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1058 Crosstalk Between Commensal Bacteria and Colonic Epithelium in Gnotobiotic Rats: A Key Role of Acetate
Methods: We genetically modified human effector T cells to express an antibody-based chimeric receptor (CAR) directed against Her2/neu as well as CD24, one of the putative PAC cancer stem cell antigens. The antitumor reactivity of the engineered redirected Tlymphocytes ("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 a target. The Capan-1 was grafted either subcutaneously or orthotopically into the murine pancreas 21 to 28 days before T-bodies treatment. Bioluminescence imaging and measurements were used for the assessment of the anti-tumoral and anti-metastatic effect of the Tbodies treatment. Results: Both CD24 and Her2/neu T-bodies underwent specific activation and cytolysed PAC cells In Vitro. Intra-tumoral injection of T-bodies, a realistic procedure in patients, completely eliminated the tumors in most of the treated animals. Systemic Tbody administration effectively reduced tumor size and prolonged the survival of mice with orthotopically transplanted PAC tumors. More than 50% of animals receiving a single split dose of human Her2/neu or CD24 T-bodies appeared disease-free for more than 2 months after 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 of most of the primary PAC xenografts as well as of metastases in the liver and draining lymph nodes. Conclusions: Our results suggest CAR-redirected lymphocytes ("T-bodies") as an efficient cell-therapy modality for intra-tumoral and systemic treatment of localized and disseminated pancreatic adenocarcinoma.
Crosstalk Between Commensal Bacteria and Colonic Epithelium in Gnotobiotic Rats: A Key Role of Acetate Laura Wrzosek, Sylvie Miquel, Marie-Louise Noordine, Stephan Bouet, Chantal Bridonneau, Catherine Philippe, Véronique Robert, Claire Cherbuy, Philippe Langella, Muriel Thomas The gastrointestinal tract (GIT) of mammals is colonized by a community of micro-organisms reaching levels as high as 1011 bacteria/g of contents in the colon. This microbiota is involved in intestinal homeostasis and epithelium structure through the control of proliferation and differentiation processes (Cherbuy et al., 2010). Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, dominant members of microbiota, are commensal model bacteria to study host-microbiota interactions. The objective of our work was to understand the effects of these two bacteria on colonic epithelium homeostasis in gnotobiotic rats and underlying this 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. Colonic epithelium morphology, crypt depth, cell cycle-related proteins and goblet cells markers were 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 after inoculation. In Ino-Bt rats, no effect was observed neither on crypt depth nor on proliferation markers whereas Krüppel Like Factor 4 protein (KLF4), a transcription factor involved in goblet 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 the colon of Ino-Bt compared to GF rats was observed. In Ino-Bt+Fp rats, there was no induction of KLF4 protein, suggesting that the presence of F. prausnitzii impaired the mucus-stimulatory effect triggered by B. thetaiotaomicron. Short Chain Fatty Acids (SCFA) composition was determined 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 of propionate (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 to Ino-Bt rats (3.0 μmol/g). Using HT29-MTX cells, a cell line producing mucus, we showed that acetate was able to increase significantly KLF4 protein whereas butyrate and propionate have no effect on KLF4 level. All of these results suggested that acetate, a bacterial metabolite produced by B. thetaiotaomicron, could mediate a bacterial signal to promote goblet cells differentiation pathway in the GIT of mono-associated rats. In Crohn's disease patients, a diminished prevalence of F. prausnitzii (Martinez-Medina et al., 2006) and an increase of mucus production (Gersemann et al., 2009) have been often reported. Our observations in gnotobiotic rats are coherent with reports made in patients and may provide mechanistic clues to understand the pathophysiology of inflammatory bowel diseases.
