AGA Abstracts fluid secretion in these pancreatic ductal organoids. We now have the ability to monitor real- time CFTR function of human pancreatic ductal epithelium using the fluid secretion assay. 72 A NOVEL PREDICTIVE ORGANOID CULTURE SYSTEM FROM PANCREATIC CANCER PATIENTS - PERSONALIZED MEDICINE IN REAL- TIME Zahra Dantes, Matthias Wirth, Karin Feldmann, Alice Nomura, Alexander Muckenhuber, Katja Steiger, Oliver Strobel, Marc martignoni, Gueralp Ceyhan, Jens Siveke, Stefan von Delius, Wilko Weichert, Rickmer Braren, Anil K. Rustgi, Roland Schmid, Dieter Saur, Günter Schneider, Maximillian Reichert Introduction: By 2030, it is estimated that the number of cancer deaths caused by pancreatic ductal adenocarcinoma (PDAC) will surpass colorectal and breast cancer. One reason for this development is resistance to standard of care chemotherapy. Molecular and morphologi- cal tumor heterogeneity results in varying response to treatment and poor clinical outcome. Therefore, better and more efficient strategies for personalized oncology are urgently needed to improve patient outcome. Materials and Methods: Due to the immense costs of established organoid media supplements, we have generated a more cost-effective alternative media- composition that can be used to generate patient-derived PDAC organoids successfully. We validated our modified normal feeding media (NFMm), in comparison to the established NFM media (Boj et al., 2015), using multilayered bioassays assessing take rate, proliferation, viability, morphology, activation of oncogenic pathways, and most importantly sensitivity toward chemotherapy. Results: Utilizing three dimensional organoid culture derived from surgical specimens and endoscopic fine needle aspirations, we are currently expanding the patient-derived organoid repository at our institution. Our organoid lines withstand freeze- thaw cycles and functional assays can be implemented within 2-6 weeks after isolation. Drug-screens are performed using conventional chemotherapeutic drugs as well as targeted therapeutics directed against PDAC core pathways and individual vulnerabilities. We provide evidence that the organoid biology mimics the biology of PDAC in situ. For instance, it has been demonstrated that mutations in DNA-repair genes (BRCA1, BRCA2, PALB2) display increased sensitivity towards platinum-based chemotherapy and PARP inhibition. Indeed, one study patient with familial pancreatic cancer due to a germline PALB2 mutation showed an outstanding clinical response towards a platinum-based chemotherapy. Organoids that have been derived after therapy have been treated with PARP inhibitors. Remarkably, this PALB2-mutated organoid line shows a significant response to Orapalib with an IC 50 0.16 nM, whereas PALB2-wildtype organoids show no response even at a concentration of 1000 nM. These individualized approaches have now been expanded to organoids derived from specific subtypes of PDAC including quasimesenchymal (QM), exocrine-like, and classical. Interestingly, the organoids derived from the QM-subtype are more sensitive towards taxol treatment consistent with recent reports (Noll et al., 2016). Conclusions: Taken together, our novel organoid culture system appears to be predictive for clinical response. In addition, the modified, affordable culture protocol makes this model system accessible to the entire scientific community in the field of pancreatic cancer and might be useful clinically at a large scale. 73 PIEZO1, A MECHANICALLY ACTIVATED ION CHANNEL, MEDIATES PRESSURE-INDUCED PANCREATITIS Joelle Romac, Rafiq A. Shahid, Steven Vigna, Rodger A. Liddle The pancreas is exquisitely sensitive to mechanical injury. Merely touching the pancreas can initiate a series of cellular events leading to premature zymogen activation and eventually pancreatitis. Why the pancreas is so sensitive to mechanical stress and the mechanism by which mechanical force causes pancreatitis is unknown. In the current study, we demon- strated that pancreatic acinar cells express the mechanoreceptor Piezo1. We were able to induce pancreatitis by increasing pressure within the pancreatic duct (15 mmHg), under conditions that resemble the clinical condition of endoscopic retrograde cholangiopancrea- tography (ERCP). To determine if this effect could be through activation of a mechano- sensitive receptor, we infused