Healthy Colon Tissue Research Articles

Abstract A123: Modulation of immune cell trafficking into human colorectal cancer by gut microbiota

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related death. CRC infiltration by immune cells, including cytotoxic CD8+ T cells (CTLs), IFN-gamma-producing T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs) and CD16+ MPO+ neutrophils, is associated with favorable prognosis. However, chemokines driving these cell populations into the tumor site, their cellular sources and their microenvironmental triggers remain to be elucidated. We investigated the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis. Analysis of freshly excised specimens of CRC and adjacent healthy colonic tissues revealed that tumor infiltration by beneficial immune cells is associated with the expression of four main chemokine patterns: 1) CCL3, CCL5, CCL8, CXCL9, CXCL10, and CXCL12 for CTLs; 2) CCL5, CCL22, CXCL9 and CXCL12 correlating with CRC infiltration by Th1; 3) CCL22 and CXCL12 attracting Tregs; 4) CXCL2 and CXCL5, promoting chemotaxis of CD16+ MPO+ myeloid cells. Most of identified chemokines were found to be expressed, although at different levels, in primary CRC cells purified from tumor specimens and from xenografts generated upon injection of CRC cells in immunodeficient mice. Notably, chemokine expression levels in orthotopic xenografts, developed upon intracecal injection of tumor cells, were significantly higher as compared to those of subcutaneous tumors. Antibiotic treatment of tumor bearing mice drastically reduced chemokine expression in orthotopic xenografts, thus suggesting a role for commensal bacteria in chemokines induction in tumor cells. Importantly, human CRC samples characterized by high expression of chemokine and immune cell markers, displayed significantly higher bacterial loads, as compared to samples showing low chemokine expression and immune cell infiltration. Furthermore, bacterial loads significantly correlated with expression of CCL3 and CCL5, and Th1 infiltration. These data suggest that stimuli derived from gut flora components may trigger chemokine production in CRC tissues thus leading to tumor infiltration by beneficial immune cells. Note: This abstract was not presented at the conference. Citation Format: Eleonora Cremonesi, Francesca Amicarella, Jesus Francisco Glaus Garzon, Valeria Governa, Manuele Giuseppe Muraro, Valentina Mele, Elisabetta Padovan, Daniel Oertli, Paul Zajac, Giulio Cesare Spagnoli, Lubor Borsig, Giandomenica Iezzi. Modulation of immune cell trafficking into human colorectal cancer by gut microbiota [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A123.

Matricellular protein SPARCL1 regulates tumor microenvironment-dependent endothelial cell heterogeneity in colorectal carcinoma.

Different tumor microenvironments (TMEs) induce stromal cell plasticity that affects tumorigenesis. The impact of TME-dependent heterogeneity of tumor endothelial cells (TECs) on tumorigenesis is unclear. Here, we isolated pure TECs from human colorectal carcinomas (CRCs) that exhibited TMEs with either improved (Th1-TME CRCs) or worse clinical prognosis (control-TME CRCs). Transcriptome analyses identified markedly different gene clusters that reflected the tumorigenic and angiogenic activities of the respective TMEs. The gene encoding the matricellular protein SPARCL1 was most strongly upregulated in Th1-TME TECs. It was also highly expressed in ECs in healthy colon tissues and Th1-TME CRCs but low in control-TME CRCs. In vitro, SPARCL1 expression was induced in confluent, quiescent ECs and functionally contributed to EC quiescence by inhibiting proliferation, migration, and sprouting, whereas siRNA-mediated knockdown increased sprouting. In human CRC tissues and mouse models, vessels with SPARCL1 expression were larger and more densely covered by mural cells. SPARCL1 secretion from quiescent ECs inhibited mural cell migration, which likely led to stabilized mural cell coverage of mature vessels. Together, these findings demonstrate TME-dependent intertumoral TEC heterogeneity in CRC. They further indicate that TEC heterogeneity is regulated by SPARCL1, which promotes the cell quiescence and vessel homeostasis contributing to the favorable prognoses associated with Th1-TME CRCs.

