblasts to identify genes potentially implicated in their protumorigenic properties. We report that the most differentially expressed gene, Saa3, a member of the serum amyloid A (SAA) apolipoprotein family, is a key mediator of the protumorigenic activity of PDGFRα+ CAFs. Whereas Saa3-competent CAFs stimulate the growth of tumor cells in an orthotopic model, Saa3-null CAFs inhibit tumor growth. Saa3 also plays a role in the cross talk between CAFs and tumor cells. Ablation of Saa3 in pancreatic tumor cells makes them insensitive to the inhibitory effect of Saa3-null CAFs. As a consequence, germline ablation of Saa3 does not prevent PDAC development in mice. The protumorigenic activity of Saa3 in CAFs is mediated by Mpp6, a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases (MAGUK). Finally, we interrogated whether these observations could be translated to a human scenario. Indeed, SAA1, the ortholog of murine Saa3, is overexpressed in human CAFs. Moreover, high levels of SAA1 in the stromal component correlate with worse survival. These findings support the concept that selective inhibition of SAA1 in CAFs may provide potential therapeutic benefit to PDAC patients.

Elevated glypican-1 expression is associated with an unfavorable prognosis in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in humans, with a 5‐year survival rate of <5%. Recently, glypican‐1 (GPC1)‐expressing circulating exosomes were found to be a promising diagnostic tool for PDAC. However, the aberrant expression of GPC1 has not been systematically evaluated in large‐scale clinical samples of PDAC. Here, we performed a comprehensive analysis of GPC1 mRNA and protein expression features. Included in this study were 178 PDAC patients from the cancer genome atlas (TCGA) and 186 subjects whose tissues were used in immunohistochemical staining assays. We demonstrated that GPC1 mRNA was silenced in normal pancreata; however, it was re‐expressed in PDAC tissues probably because of the promoter hypomethylation. The GPC1 protein was barely expressed in the normal and adjacent noncancerous pancreata. In tumor tissues, 59.7% (111/186) of the detected samples showed positive expression. Notably, GPC1 was elevated in 63.6% (34/55) of early stage cases. High levels of GPC1 were associated with poorer differentiation and larger tumor diameters. Kaplan–Meier analysis showed a significant difference in overall survival between the groups categorized by GPC1 expression (P = 0.0028). Multivariate analyses indicated that GPC1 was a significant risk factor for poor overall survival with a 1.82‐fold increase in the hazard ratio (P = 0.0022). In conclusion, during pancreatic tumorigenesis, GPC1 was ectopically expressed and served as an independent poor prognostic factor. Our findings highlighted the alluring prospect of GPC1 as an early diagnostic and prognostic marker as well as a therapeutic target for PDAC.

Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

Little is known about the origin of pancreatic intraductal papillary mucinous neoplasms (IPMN). Pancreatic duct glands (PDGs) are gland-like outpouches budding off the main pancreatic ducts that function as a progenitor niche for the ductal epithelium; they express gastric mucins and have characteristics of side-branch IPMNs. We investigated whether PDGs are a precursor compartment for IPMNs and the role of Trefoil factor family 2 (TFF2)-a protein expressed by PDGs and the gastric mucosa that are involved in epithelial repair and tumor suppression. We obtained pancreatectomy specimens from 20 patients with chronic pancreatitis, 13 with low-grade side-branch IPMNs, and 15 patients with PDAC; histologically normal pancreata were used as controls (n= 18). Samples were analyzed by immunohistochemistry to detect TFF1 and TFF2 and cell proliferation. We performed mitochondrial DNA mutational mapping studies to determine the cell lineage and fate of PDG cells. Pdx1-Cre;LSL-KRASG12D (KC) mice were bred with TFF2-knockout mice to generate KC/Tff2-/- and KC/Tff2+/- mice. Pancreata were collected and histologically analyzed for formation of IPMN, pancreatic intraepithelial neoplasias, and PDAC, in addition to proliferation and protein expression. Human pancreatic ductal epithelial cells and PDAC cell lines were transfected with vectors to overexpress or knock down TFF2 or SMAD4. Histologic analysis of human samples revealed gastric-type IPMN to comprise 2 molecularly distinct layers: a basal crypt segment that expressed TFF2 and overlying papillary projections. Proliferation occurred predominantly in the PDG-containing basal segments. Mitochondrial mutation mapping revealed a 97% match between the profiles of proliferating PDG cells and their overlying nonproliferative IPMN cells. In contrast to KC mice, 2-month-old KC/Tff2+/- and KC/Tff2-/- mice developed prominent papillary structures in the duct epithelium with cystic metaplasia of the PDG, which resembled human IPMN; these expressed gastric mucins (MUC5AC and MUC6), but not the intestinal mucin MUC2. KC/TFF2-knockout mice developed a greater number and higher grade of pancreatic intraepithelial neoplasias than KC mice, and 1 mouse developed an invasive adenocarcinoma. Expression of TFF2 reduced proliferation of PDAC cells 3-fold; this effect required up-regulation and activation of SMAD4. We found expression of TFF2 to be down-regulated in human PDAC by hypermethylation of its promoter. In histologic analyses of human IPMNs, we found PDGs to form the basal segment and possibly serve as a progenitor compartment. TFF2 has tumor-suppressor activity in the mouse pancreas and prevents formation of mucinous neoplasms.