1061 Novel Genetic/MicroRNA Evidence That the Microscopically Normal Duodenal Mucosa is Encompassed by Pancreatic Cancer Extended Field Carcinogenesis Dhananjay Kunte, Ramesh K. Wali, Randall Brand, Shailesh Bajaj, Mart DeLaCruz, Amir C. 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 clinically insidious and difficult/dangerous to access with ERCP having significant complications (i.e. pancreatitis). One increasingly common strategy is exploitation of field carcinogenesis. In the colon, the rectum is a well established site for field effect. However, there is evidence that field carcinogenesis may occur outside the organ. For instance, in lung cancer the buccal mucosa is in the tobacco smoke field of injury (Steiling et al, Can Prev Res 2008). For pancreatic cancer, there is clear evidence of diffuse ductal dysplasia (PanIN). There has been some emerging data that the duodenum may represent a field effect site (possibly secondary to pancreatic juices). Indeed, our group has noted that via optical techniques, there are micro-architectural alterations in the peri-ampullary duodenum (reviewed in Gastro 2011). Since biological corroboration has been lacking, we evaluated duodenal microRNA and gene expression profiles among the pancreatic cancer patients and matched controls. Methods: Patients: 32 patients (16 cancer, 16 age/gender matched controls) had 2 peri-ampullary biopsies with RNA isolated with Ribopure kit (Ambion). The total RNA was then reverse transcribed 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 expression studies, RNA was reverse transcribed using High Capacity Reverse Transcription kit (ABI) and preamplified using custom pre-amplification primers and then the pre-amplified cDNA was analyzed using custom made Taqman gene expression arrays (ABI). The data analysis was done using the SDS RQ manager 2.1 and Realtime Statminer software programs. Results: Comparative analysis of the miRNA profiles of duodenal biopsies from patients with and without pancreatic cancer revealed a significant modulation of 18 miRNAs out of which 10 were significantly upregulated and 8 were downregulated. Importantly several of the dysregulated miRNAs have been reported in pancreatic cancer. In the gene expression analysis, 14 biomarkers were found to be significantly upregulated (Table.1). The duodenal genetic biomarkers showed impressive diagnostic performance with AUROC >0.8. Conclusion: We provide, for the first time, powerful genetic and epigenetic evidence that the duodenum is a site of extended field carcinogenesis of pancreatic cancer. This may open up novel diagnostic and screening approaches that do not necessitate direct instrumentation of the pancreatic duct. Future studies will be designed to optimize the biomarker panel (genomic and/or microRNA) and elucidate the biological implications Table1: Significantly modulated genetic biomarkers in duodenal mucosa
1059 Negative Crosstalk Between the Extracellular Calcium Receptor and β-Catenin in Colonic Epithelium Osvaldo Rey, Wenhan Chang, Daniel Bikle, Nora Rozengurt, Enrique Rozengurt Background: Signaling pathways activated by G protein-coupled receptors (GPCRs) expressed in the gastrointestinal (GI) tract play a critical role in the regulation of multiple functions of the digestive system, including cell proliferation, differentiation, inflammation and promotion of colorectal cancer (CRC). The extracellular Ca2+-sensing receptor (CaSR), a member of the C family of GPCRs, is expressed in many tissues and organs not directly involved in the control of Ca2+ homeostasis, including both surface and crypt epithelial cells in rodent and human colons. Interestingly, the expression of the CaSR is greatly reduced or completely lost in CRC again suggesting that signaling pathways activated by this receptor negatively control cellular proliferation and CRC development. However, there is no definitive evidence for a physiological role of the CaSR in the regulation of normal and/or abnormal intestinal epithelial cell proliferation In Vivo. Results: In order to examine the role of the CaSR in colonic epithelial cell proliferation, we used a recently developed Casr intestinal-specific knock out mice model. Our results demonstrate that genetic ablation of this receptor led to hyperproliferation of colonic epithelial cells (Ki67 labeling), expansion of the proliferative zone, increase in the size of colonic crypts (length and number of cells/crypt) and enhanced β-catenin nuclear localization, a regulator of genes that control intestinal cell proliferation and differentiation. To elucidate the mechanism(s) by which CaSR inhibits colonic cell proliferation, we examined multi-site phosphorylation of β-catenin, a post-translational modification that plays a critical role in its degradation, nuclear translocation and transcriptional activity. We demonstrate that stimulation of the CaSR by either extracellular Ca2+ (5 mM) 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, we detected by immunofluorescence a reduced nuclear reactivity of the phosphoSer552 antibody as well as a marked redistribution of β-catenin to the plasma membrane. These changes in phosphorylation and intracellular distribution were associated with a 40% downregulation of β-catenin mediated transcriptional activation (TCF/LEF-1 luciferase reporter TOP-Flash assay). Conclusion: These studies demonstrate that signaling pathways emanating from the CaSR control colonic epithelial cells proliferation In Vivo and suggest that the mechanism involves regulation of β-catenin phosphorylation. 1060 Redirecting Effector Lymphocytes for Adoptive Cell Therapy of Pancreatic Adenocarcinoma in Mice Amit Maliar, Charlotte Servais, Tova Waks, Zelig Eshhar Background & Aims: Pancreatic adenocarcinoma (PAC) is a common malignancy that is often diagnosed at an advanced and inoperable stage; it is usually incurable, and the standard systemic treatments are generally ineffective for metastatic disease. We have investigated, in an animal model, whether adoptive cell therapy (ACT) using redirected, tumor-specific T cells could be applied for the treatment of primary and metastatic pancreatic adenocarcinoma.