Dielectric characterization of healthy and malignant colon tissues in the 0.5–18 GHz frequency band

Several reports over the last few decades have shown that the dielectric properties of healthy and malignant tissues of the same body organ usually show different values. However, no intensive dielectric studies of human colon tissue have been performed, despite colon cancer’s being one of the most common types of cancer in the world. In order to provide information regarding this matter, a dielectric characterization of healthy and malignant colon tissues is presented. Measurements are performed on ex vivo surgery samples obtained from 20 patients, using an open-ended coaxial probe in the 0.5–18 GHz frequency band. Results show that the dielectric constant of colon cancerous tissue is 8.8% higher than that of healthy tissues (p = 0.002). Besides, conductivity is about 10.6% higher, but in this case measurements do not have statistical significance (p = 0.038). Performing an analysis per patient, the differences in dielectric constant between healthy and malignant tissues appear systematically. Particularized results for specific frequencies (500 MHz, 900 MHz, 2.45 GHz, 5 GHz, 8.5 GHz and 15 GHz) are also reported. The findings have potential application in early-stage cancer detection and diagnosis, and can be useful in developing new tools for hyperthermia treatments as well as creating electromagnetic models of healthy and cancerous tissues.

Does the Diversity of the Microbiome Reflect Possible Colonic Polyps in a Multi-ethnic Population?

Introduction: Colorectal cancer (CRC) is the third most common cause of malignancy-related death in the USA. The etiology of colon cancer is multifactorial; however, linkage to alterations in the gut microbiome has been reported. Hypotheses for oncogenesis include the induction of a chronic inflammatory state, cell cycle dysregulation and exposure to carcinogenic compounds, any of which might reflect a disordered microbiome. In this pilot study, we aimed to assess colonic and fecal microbiota with respect to presence of colonic polyps in a multiethnic population. Methods: Asymptomatic patients at average risk for CRC referred for screening colonoscopy were prospectively enrolled and consented. A detailed history was obtained via a standardized validated questionnaire. Stool was collected prior to (baseline, pre-preparation) and 3 weeks after colonoscopy. Biopsies were obtained from normal-appearing sigmoid mucosa in all subjects and normal-appearing mucosa within 5 cm of any polyp. DNA was purified from stool and tissue using standard extraction methods and bacterial sequencing and taxa identified using high throughput (Ilumina) 16S rRNA gene (V4 region) sequencing and analysis using the Qiime pipeline. Results: Demographics are presented in Table 1. In the 17 non-polyp controls, α and β diversity were similar in the pre-colonoscopy and the follow-up samples, 3 weeks later, indicating that the microbiota had normalized after the procedure. Analysis of gut microbiota showed lower α-diversity at baseline in subjects with polyps compared to controls, by the PD- whole-tree (p=0.014) metric. In the second sample, after polyp removal, α-diversity trended toward normalization but differences were not significant (p=0.6). Healthy colonic tissue showed higher α-diversity in subjects with adenomatous polyps compared with those with hyperplastic or no polyps (p=0.025). In subjects with polyps, α-diversity was higher in healthy tissue compared to tissue obtained within proximity of a polyp (p=0.02). There were no significant differences in β-diversity between subjects with or without polyps at any time.Table 1: Demographics and clinical features of 34 study subjectsConclusion: In our population, the lowered α-diversity (richness) prior to colonoscopy in the subjects with polyps and in tissue near a polyp suggests that the microbiota changes might be secondary to the neoplastic lesion. Larger studies are needed to question this finding in other multi-ethnic populations. Supported in part by 1R24OD01A340-01 from NIH.

Quantitative analysis of mucosal oxygenation using ex vivo imaging of healthy and inflamed mammalian colon tissue.

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O2 consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O2 in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O2 sensitive probe Pt-Glc and high-resolution imaging of O2 concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50-60μm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O2 values in normal mouse tissue ranged between 5 and 35μM (4-28Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O2 consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.

A model of the spatial tumour heterogeneity in colorectal adenocarcinoma tissue.