Abstract 4940: Use of whole slide digital image analysis for determination of expression of Twist-1 in normal panceata and stage IV pancreatic ductal adenocarcinomas

Abstract Pancreatic cancer is the 4th most common cause of cancer deaths in this country. Close to 50,000 deaths occur every year. This is due to the fact that most patients are not diagnosed until presentation at a stage IV. In a recent study, it is estimated that more than 10 years go by between the first genetic mutation and the formation of invasive cancer. More mutations occur in metastatic lesions after an additional 6 years. Both SNAIL and TWIST have gained more attention as biomarkers because that contribute to E-cadherin dysregulation that allows the tumor cells to invade. In this study we evaluated 17 cases of pancreatic ductal adenocarcinoma using twist-1 antibody to evaluate tumor cell expression in these cases. All paraffin blocks were retrieved from the IU pathology department files via an IRB approved protocol. The unstained slides were stained with primary antibody Twist-1 using the DAKO Flex system. Control sections were treated with an isotype control using the same concentration as primary antibody to verify the staining specificity. The Aperio whole slide system was used to image the slides. Computer-assisted morphometric analysis of digital images was carried out using the Aperio Image scope software using the positive pixel algorithm. The algorithm was used to quantify the Twist-1 positivity of normal pancreas, and pancreatic ductal adenocarcinoma. This algorithm is the only FDA approved algorithm for human clinical trials. The immunostaining of Twist-1 was found only in the ductal epithelial carcinoma cells in the pancreas. There was no staining in the stroma in any of the normal pancreas and ductal pancreatic carcinoma cases. We analyzed 5 normal pancreata, and 12 pancreatic ductal adenocarcinomas. The data from the image analysis was 3.38% expression in normal pancreata and 11.44% expression in the pancreatic ductal adenocarcinoma. In summary, the image analysis data demonstrates the overexpression of Twist-1 in stage IV pancreatic adenocarcinomas compared to the normal pancreas. This suggests that this marker may be a predictor of tumor invasiveness and metastasis. Citation Format: Max Jacobsen, George Sandusky, Kavita Shah. Use of whole slide digital image analysis for determination of expression of Twist-1 in normal panceata and stage IV pancreatic ductal adenocarcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4940.

Generation of functional organs from pluripotent stem cells

Hematopoietic stem cells (HSCs) have played a major role in stem cell biology, providing many conceptual ideas and models. Among them is the concept of the "niche", a special bone-marrow microenvironment that by exchanging cues regulates stem-cell fate. The HSC niche also plays an important role in HSC transplantation. Successful engraftment of donor HSCs depends on myeloablative pretreatment to empty the niche. The concept of the stem-cell niche has now been extended to the generation of organs. We postulated that an empty "organ niche" exists in a developing animal when development of an organ is genetically disabled. This organ niche should be developmentally compensated by blastocyst complementation using wild-type primary stem cells (PSCs). We proved the principle of organogenesis from xenogeneic PSCs in an embryo unable to form a specific organ, demonstrating the generation of functionally normal rat pancreas by injecting rat PSCs into pancreatogenesis-disabled mouse embryos. This principle has held in pigs. When pancreatogenesis-disabled pig embryos underwent complementation with blastomeres from wild-type pig embryos to produce chimeric pigs, the chimeras had normal pancreata and survived to adulthood. Demonstration of the generation of a functional organ from PSCs in pigs is a very important step toward generation of human cells, tissues, and organs from individual patients' own PSCs in large animals.

Abstract A25: Heterozygous deletion of Dicer promotes anoikis and reduces pancreatic cancer metastasis in vivo