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AGA Abstracts
AGA Abstracts
1058
Crosstalk Between Commensal Bacteria and Colonic Epithelium in Gnotobiotic Rats: A Key Role of Acetate Laura Wrzosek, Sylvie Miquel, Marie-Louise Noordine, Stephan Bouet, Chantal Bridonneau, Catherine Philippe, Véronique Robert, Claire Cherbuy, Philippe Langella, Muriel Thomas The gastrointestinal tract (GIT) of mammals is colonized by a community of micro-organisms reaching levels as high as 1011 bacteria/g of contents in the colon. This microbiota is involved in intestinal homeostasis and epithelium structure through the control of proliferation and differentiation processes (Cherbuy et al., 2010). Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, dominant members of microbiota, are commensal model bacteria to study host-microbiota interactions. The objective of our work was to understand the effects of these two bacteria on colonic epithelium homeostasis in gnotobiotic rats and underlying this 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. Colonic epithelium morphology, crypt depth, cell cycle-related proteins and goblet cells markers were 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 after inoculation. In Ino-Bt rats, no effect was observed neither on crypt depth nor on proliferation markers whereas Krüppel Like Factor 4 protein (KLF4), a transcription factor involved in goblet 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 the colon of Ino-Bt compared to GF rats was observed. In Ino-Bt+Fp rats, there was no induction of KLF4 protein, suggesting that the presence of F. prausnitzii impaired the mucus-stimulatory effect triggered by B. thetaiotaomicron. Short Chain Fatty Acids (SCFA) composition was determined 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 of propionate (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 to Ino-Bt rats (3.0 μmol/g). Using HT29-MTX cells, a cell line producing mucus, we showed that acetate was able to increase significantly KLF4 protein whereas butyrate and propionate have no effect on KLF4 level. All of these results suggested that acetate, a bacterial metabolite produced by B. thetaiotaomicron, could mediate a bacterial signal to promote goblet cells differentiation pathway in the GIT of mono-associated rats. In Crohn's disease patients, a diminished prevalence of F. prausnitzii (Martinez-Medina et al., 2006) and an increase of mucus production (Gersemann et al., 2009) have been often reported. Our observations in gnotobiotic rats are coherent with reports made in patients and may provide mechanistic clues to understand the pathophysiology of inflammatory bowel diseases.
1061 Novel Genetic/MicroRNA Evidence That the Microscopically Normal Duodenal Mucosa is Encompassed by Pancreatic Cancer Extended Field Carcinogenesis Dhananjay Kunte, Ramesh K. Wali, Randall Brand, Shailesh Bajaj, Mart DeLaCruz, Amir C. 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 clinically insidious and difficult/dangerous to access with ERCP having significant complications (i.e. pancreatitis). One increasingly common strategy is exploitation of field carcinogenesis. In the colon, the rectum is a well established site for field effect. However, there is evidence that field carcinogenesis may occur outside the organ. For instance, in lung cancer the buccal mucosa is in the tobacco smoke field of injury (Steiling et al, Can Prev Res 2008). For pancreatic cancer, there is clear evidence of diffuse ductal dysplasia (PanIN). There has been some emerging data that the duodenum may represent a field effect site (possibly secondary to pancreatic juices). Indeed, our group has noted that via optical techniques, there are micro-architectural alterations in the peri-ampullary duodenum (reviewed in Gastro 2011). Since biological corroboration has been lacking, we evaluated duodenal microRNA and gene expression profiles among the pancreatic cancer patients and matched controls. Methods: Patients: 32 patients (16 cancer, 16 age/gender matched controls) had 2 peri-ampullary biopsies with RNA isolated with Ribopure kit (Ambion). The total RNA was then reverse transcribed 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 expression studies, RNA was reverse transcribed using High Capacity Reverse Transcription kit (ABI) and preamplified using custom pre-amplification primers and then the pre-amplified cDNA was analyzed using custom made Taqman gene expression