BackgroundThere have been great advancements in the field of digital pathology. The surge in development of analytical methods for such data makes it crucial to develop benchmark synthetic datasets for objectively validating and comparing these methods. In addition, developing a spatial model of the tumour microenvironment can aid our understanding of the underpinning laws of tumour heterogeneity.ResultsWe propose a model of the healthy and cancerous colonic crypt microenvironment. Our model is designed to generate synthetic histology image data with parameters that allow control over cancer grade, cellularity, cell overlap ratio, image resolution, and objective level.ConclusionsTo the best of our knowledge, ours is the first model to simulate histology image data at sub-cellular level for healthy and cancerous colon tissue, where the cells have different compartments and are organised to mimic the microenvironment of tissue in situ rather than dispersed cells in a cultured environment. Qualitative and quantitative validation has been performed on the model results demonstrating good similarity to the real data. The simulated data could be used to validate techniques such as image restoration, cell and crypt segmentation, and cancer grading.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1126-2) contains supplementary material, which is available to authorized users.

Post-translational modifications of α-synuclein contribute to neurodegeneration in the colon of elderly individuals.

Synucleinopathies and abnormalities in the nerves of the enteric nervous system are hypothesized to be involved in age-associated motility disorders. The aim of the present study was to investigate the expression of various antigens, including α-synuclein (Syn) and its post-translational modified forms, in the human colon at various ages. In addition, the study aimed to correlate the expression of Syn with neurodegeneration. Immunohistochemistry was used to detect the expression of neurofilament (NF), Syn, as well as its nitrated (N) form in the healthy colonic tissue of 12 young (34.08±5.12 years), 10 middle-aged (51.80±3.52 years), and 11 elderly (75.82±7.70 years) individuals. To the best of our knowledge, the current study is the first to demonstrate the presence of N-Syn in the colonic tissue. N-Syn was identified in the upper layer of the mucosa and submucosa layer. Furthermore, Syn (wild-type) was present in the mucosa and submucosa. The number of NF-positive neurons in the submucosal layer declined significantly with age (P<0.01). In addition, Syn and N-Syn significantly increased during aging (P<0.01). Furthermore, a negative correlation was identified between neuron number and synucleinopathies, indicating the abnormal accumulation of both wild-type Syn and N-Syn in the mucosa, submucosa, muscle layer and myenteric plexus. The present study demonstrates that the Syn pathology may be linked to colic neuronal degeneration during normal aging, and this link may cause functional deficits.

Expression of enterotoxigenic Bacteroides fragilis and polyketide synthase gene-expressing Escherichia coli in colorectal adenoma patients

To investigate the distribution of various bacteria in adenoma tissue of colorectal adenoma (T/CRA), normal colonic mucosa tissue adjacent to the adenoma (N/CRA), and healthy colonic mucosa tissue (N/H) by comparing the number of total bacteria, Bacteroides fragilis (BF), enterotoxigenic Bacteroides fragilis (ETBF), polyketide synthase (pks) gene-expressing Escherichia coli(E.coli)(pks(+) E. coli)among the above 3 types of tissues. A total of 36 patients diagnosed with colorectal adenoma by colonoscopy and pathology in Department of Gastroenterology, Peking University People's Hospital from September 2011 to September 2013 were selected into this study. T/CRA and N/CRA tissues from the 36 patients and N/H tissues from 18 healthy controls were collected for DNA extraction. The number of total bacteria, BF, ETBF, pks(+) E. coli was detected by quantitative real time PCR, and their correlation with colorectal adenoma was analyzed. (1) The number of total bacteria decreased gradually from N/H, N/CRA, to T/CRA, with the median values being 3.18×10(8,) 1.57×10(8,) and 7.91×10(7) copies/g, respectively, and with significant difference among the three groups and between each two groups (all P<0.01). (2) The content of BF decreased gradually from N/H, N/CRA, to T/CRA, the median values being 6.03×10(5,) 4.28×10(4,) and 5.48×10(3) copies/g, respectively, and with significant difference among the three groups and between each two groups (all P<0.01). (3) The toxin content produced by ETBF increased from N/H, N/CRA, to T/CRA, the relative expression being 1.73±0.30, 6.15±1.52, and 8.54±1.80, respectively. Significant difference was found between the T/CRA and N/H tissue (P=0.003), but not between any other two groups. (4) The expression of clbB in pks(+) E.coli was highest in T/CRA colonic tissue (2.96±0.28), followed by the N/CRA (2.79±0.19) and N/H tissue (1.06±0.08). Significant difference was found between T/CRA and N/H tissues, as well as between N/CRA and N/H tissues (both P<0.001), but not between T/CRA and N/CRA tissues. The number of total bacteria is markedly reduced in the colonic mucosa of CRA patients compared to normal people, while the expressions of ETBF and pks(+) E.coli are significantly increased. Such changes in total bacterial, ETBF and pks(+) E.coli concentrations in colonic mucosa may be related to the tumorigenesis of colorectal adenoma.

