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Crosstalk Between Commensal Bacteria and Colonic Epithelium inGnotobiotic Rats: A Key Role of AcetateLaura Wrzosek, Sylvie Miquel, Marie-Louise Noordine, Stephan Bouet, ChantalBridonneau, Catherine Philippe, Véronique Robert, Claire Cherbuy, Philippe Langella,Muriel Thomas

The gastrointestinal tract (GIT) of mammals is colonized by a community of micro-organismsreaching levels as high as 1011 bacteria/g of contents in the colon. This microbiota is involvedin intestinal homeostasis and epithelium structure through the control of proliferation anddifferentiation processes (Cherbuy et al., 2010). Bacteroides thetaiotaomicron and Faecalibacter-ium prausnitzii, dominant members of microbiota, are commensal model bacteria to studyhost-microbiota interactions. The objective of our work was to understand the effects ofthese two bacteria on colonic epithelium homeostasis in gnotobiotic rats and underlyingthis crosstalk using In Vitro models. Germ-free rats (GF) were orally inoculated with B.thetaiotaomicron either alone or with a mix of B. thetaiotaomicron and F. prausnitzii. Colonicepithelium morphology, crypt depth, cell cycle-related proteins and goblet cells markerswere analyzed by histological staining, immunohistochemistry and Western blot in GF (n=6), mono-associated (Ino-Bt; n=12) or di-associated (Ino-Bt+Fp; n=6) rats 30 days afterinoculation. In Ino-Bt rats, no effect was observed neither on crypt depth nor on proliferationmarkers whereas Krüppel Like Factor 4 protein (KLF4), a transcription factor involved ingoblet cells differentiation, was significantly increased (2.5 ±0.8-fold) compared to GF rats.In parallel with this KLF4 induction, a significant increase of goblet cells per crypt in thecolon of Ino-Bt compared to GF rats was observed. In Ino-Bt+Fp rats, there was no inductionof KLF4 protein, suggesting that the presence of F. prausnitzii impaired the mucus-stimulatoryeffect triggered by B. thetaiotaomicron. Short Chain Fatty Acids (SCFA) composition wasdetermined on caecal contents by gas chromatograph to assess bacterial metabolic activity.In Ino-Bt rats, acetate was the major SCFA detected (5.8 μmol/g) with a low level ofpropionate (1.0 μmol/g) and no butyrate. In Ino-Bt+Fp rats, butyrate was the major SCFA(1.3 μmol/g) and acetate concentration was significantly decreased (two-fold) compared toIno-Bt rats (3.0 μmol/g). Using HT29-MTX cells, a cell line producing mucus, we showedthat acetate was able to increase significantly KLF4 protein whereas butyrate and propionatehave no effect on KLF4 level. All of these results suggested that acetate, a bacterial metaboliteproduced by B. thetaiotaomicron, could mediate a bacterial signal to promote goblet cellsdifferentiation pathway in the GIT of mono-associated rats. In Crohn's disease patients, adiminished prevalence of F. prausnitzii (Martinez-Medina et al., 2006) and an increase ofmucus production (Gersemann et al., 2009) have been often reported. Our observations ingnotobiotic rats are coherent with reports made in patients and may provide mechanisticclues to understand the pathophysiology of inflammatory bowel diseases.

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Negative Crosstalk Between the Extracellular Calcium Receptor and β-Cateninin Colonic EpitheliumOsvaldo Rey, Wenhan Chang, Daniel Bikle, Nora Rozengurt, Enrique Rozengurt

Background: Signaling pathways activated by G protein-coupled receptors (GPCRs) expre-ssed in the gastrointestinal (GI) tract play a critical role in the regulation of multiple functionsof the digestive system, including cell proliferation, differentiation, inflammation and promo-tion of colorectal cancer (CRC). The extracellular Ca2+-sensing receptor (CaSR), a memberof the C family of GPCRs, is expressed in many tissues and organs not directly involved inthe control of Ca2+ homeostasis, including both surface and crypt epithelial cells in rodentand human colons. Interestingly, the expression of the CaSR is greatly reduced or completelylost in CRC again suggesting that signaling pathways activated by this receptor negativelycontrol cellular proliferation and CRC development. However, there is no definitive evidencefor a physiological role of the CaSR in the regulation of normal and/or abnormal intestinalepithelial cell proliferation In Vivo. Results: In order to examine the role of the CaSR incolonic epithelial cell proliferation, we used a recently developed Casr intestinal-specificknock out mice model. Our results demonstrate that genetic ablation of this receptor ledto hyperproliferation of colonic epithelial cells (Ki67 labeling), expansion of the proliferativezone, increase in the size of colonic crypts (length and number of cells/crypt) and enhancedβ-catenin nuclear localization, a regulator of genes that control intestinal cell proliferationand differentiation. To elucidate the mechanism(s) by which CaSR inhibits colonic cellproliferation, we examined multi-site phosphorylation of β-catenin, a post-translationalmodification that plays a critical role in its degradation, nuclear translocation and transcrip-tional activity. We demonstrate that stimulation of the CaSR by either extracellular Ca2+ (5mM) or the allosteric activator R-568 (100 nM) produced a striking and time-dependent(30 min to 4 h) decrease (≈ 60%) in the phosphorylation of β-catenin at Ser552 and Ser675

in NCM-460 human-colon derived epithelial cells expressing the CaSR. Concomitantly, wedetected by immunofluorescence a reduced nuclear reactivity of the phosphoSer552 antibodyas well as a marked redistribution of β-catenin to the plasma membrane. These changes inphosphorylation and intracellular distribution were associated with a 40% downregulationof β-catenin mediated transcriptional activation (TCF/LEF-1 luciferase reporter TOP-Flashassay). Conclusion: These studies demonstrate that signaling pathways emanating from theCaSR control colonic epithelial cells proliferation In Vivo and suggest that the mechanisminvolves regulation of β-catenin phosphorylation.