arrays (ABI). The data analysis was done using the SDS RQ manager 2.1 and Realtime Statminer software programs. Results: Comparative analysis of the miRNA profiles of duodenal biopsies from patients with and without pancreatic cancer revealed a significant modulation of 18 miRNAs out of which 10 were significantly upregulated and 8 were downregulated. Importantly several of the dysregulated miRNAs have been reported in pancreatic cancer. In the gene expression analysis, 14 biomarkers were found to be significantly upregulated (Table.1). The duodenal genetic biomarkers showed impressive diagnostic performance with AUROC >0.8. Conclusion: We provide, for the first time, powerful genetic and epigenetic evidence that the duodenum is a site of extended field carcinogenesis of pancreatic cancer. This may open up novel diagnostic and screening approaches that do not necessitate direct instrumentation of the pancreatic duct. Future studies will be designed to optimize the biomarker panel (genomic and/or microRNA) and elucidate the biological implications Table1: Significantly modulated genetic biomarkers in duodenal mucosa
1059 Negative Crosstalk Between the Extracellular Calcium Receptor and β-Catenin in Colonic Epithelium Osvaldo Rey, Wenhan Chang, Daniel Bikle, Nora Rozengurt, Enrique Rozengurt Background: Signaling pathways activated by G protein-coupled receptors (GPCRs) expressed in the gastrointestinal (GI) tract play a critical role in the regulation of multiple functions of the digestive system, including cell proliferation, differentiation, inflammation and promotion of colorectal cancer (CRC). The extracellular Ca2+-sensing receptor (CaSR), a member of the C family of GPCRs, is expressed in many tissues and organs not directly involved in the control of Ca2+ homeostasis, including both surface and crypt epithelial cells in rodent and human colons. Interestingly, the expression of the CaSR is greatly reduced or completely lost in CRC again suggesting that signaling pathways activated by this receptor negatively control cellular proliferation and CRC development. However, there is no definitive evidence for a physiological role of the CaSR in the regulation of normal and/or abnormal intestinal epithelial cell proliferation In Vivo. Results: In order to examine the role of the CaSR in colonic epithelial cell proliferation, we used a recently developed Casr intestinal-specific knock out mice model. Our results demonstrate that genetic ablation of this receptor led to hyperproliferation of colonic epithelial cells (Ki67 labeling), expansion of the proliferative zone, increase in the size of colonic crypts (length and number of cells/crypt) and enhanced β-catenin nuclear localization, a regulator of genes that control intestinal cell proliferation and differentiation. To elucidate the mechanism(s) by which CaSR inhibits colonic cell proliferation, we examined multi-site phosphorylation of β-catenin, a post-translational modification that plays a critical role in its degradation, nuclear translocation and transcriptional activity. We demonstrate that stimulation of the CaSR by either extracellular Ca2+ (5 mM) 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, we detected by immunofluorescence a reduced nuclear reactivity of the phosphoSer552 antibody as well as a marked redistribution of β-catenin to the plasma membrane. These changes in phosphorylation and intracellular distribution were associated with a 40% downregulation of β-catenin mediated transcriptional activation (TCF/LEF-1 luciferase reporter TOP-Flash assay). Conclusion: These studies demonstrate that signaling pathways emanating from the CaSR control colonic epithelial cells proliferation In Vivo and suggest that the mechanism involves regulation of β-catenin phosphorylation. 1060 Redirecting Effector Lymphocytes for Adoptive Cell Therapy of Pancreatic Adenocarcinoma in Mice Amit Maliar, Charlotte Servais, Tova Waks, Zelig Eshhar Background & Aims: Pancreatic adenocarcinoma (PAC) is a common malignancy that is often diagnosed at an advanced and inoperable stage; it is usually incurable, and the standard systemic treatments are generally ineffective for metastatic disease. We have investigated, in an animal model, whether adoptive cell therapy (ACT) using redirected, tumor-specific T cells could be applied for the treatment of primary and metastatic pancreatic adenocarcinoma.
S-187
AGA Abstracts
AGA Abstracts
1058