Dysregulated Lysine Acetyltransferase 2B Promotes Inflammatory Bowel Disease Pathogenesis Through Transcriptional Repression of Interleukin-10.

Accumulating evidence supports epigenetic modifications in mediating intestinal immunity in inflammatory bowel disease [IBD] pathogenesis. This study aimed to identify key dysregulated epigenetic modulators and the molecular downstream pathways in IBD. Expression of 116 well-defined epigenetic modulators was profiled and validated in 96 intestinal tissues from patients with Crohn's disease [CD], ulcerative colitis [UC], and healthy controls using quantitative reverse transcriptase polymerase chain reaction [QRT-PCR], western blot, and immunohistochemistry. Dysregulation of histone modifications and IBD-related cytokines were examined by chromatin immunoprecipitation, luciferase activity, and gene expression analyses in normal colonic epithelial cell line, NCM460, upon small-molecule inhibition or RNA interference, followed by validation in primary colonic tissues. Targeted expression profiling uncovered seven differentially expressed epigenetic modulators, of which the down-regulation of lysine acetyltransferase 2B [KAT2B] mRNA and protein was the most significant and was consequently validated in inflamed CD and UC compared with healthy colonic tissues. KAT2B protein localised abundantly in nuclei of normal colonic epithelium but diminished in paired inflamed CD and UC tissues. Pharmacological inhibition of KAT2B by anacardic acid in NCM460 cells reduced the levels of histone H4 lysine 5 acetylation [H4K5ac] and interleukin-10 [IL-10] in a dose-dependent manner. Knockdown of KAT2B reduced the IL-10 promoter occupancy of KAT2B and H4K5ac, resulting in transcriptional silencing. IL-10 level was also diminished in inflamed IBD tissues. Our findings demonstrated a novel epigenetic mechanism of IL-10 dysregulation in IBD. Down-regulation of KAT2B may disrupt the innate and adaptive inflammatory responses due to the suppression of this crucial anti-inflammatory cytokine.

MiR-331-3p inhibits proliferation and promotes apoptosis by targeting HER2 through the PI3K/Akt and ERK1/2 pathways in colorectal cancer.

MicroRNAs (miRNAs) regulate cell proliferation, apoptosis and carcinogenesis by targeting related mRNAs in different types of cancer. miR-331-3p has been found to regulate the development and progression of various types of cancer cells. However, little research has been conducted on the role of miR-331-3p in colorectal cancer (CRC). The present study aimed to explore the function of miR-331-3p in CRC. We found that miR-331-3p was significantly downregulated in CRC tissues and cells compared to the level in healthy colon tissues and cells. Overexpression of miR-331-3p by transfection with pre‑miR-331-3p inhibited cell proliferation, promoted apoptosis and activated caspase-3. Furthermore, the protein expression level of apoptosis-related protein Bcl-2 was downregulated and Bax was upregulated by pre‑miR‑331-3p. Downregulation of the expression of miR-331-3p by transfection with AS-miR-331-3p had the opposite effect. Moreover, we found that HER2 was overexpressed in the CRC cell lines, and the expression level of HER2 was negatively regulated by miR‑331-3p. Additionally, knockdown of HER2 inhibited cell proliferation and phosphorylation of Akt and ERK1/2 induced by AS-miR-331-3p. Overall, we identified that miR‑331-3p is underexpressed in CRC and contributes to cell growth regulation by targeting HER2 through activating the PI3K/Akt and ERK1/2 signaling pathways.

DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer.

BackgroundColorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence.MethodsWhole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10–10 macrodissected and 5–5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed.ResultsA set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3) showed hypermethylation and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g. BCL2, MAL, PTGS2). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence.ConclusionHypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1687-x) contains supplementary material, which is available to authorized users.

Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding Km values were measured as 93.6±7.7µM for CRC cells and 84.9±9.9µM for nearby tissue; both these apparent Km (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34µM).