Abstract Previous studies have suggested that Dicer acts as a haploinsufficient tumor and metastasis suppressor. In contrast, studies of human pancreatic ductal adenocarcinoma (PDAC) tumors and cell lines demonstrate upregulation of miRNAs with progression. And yet, disagreement exists regarding which miRNA-target relationships are critical to tumor biology. We therefore sought to ascertain the effects of global miRNA suppression on pancreatic tumorigenesis using a conditional loss-of-function allele of Dicer in a mouse model of pancreatic cancer. Given the common observation of elevated miRNA expression in PDAC, we hypothesized that loss of one allele of Dicer would inhibit pancreatic tumorigenesis and prolong overall survival. Ptf1a-Cre-mediated deletion of Dicer in the pancreas demonstrated that homozygous loss is not tolerated, leading to progressive acinar cell atrophy as mice aged and suggesting that Dicer activity is required for the long-term viability of this cell type in the postnatal pancreas. However, mice with heterozygous deletion of Dicer had developmentally normal pancreata that were maintained into adulthood. To assess the impact of Dicer gene deletion on pancreatic tumorigenesis in vivo, we used the RCAS-TVA pancreatic cancer model incorporating polyoma virus middle T antigen (PyMT) expression and p53 gene deletion. We found that heterozygous deletion of Dicer did not impact overall animal survival; primary tumor size, number, or burden; or tumor histology (acinar, ductal, or poorly differentiated carcinomas). However, loss of one copy of Dicer robustly inhibits metastasis of PyMT-driven pancreatic tumors, with metastases seeding from 12.5% of DicerFL/WT tumors versus 54.5% of DicerWT/WT tumors (p&amp;lt;0.05). Intriguingly, cell lines derived from DicerWT/WT and DicerFL/WT tumors also display no differences in their proliferative rate, migration, invasion, soft agar colony formation, or survival following serum-starvation. However, DicerFL/WT cancer cell lines exhibit markedly increased sensitivity to cell death from loss of attachment (anoikis) compared to DicerWT/WT lines (50% survival vs 76% survival at 24 hours; p&amp;lt;0.01). This result provides a potential explanation for the reduced metastasis seen with DicerFL/WT tumors in our mouse model, and it invites further investigation into the precise miRNAs responsible for this phenotype. Importantly, our findings are consistent with observations in human pancreatic cancer, where upregulation of oncogenic miRNAs is commonly seen in late-stage disease. However, our data are in disagreement with published literature implicating Dicer as a tumor suppressor whose expression is decreased during carcinoma progression in other tumor contexts. This discrepancy highlights the importance of cellular context in understanding miRNA biology and the need for more thorough in vivo genetic experimentation to dissect the role of miRNAs in pancreatic carcinogenesis. Citation Format: Brian Quattrochi, Brian C. Lewis. Heterozygous deletion of Dicer promotes anoikis and reduces pancreatic cancer metastasis in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A25.

The role of SPARC expression in pancreatic cancer progression and patient survival

Background. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that has been implicated in tumor–stroma interactions in pancreatic cancer. Here we evaluated the expression of SPARC during the progression of pancreatic cancer and its correlation with survival following curative intent surgery. Methods. The expression profile of SPARC was investigated in normal pancreas, invasive adenocarcinoma, and lymph node metastasis by immunohistochemistry. Kaplan–Meier and multivariate Cox regression were used to assess for mortality risk. Results. None (0%) of 10 normal pancreata, 68 (77%) of 88 primary tumors, and 28 (67%) of 42 lymph node metastases were labeled with the SPARC antibody used in this study. SPARC was expressed exclusively in stromal cells. The survival of patients with high stromal SPARC expression was significantly worse than that of the patients with low stromal SPARC expression (11.5 vs 25.3 months; p = 0.020). Multivariate analysis showed that stromal SPARC expression (hazard ratio (HR) 2.12; p = 0.012), tumor location (body/tail) (HR 2.95; p = 0.003), and adjuvant chemotherapy (HR 0.55; p = 0.018) were significant independent risk factors. Conclusion. This study describes the pattern of SPARC expression in pancreatic neoplastic progression and supports the role of stromal SPARC expression as a prognostic factor and target for directed therapy.

KRAS and Guanine Nucleotide-Binding Protein Mutations in Pancreatic Juice Collected From the Duodenum of Patients at High Risk for Neoplasia Undergoing Endoscopic Ultrasound

Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid after secretin stimulation might identify microscopic neoplasias in the pancreatic duct system. We determined the prevalence of mutations in KRAS and guanine nucleotide-binding protein α-stimulating genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma. Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer, and 78 subjects were evaluated for pancreatic cancer (n = 30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n = 48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score. KRAS mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (P = .0005). A greater proportion of patients with pancreatic cancer had at least 1 KRAS mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, P = .002). Among screened subjects, mutations in KRAS (but not guanine nucleotide-binding protein α-stimulating) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients older than age 50 years had KRAS mutations (54.6%) than younger patients (36.3%) (P = .032); the older subjects also had more mutations in KRAS (P = .02). Mutations in KRAS are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, KRAS mutations were detected in pancreatic juice from 19% of controls. Mutations detected in individuals without pancreatic abnormalities, based on imaging analyses, likely arise from small pancreatic intraepithelial neoplasia lesions. ClinicalTrials.gov no: NCT00438906 and NCT00714701.