1060

Redirecting Effector Lymphocytes for Adoptive Cell Therapy of PancreaticAdenocarcinoma in MiceAmit Maliar, Charlotte Servais, Tova Waks, Zelig Eshhar

Background & Aims: Pancreatic adenocarcinoma (PAC) is a common malignancy that isoften diagnosed at an advanced and inoperable stage; it is usually incurable, and the standardsystemic treatments are generally ineffective for metastatic disease. We have investigated, inan animal model, whether adoptive cell therapy (ACT) using redirected, tumor-specific Tcells could be applied for the treatment of primary and metastatic pancreatic adenocarcinoma.

S-187 AGA Abstracts

Methods: We genetically modified human effector T cells to express an antibody-basedchimeric receptor (CAR) directed against Her2/neu as well as CD24, one of the putativePAC cancer stem cell antigens. The antitumor reactivity of the engineered redirected T-lymphocytes ("T-bodies") that specifically recognize their targets independently of MHC,was evaluated in SCID mice using a luciferase labeled human PAC cell line Capan-1 as atarget. The Capan-1 was grafted either subcutaneously or orthotopically into the murinepancreas 21 to 28 days before T-bodies treatment. Bioluminescence imaging and measure-ments were used for the assessment of the anti-tumoral and anti-metastatic effect of the T-bodies treatment. Results: Both CD24 and Her2/neu T-bodies underwent specific activationand cytolysed PAC cells In Vitro. Intra-tumoral injection of T-bodies, a realistic procedurein patients, completely eliminated the tumors in most of the treated animals. Systemic T-body administration effectively reduced tumor size and prolonged the survival of mice withorthotopically transplanted PAC tumors. More than 50% of animals receiving a single splitdose of human Her2/neu or CD24 T-bodies appeared disease-free for more than 2 monthsafter initiation of treatment. T-bodies were effective even in mice with high tumor burden.A further course of T-bodies treatment resulted in a complete response and elimination ofmost of the primary PAC xenografts as well as of metastases in the liver and draining lymphnodes. Conclusions: Our results suggest CAR-redirected lymphocytes ("T-bodies") as anefficient cell-therapy modality for intra-tumoral and systemic treatment of localized anddisseminated pancreatic adenocarcinoma.

1061

Novel Genetic/MicroRNA Evidence That the Microscopically Normal DuodenalMucosa is Encompassed by Pancreatic Cancer Extended Field CarcinogenesisDhananjay Kunte, Ramesh K. Wali, Randall Brand, Shailesh Bajaj, Mart DeLaCruz, AmirC. Patel, Wentao Qi, Yolanda E. Stypula, Tina P. Gibson, Hemant K. Roy

Background: Pancreatic cancer is one of the most lethal cancers largely because it is clinicallyinsidious and difficult/dangerous to access with ERCP having significant complications (i.e.pancreatitis). One increasingly common strategy is exploitation of field carcinogenesis. Inthe colon, the rectum is a well established site for field effect. However, there is evidencethat field carcinogenesis may occur outside the organ. For instance, in lung cancer the buccalmucosa is in the tobacco smoke field of injury (Steiling et al, Can Prev Res 2008). Forpancreatic cancer, there is clear evidence of diffuse ductal dysplasia (PanIN). There has beensome emerging data that the duodenum may represent a field effect site (possibly secondaryto pancreatic juices). Indeed, our group has noted that via optical techniques, there aremicro-architectural alterations in the peri-ampullary duodenum (reviewed in Gastro 2011).Since biological corroboration has been lacking, we evaluated duodenal microRNA and geneexpression profiles among the pancreatic cancer patients and matched controls. Methods:Patients: 32 patients (16 cancer, 16 age/gender matched controls) had 2 peri-ampullarybiopsies with RNA isolated with Ribopure kit (Ambion). The total RNA was then reversetranscribed using Taqman miRNA kit using Megaplex RT Primer set A (Applied Biosystems).The cDNA was then diluted and loaded into Taqman Low Density Array (TLDA) for miRNA(ABI). Real time RT-PCR was performed on ABI Prism 7900HT system. For gene expressionstudies, RNA was reverse transcribed using High Capacity Reverse Transcription kit (ABI)and preamplified using custom pre-amplification primers and then the pre-amplified cDNAwas analyzed using custom made Taqman gene expression arrays (ABI). The data analysiswas done using the SDS RQmanager 2.1 and Realtime Statminer software programs. Results:Comparative analysis of the miRNA profiles of duodenal biopsies from patients with andwithout pancreatic cancer revealed a significant modulation of 18 miRNAs out of which10 were significantly upregulated and 8 were downregulated. Importantly several of thedysregulated miRNAs have been reported in pancreatic cancer. In the gene expressionanalysis, 14 biomarkers were found to be significantly upregulated (Table.1). The duodenalgenetic biomarkers showed impressive diagnostic performance with AUROC >0.8. Conclu-sion: We provide, for the first time, powerful genetic and epigenetic evidence that theduodenum is a site of extended field carcinogenesis of pancreatic cancer. This may openup novel diagnostic and screening approaches that do not necessitate direct instrumentationof the pancreatic duct. Future studies will be designed to optimize the biomarker panel(genomic and/or microRNA) and elucidate the biological implicationsTable1: Significantly modulated genetic biomarkers in duodenal mucosa

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