CD163L1 and CLEC5A discriminate subsets of human resident and inflammatory macrophages in vivo

Macrophages (Mϕ) can be differentiated and polarized in vitro from human CD14(+) monocytes under the influence of GM-CSF (GM-Mϕ) and M-CSF (M-Mϕ). GM-Mϕs are proinflammatory and M-Mϕs have an anti-inflammatory phenotype. We found selective expression of the lectin C-type lectin domain family 5 member A (CLEC5A) transcripts in GM-Mϕs and the scavenger receptor CD163 molecule-like 1 (CD163L1) in M-Mϕs by microarray assay. In vitro, CD163L1 expression was induced by IL-10 and M-CSF and CLEC5A by inflammatory cytokines and cell adherence. In secondary lymphoid organs, their respective expression was restricted to CD68(+)/CD163(+) Mϕs that preferentially produced either TNF (CLEC5A(+)) or IL-10 (CD163L1(+)). Mϕs from healthy liver and colon tissue were mostly CD163L1(+), and CLEC5A(+) cells were scarce. In contrast, CLEC5A(+) Mϕs were abundant in the intestinal lamina propria from patients with inflammatory bowel disease (IBD), with higher numbers of CLEC5A(+)CD163L1(+) found compared with those in secondary lymphoid organs. CLEC5A(+) cells were CD14(+)CD209(-)CD11b(+)CD11c(+)TNF(+)IL-10(+), and single positive CD163L1(+) cells were CD14(-)CD209(+)CD11b(-)CD11c(-)TNF(-)IL-10(+) in healthy donors and had lost the ability to produce IL-10 and to express CD209 in those with IBD. In melanomas, CLEC5A(+) tumor-associated Mϕs (TAMs) were not detected in 42% of the cases evaluated, but CD163L1(+) TAMs were found in 100%. Similar to IBD, CD163L1(+) TAMs expressed high levels of CD209 and produced significant amounts of IL-10, and CLEC5A(+) TAMs were CD14(hi) and produced enhanced levels of TNF in metastases. Overall, these results suggest that CD163L1 expression is associated with tissue-resident Mϕs with an anti-inflammatory or anergic phenotype and that CLEC5A(+) Mϕs exhibit TNF-producing ability and might display a proinflammatory effect.

MicroRNA-214 functions as a tumor suppressor in human colon cancer via the suppression of ADP-ribosylation factor-like protein 2.

microRNAs (miRNAs/miRs) are a conserved class of endogenous, short non-coding RNAs that post-transcriptionally regulate the expression of genes involved in diverse cellular processes. miR-214 has been reported to be associated with several cancers, including human colon cancer. However, the function of miR-214 in colon cancer development is poorly understood. In the current study, miR-214 was demonstrated to be downregulated in colon cancer tissues compared with healthy colon tissues. Functional studies showed that miR-214 overexpression results in the inhibition of cell viability, colony formation and proliferation, and the induction of cell apoptosis. ADP-ribosylation factor-like protein 2 (ARL2) is predicted to be a target candidate of miR-214. A luciferase reporter assay, western blot analysis and quantitative polymerase chain reaction were performed, which revealed that miR-214 negatively regulates ARL2 expression by targeting its 3′ untranslated region directly. In conclusion, the results of the present study revealed that miR-214 suppresses colon cancer cell growth via the suppression of ARL2, and indicated that miR-214 may present a significant potential therapeutic target for colon cancer.

Abstract 1603: Estrogen metabolites and colorectal cancer risk in postmenopausal women in the Breast and Bone Follow-up to the Fracture Intervention Trial (B∼FIT)