into the pancreatic duct the Piezo1 antagonist GsMTx4 which blocked pressure-induced pancreatitis. We then sought to determine if selective activation of a mechanoreceptor in the pancreas was sufficient to cause pancreatitis. Therefore, we infused the Piezo1 agonist, Yoda1 into the pancreatic duct at a pressure that does not cause pancreatitis (5 mmHg) and discovered that Yoda1 produced profound pancreatic inflammation. Furthermore, in pancreatic acinar cells, in vitro, Yoda1 stimulated calcium influx and induced pancreatic injury with accompanying NF-kB activation and generation of IL-6 and TNF-alpha; effects that were all calcium-dependent. Finally, selective acinar cell-specific genetic deletion of Piezo1 protected mice against pressure-induced pancreatitis. Our studies demonstrate that activation of mechanoreceptors in pancreatic acinar cells is a previously unrecognized mechanism for pancreatitis and may explain why pancreatitis devel- ops following application of pressure to the gland. These findings also demonstrate that Piezo1 activation can exert pathophysiological effects and suggest that Piezo1 may be a novel target for preventing pancreatitis in which manipulation of the gland is anticipated (e.g., ERCP or pancreatic surgery). 74 A NOVEL TRAIL-BASED THERAPY FOR CHRONIC PANCREATITIS Seulki Lee, Yumin Oh, Ogyi Park, Jong-Sung Park, Adriana Bora, Vikesh Singh, Bin Gao, Pankaj J. Pasricha Background: Chronic pancreatitis (CP) is characterized by a destruction of pancreas function resulting in acinar atrophy and fibrosis with diabetes, mal-digestion and symptoms of persistent abdominal pain. An essential pathological feature of CP is the loss of exocrine and endocrine function by a prominent fibrosis. CP is mainly orchestrated by activation of pancreatic stellate cells (PSCs) which produce various fibrogenic and growth factors to S-18 AGA Abstracts facilitate the accumulation of extracellular matrix. We, therefore, hypothesized that selective blocking or depletion of aPSCs will offer an antifibrotic treatment of CP, and consequently, will alleviate the CP-associated complications including functional loss and pain. We discov- ered that TRAIL selectively blocks priamry human PSC activation and induces apoptosis in aPSCs that express its cognate death receptors (DRs) while sparing normal tissues. To this end, we developed an engineered, recombinant human TRAIL for CP. Methods: Primary human PSCs were culture-activated, {2 days (quiescent) and 7 days (activated)}, and flow cytometry, caspase activities, Western blot, and qRT-PCR were performed to analyze the efficacy of TRAIL PEG . Immunofluorescence was used to validate the clinical feasibility of TRAIL signaling on human CP sections by DRs/α-SMA dual-staining. Sprague-Dawley(SD) rats were fed with ethanol/Lieber-Decarli (LD) diet and received intraperitoneal injections of cerulein every week from CP subjects. CP rats were treated with TRAIL PEG or vehicle and the pancreas specimen was assessed by the pathophysiological score, immunohistochemistry, Western blotting, and gene expression by qRT-PCR (Acta2, Col1a2, Dr4, Dr5, and Tgf-b). Results: We investigated the effects of TRAIL PEG in culture- or EtOH-activated PSCs, and the role of TRAIL signaling in DR-mediated apoptosis in aPSCs. Activated PSCs highly expresses fibrogenic markers including α-SMA and collagen with a membrane expression of DR5. By a TRAIL PEG treatment, selective apoptosis was clearly observed by caspase-3/8 activation with the Death-Inducing Signaling Complex (DISC) formation, whereas changes were minimal in qPSCs. Clinical samples for CP patient showed that fibrotic tissue had significant levels of the double positive area of α-SMA + DR + . To validate the efficacy of TRAIL PEG in SD rats, CP was induced by LD diet and cerulein i.p. injection and treated with TRAIL PEG . We found that systemically administered long-acting TRAIL PEG in CP animal models selectively induces apoptosis in α-SMA + DR + cells and reversed fibrosis resulting in a downregulation of multiple fibrogenic factors without off-target toxicity. Conclusions: The therapeutic potential to target a TRAIL signaling for selectively eliminating aPSCs can possibly provide a noninvasive method to address the highly unmet need in CP patients as a novel approach. 