AGA Abstracts 761 Weight Loss in Obesity Reduces Colorectal Inflammation With Upregulation of FOXP3 Regulatory T Cells Swaroop Pendyala, Michaela M. Ennis,Judilyn Fuentes-Duculan, Peter R. Holt Introduction: In obesity,adipose tissue shows evidence of inflammation with increased numbersof macrophages and T cellsbut reduced Forkhead box protein 3(FOXP3) T regulatory cells. Aim:To evaluate the effects of diet-induced weight loss on inflammatory markers and infiltrating cell numbers in colorectal mucosal biopsies of obese women. Methods: 10 healthy pre-menopausal obese women (mean age 43 ± 8 years) were fed a very low calorie diet (VCLD) providing approximately 800 kcals/day in a closely monitored inpatient dietary intervention study to achieve a 10% weight loss. At baseline and after diet- induced weight loss, serum and mucosal cytokines were measured by human proinflammat- ory 9-plex assay (MSD). Changes in mucosal macrophage, total T cell, regulatory T cell and dendritic subtype cell numbers were determined using CD163, CD3, FOXP3,Langerin, BDCA1 and BDCA2 antibodies. Results: The average prestudy weight of the subjects was 95.9 ± 12.1 kg, BMI was 35 ± 3 kg/m 2 and they lost 10.1 ± 1.4 % of their initial weight consuming the VLCD diet in a mean of6.5 weeks. Dietinduced weight loss decreased serum cytokines significantly TNF by 14% (p<0.05),IL-8 by 30% ( p<0.05), MCP-1 by 9.4% (p<0.1). Mucosal cytokines also fell profoundly with TNF- α by 57% (p<0.05), IL-6 by 45% (p=0.06), IL-8 by 44% ( p<0.05), IL-1 β by 38%( p<0.05) and MCP-1 by 24% (p<0.05). By immunohistochemistry diet induced weight loss reduced macrophage (CD163) numbers by 42% (p=0.02), T cells (CD3) by 28.3%(p=0.007). But T reg cells(FOXP3) increased by 25.7% (p=0.02). There were no significant changes in dendritic cell subtype numbers with weight loss. Summary: Diet induced weight loss in obese women resulted in distinct reduc- tions in circulating and mucosal cytokines. Weight loss also reduced mucosal macrophages and total T cells,but was accompanied by an increase in FOXP3 T reg cells. Conclusions: Obesity is associated with fewer FOXP3 T reg cells in the colorectal epithelium which may contribute to the inflammation in the mucosa. This is reversible by diet-induced weight loss lowering inflammation and thus the risk of colorectal neoplasia 762 Novel Hydrogen Peroxide (H2O2) Sensing Probe for In Vivo Cellular Imaging in Colorectal Cancer VaniJ. Konda,Bryan C. Dickinson, Urszula Dougherty, Reba Mustafi, Alessandro Fichera, Irving Waxman, Tijana Rajh, Christopher J. Chang,Marc Bissonnette Background: Although hydrogen peroxide (H2O2) plays critical roles in cell signaling, In Vivo assessment of H2O2 by traditional laboratory methods is difficult. Recent advances in H2O2 sensitive and selective fluorescent probes have made possible detection of this labile EGFR second messenger In Vivo. The aim of this study was to detect In Vitro EGF-induced H2O2 in cell culture and In Vivo generation of H2O2 in a murine model of colon cancer using confocal laser scanning microscopy and recently developed boronate-based H2O2 sensitive probes. Methods: For In Vitro studies, three H202 sensing probes, Peroxy Yellow 1 (PY1),Peroxy Orange 1 (PO1) and Peroxyfluor-6 acetoxymethyl ester (PF6-AM) were studied in cell culture in HCT116 colon cancer cells. Cells were pre-incubated with the probe for 20 min in a 6-well plate that was then positioned on the stage of a confocal spinning disk laser-scanning microscope. EGF (100 ng/ml) was added and images captured every 30 sec for 30 min. For In Vivo studies, tumors were induced by treating C57Bl6 mice with azoxymethane (AOM, 10 mg/kg body wt) x 2 wks followed by 5 days dextran sulfate sodium (DSS). DSS cycle was repeated twice and mice imaged 22 wks after 1st AOM. A confocal laser scanning unit with a miniprobe (Cellvizio Laser Scanning unit and UHD probe,Mauna Kea Technologies, Paris) was used for In Vivo imaging. Prior to imaging, mice were pre-treated with 50 mM sodium peroxovanadate i.p. to inhibitendogenous phosphatases. Hydrogen peroxide sensing probes were administered by tail vein injection. To image tumors In Vivo we used an open laparotomy approach to identify tumors from the serosal side and then position the intraluminal CLE probe directly on the tumor. Results: In Vitro HCT116 cell studies: The PF6-AM signal increased in the cytoplasm from baseline (~15,000 to ~30,000 within 20 min following EGF stimulation and then remained stable for up to 45 min. The PY1 signal increased from baseline (~9,000) to ~20,000 with a similar kinetic pattern. Fluorescence of the PO1 probe did not increase following EGF addition. In Vivo mouse tumor studies (n=2): Both intravenous PF6-AM and PY1 fluoresced within tumors as detected using a confocal laser scanning endomicroscopy probe. Signals were heterogeneous within tumors and absent in normal-appearing adjacent mucosa. The back- ground was brighter with the PF6-AM probe, whereas more tumor structure could be appreciated with the PY1 probe. Conclusions: The role ofsecond messenger hydrogen peroxide has been challenging to determine in colorectal tumorigenesis. Recent advances in H2O2 sensitive fluorescent probes now provide powerful new tools to dissect the role of H2O2 in signaling in colonic tumorigenesis. Among the family of boronate-containing H2O2 sensing probes tested here, PY1 may be most suitable for In Vivo imaging of colorec- taltumors. 763 Human and Mouse Gastrointestinal Tumor Distribution is Selected According to a Basal Wnt Signalling Gradient Pedro Rodenas-Cuadrado, Kimberley Howarth, Annabelle Lewis, Sreelakshmi Mallappa, Rosemary Jeffery, Manuel Rodriguez-Justo, Susan K. Clark,James E. East,Simon Travis, Ian Tomlinson, Simon Leedham Introduction: It is established that Adenomatous polyposiscoli (APC)mutations occur non-randomly with respect to each other, resulting in characteristic genotype-phenotype correlations in colorectal cancer (CRC). We have recently compared two mouse polyposis models (ApcMin and Apc1322T mice) to demonstrate that ‘just-right’ levels of Wnt signaling underpin the severity and distribution of lesions in mouse intestinal neoplasia. We have also shown that human APC mutation spectra varies according to the anatomical distribution of colorectal lesions. We hypothesise that an intestinal gradient of basal Wnt signaling and S-128 AGA Abstracts stem cell number may select for optimal mutation spectra and influence gastrointestinal tumordistribution Methods: Wnt targetgene activity in individual smallintestinal and colonic whole mount crypts was assessed using qRT-PCR. Stem cell number was counted using in-situ hybridization for Lgr5. The regional intestinal effect of increased Wnt signali was examined in the mouse using a stabilized beta catenin transgenic model (Villin-CreE Catnb lox(ex3)/lox(ex3)) and the human by comparing upper gastrointestinal (UGI) and colonic APC mutation spectra in FAP and sporadic CRC patients. Results: Wnt target gene activity and stem cell number were greatest in the proximal small intestine (SI) decreasing steadily through to the colon. The BMP2 gradient was inversely correlated. The stabilized beta catenin transgenic mice had a striking phenotype with lesion distribution mirroring this gradient - thus the proximal SI was carpeted with dysplastic lesions, whereas the col was polyp free. However stem cell number was increased in the transgenic mouse colon. Human FAP associated UGI APC mutations retained more beta-catenin binding 20 amino acid repeats (AAR) than the same patient's colonic lesions. Sporadic CRC lesion distribut tended to vary according to the number of 20AAR's retained with left sided lesions requir fewer 20AAR's than right sided. Conclusion: The ‘just right’ hypothesis proposes a threshold of wnt signaling should be reached but not exceeded in order for polyp formation to occu We have demonstrated a physiological SI to colonic gradient of wntsignaland stem cell number. Thus the wntsignalthreshold gap varies throughout the intestinal tract.Beta- catenin stabilization in the mouse provides sufficient wntsignalperturbation to initiate tumour formation in the small intestine but is insufficient to initiate dysplasia in the colon despite increasing the number of intestinal stem cells. The physiological wnt gradient de mines the distribution of sporadic and polyposis-associated tumours by selecting for the optimal mutation spectra in each lesion- thus left sided lesions select for common mutat that provide a greater wnt perturbation than their more proximal counterparts. 