75 SIRTUIN 3 GENETIC ABLATION CAUSES MITOCHONDRIAL DYSFUNCTION AND WORSENS ACUTE PANCREATITIS Sudarshan R. Malla, Natalia Shalbueva, Yueqiu Qin, Richard Waldron, Jingzhen Yuan, Sophie Gretler, Olga A. Mareninova, Stephen J. Pandol, Anna Gukovskaya Background & Aims: We have shown that mitochondrial dysfunction is a key pathologic event of experimental nonalcoholic and alcoholic pancreatitis. We further found that alcoholic pancreatitis reduced the pancreatic level of sirtuin 3 (SIRT3), mitochondrial resident protein deacetylase implicated in ATP production. To elucidate the role of sirtuins in pancreatitis and the mechanisms of alcohol's effects on pancreas, we investigated the effect of SIRT3 genetic ablation on mitochondrial functions and the disease severity. Methods: Acute pancre- atitis was induced in wild type and SIRT3 null (KO) mice by i.p. injections of cerulein or L-arginine. Measurements of mitochondrial membrane potential were performed in isolated pancreatic mitochondria and acinar cells using TPP+ electrode and the mitochondrial mem- brane potential-sensitive fluorescent dye TMRM. Protein acetylation was measured in whole tissue and mitochondrial fractions by immunoblot against acetylated lysine and mass spec- trometry. Results: Pancreatic mitochondria from SIRT3 KO mice failed to maintain mitochon- drial membrane potential, and exhibited greater depolarization in response to CCK (compared to wild type). SIRT3 ablation deranged mitochondrial dynamics, manifested by a decrease in the mitochondrial fusion mediator, MFN1, and increase in the fragmentation mediator DRP1. In addition, SIRT3 deficiency perturbed mitochondrial biogenesis as evidenced by decreased level of the key transcription factor PGC-1alpha. Mass spectrometry analysis showed hyperacetylation of several mitochondrial proteins, including adenine nucleotide translocase which facilitates the exchange of ADP and ATP across the mitochondrial inner membrane, and the enzymes of tricarboxylic acid (TCA) cycle. In particular, the hyperacetyla- tion of isocitrate dehydrogenase we observed in SIRT3 deficient pancreas resulted in decreased activity of this enzyme, which is responsible for catalyzing the conversion of isocitrate to alpha-ketoglutarate critical for the TCA cycle. At the same time, SIRT3 ablation increased the activities of electron transport chain (ETC) complexes I and III in pancreatic mitochondria, which likely serves as a compensatory feedback mechanism. SIRT3 genetic deletion worsened experimental pancreatitis induced by both cerulein and L-arginine, manifested by increased serum amylase and lipase, necrosis, and neutrophilic infiltration. Conclusions: SIRT3 genetic ablation causes mitochondrial dysfunction in pancreas, evidenced by loss of mitochondrial membrane potential, aberrant mitochondrial dynamics, changes in acetylation and activities of the enzymes of TCA cycle and ETC complexes. It aggravates experimental pancreatitis. The results indicate an important role of mitochondrial proteins' acetylation in nonalcoholic and alcoholic pancreatitis. 76 EGFR INDUCES ILF3 AND G9A EXPRESSIONS TO MAINTAIN OCT4- MEDIATED CANCER STEMNESS PROPERTY IN EGFR-POSITIVE CANCERS Chun-Chia Cheng, Ai-Sheng Ho Cancer stem cells survive and lead to tumor recurrence in tumor therapeutic treatments. Colorectal and lung cancer cells highly express epidermal growth factor receptor (EGFR), which is critical for tumor progression. We hypothesized that EGFR may play a pivotal role in the induction of cancer stemness cells (CSCs). The aim of this study attempted to investigate the molecular mechanism of EGFR-mediated cancer stemness, and to discover the potent therapeutic agents. A high expression level of cancer stemness markers, ALDH1, CD133, Oct4, and Nanog was observed in tumorspheres derived from colorectal HCT116 and lung HCC827 cells. External EGF further elevated the expression of Oct4 in HCC827 cells. Moreover, chemical inhibition and genetic knockdown of EGFR reduced the Oct4 expression and also tumorsphere formation. We found that inhibitors targeting to EGFR (afatinib), ILF3 (YM155), and G9a (UNC0642) are potential to reduce the formations of tumorspheres. The results revealed that EGFR triggered the formation of tumorspheres through elevating the activations of G9a-mediated stemness genes. In addition, YM155 effectively inhibited tumor growth and formation of tumorspheres as an anti-cancer agent