764 Gender Based Tumor Characterization in the Carcinogen (Azoxymethane) Treated and Germ Line [APC Mutation, Polyposis in Rat (Pirc)] Models of Colorectal Carcinogenesis Mart DeLaCruz, Ramesh K. Wali,Dhananjay Kunte, Ashish K.Tiwari,Tina P. Gibson, Jeffrey T. Brasky, Preethi Subramanian, Yolanda Stypula, Hemant K. Roy Background: There are significant differences in location, MSI-status, onset and prognosis of colorectal cancers between men and women. We have recently noted that these differ may have important clinical ramifications but have been heretofore underappreciated (JA 2009).Since the biological differences are difficult to elucidate from epidemiology, animal models are of importance with the current mainstay being the azoxymethane (AOM)-trea ratand APC mutated MIN mouse. Unfortunately, the MIN mouse develops mainly small boweladenomas (<5% in colon). Recently, a germline APC mutated rat modelhas been developed, the polyposis in rat colon (Pirc) rat (PNAS 2007), which develops more colonic lesions. We,in the current study,have demonstrated the role of gender in the colorectal carcinogenesis initiation→progression by using AOM treated and Pirc F344 rat modelof carcinogenesis. Methods: Age-matched male (n=14) and female (n=15) F344 rats were randomized to either AOM (15mg/kg, two injections) or saline injections. Age-matched m (n=6) and female (n=6) Pirc rats (with respected controls) were housed for ~30 weeks b euthanization. The development of lesions in colon was monitored through frequent In V colonoscopic evaluation at different time points in both AOM and Pirc rats. Results: In V colonoscopy revealed colonic lesions at16 weeks and 28 weeks post AOM treatment; and at the age 16 weeks and 28 weeks in Pirc rats in males and females respectively, indicat late initiation in females. Ultimately, there was increased incidence of tumors in AOM tre male rats (2.0+/-1.67; 8 out of 9 rats developed tumors) as compared to females (2+/-1. only 3 out of 10 developed tumors). As expected, all the Pirc rats developed tumors due to presence of germ line mutation. However, male Pirc rats developed significantly higher number oftumors (9.83+/-1.5) as compared to females (1.66+/-0.66), underscoring the gender differences associated with the progression. Regardless of gender, majority of tum in AOM treated rats appeared in distal colon.Interestingly though, the pattern of tumor location (60% distal and 40% proximal) as well as increased tumor size despite less tumo burden in female Pirc rats as compared to male Pirc rats (90% distal and 10% proximal) reflected similarities between natural history ofcolon carcinogenesis in humans and Pirc rats. Conclusion: This is the first temporal phenotyping of Pirc rat model of colon carcin esis and more so from gender perspective. Furthermore, from a gender perspective, we demonstrate, herein,thatthe Pirc rats appears to be the superior model for investigating the biological basis ofgendereffectin colon carcinogenesis as manifested by proximal distribution and recapitulation of genetic events in colon carcinogenesis. 765 A Novel Potential of Cyclooxygenase-2 Specific Inhibitors to Prevent Intri Epigenetic Dysregulation and Aberrant Cancer Stem Cell Biology Ying Jin,Masahiko Tsujii, Eri Shiraishi, Satoshi Hiyama, Takuya Inoue, Shunsuke Yamamoto, Motohiko Kato, Akira Mukai, Takahiro Inoue, Yoshito Hayashi, Tomofumi Akasaka, JumpeiKondo,Takuya Yamada, Hideharu Ogiyama, Shinichiro Shinzaki, Kenji Watabe, Tsutomu Nishida, Sachiko Nakajima, HidekiIijima,Shusaku Tsutsui, Tetsuo Takehara, Norio Hayashi [Background] Recent evidence has suggested the existence of a small population of cancer stem cells with self-renewal, pluripotency chemo- and radio-resistant potentials. Reprogr ming provides multiple routes for achieving stemness. Epigenetic dysregulation is also reported to be important for carcinogenesis, and global hypomethylation and gene-speci CpG island hypermethylation are progressively accumulated during aging and directly co tribute to tumorigenesis. Aberrant DNA methylation is intricately involved in cancer stem cell biology; hypomethylation is shown to be related to reprogramming and DNA methylt ferases (DNMTs) are indispensable for stem cell maintenance. Cyclooxygenase-2 (COX-2) has been revealed to play an important role in carcinogenesis. We have reported that CO 2 has an effect on promoter methylation status and cancer stem cell biology through PG production in colon cancer cells. However, the precise mechanism is still obscure. The ai of this study is to investigate how COX-2 is involved in cancer stem cell biology. [Method