Abstract Background: A role for estrogen in colon carcinogenesis is suggested by the lower incidence of colorectal cancer (CRC) in women than men; the protective effect of exogenous hormones on risk; and reduced levels of estrogen receptor beta (ERβ) expression, the predominant ER in the colon, in cancerous compared to healthy colonic tissue. Nonetheless, the few cohort studies of endogenous hormones have not found reduced risks associated with circulating estradiol or estrone levels in postmenopausal women. We hypothesized that specific metabolites in the estrogen metabolic pathway may preferentially bind to and activate colonic ERβ, and analyzed a panel of 15 estrogen metabolites (EM), including estradiol and estrone, in a case-cohort study of CRC. Methods: We prospectively followed 15,595 women enrolled in B∼FIT (Breast and Bone Follow-up to the Fracture Intervention Trial (FIT)) who had completed questionnaires and donated blood between 1992 and 1993 for incident CRC through December 2004. Using liquid chromatography-tandem mass spectrometry, EM were measured in serum from 197 CRC cases and a subcohort of 491 women who were not using exogenous hormones at the time of blood draw. EM were examined individually, grouped by pathway (hydroxylation at the C-2, C-4, or C-16 positions of the steroid ring), and by ratios of the groupings. Cox proportional hazard regression models adjusted for clinical site and age (grouped in ten year intervals) were used to calculate quartile-specific hazard ratios (HRs) and 95% confidence intervals (CIs). Results: Significant risks were not observed for individual EM, and HR for quartiles of total EM (sum of all EM) were elevated but not significant (HRQ4 v Q1=1.23, 95% CI=0.8-2.0, ptrend=0.53). No associations were seen for estrone (HRQ4 v Q1=1.05, 95% CI=0.6-1.7) or estradiol (HRQ4 v Q1=0.90. 95% CI=0.5-1.5). When represented as a ratio to the parent estrogens (estrone and estradiol), risks of CRC for women with higher levels of 2-pathway EM or 4-pathway EM were reduced in each quartile, but neither HR nor trends were significant (ratio 2-pathway: parent, HRQ4 v Q1=0.88, 95 CI=0.6-1.4 ptrend=0.56; ratio 4-pathway: parent, HRQ4 v Q1=0.92, 95% CI=0.6, 1.5 ptrend=0.46). Conclusions: Levels of circulating estrone and estradiol and their metabolites are not associated with CRC risk in the B∼FIT cohort. Citation Format: Roni T. Falk, Cher M. Dallal, James V. Lacey, Douglas C. Bauer, Diana SM Buist, Jane A. Cauley, Trisha F. Hue, Andrea LaCroix, Jeffrey A. Tice, Ruth M. Pfeiffer, Xia Xu, Timothy D. Veenstra, Louise A. Brinton. Estrogen metabolites and colorectal cancer risk in postmenopausal women in the Breast and Bone Follow-up to the Fracture Intervention Trial (B∼FIT). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1603. doi:10.1158/1538-7445.AM2014-1603

Abstract 422: DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer

Abstract Background: It is well known that beside genetic factors such as mutations, epigenetic mechanisms can also contribute to colorectal cancer formation and progression. Although several genes influenced by DNA methylation and miRNA expression alteration have been identified, our knowledge of the background of gene expression alterations during CRC development remains incomplete. Aims: Our aims were to identify DNA methylation markers and miRNAs playing role in left-sided CRC development on the basis of gene expression alterations along the adenoma-carcinoma formation. Materials &amp; methods: Whole genome expression profiling was performed by using HGU133 Plus 2.0 microarrays (Affymetrix) on healthy colonic (n=49), colorectal adenoma (n=49) and left-sided CRC (n=49) biopsy samples and also on laser microdissected (LCM) epithelial and stromal cells from healthy (n=6) and CRC (n=6) samples. Methylation status of genes with gradually decreasing or increasing expression along the adenoma-carcinoma sequence were analyzed on macrodissected (n=10) and LCM (n=5) healthy colonic, adenomatous biopsy (n=10) and LCM (n=5), macrodissected (n=10) and LCM (n=5) left-sided colorectal cancer samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes with miRWALK algorithm, miRNA expression was analyzed on colorectal cancers (n=3), adenomas (n=3) and normal tissue adjacent to tumor (NAT)(n=3) samples using the Human Panel I + II with the miRCURY Universal RT microRNA PCR protocol (Exiqon). In order to investigate the potential impact of DNA methylation on the protein levels PTGDR and SFRP1 immunohistochemistry experiments were performed. Results: A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p≤0,01) in the biopsy samples along the adenoma-carcinoma sequence. COL1A2, SFRP2, SOCS3 showed hypermethylation and THBS2 showed hypomethylation both in the adenomas and tumor samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p&amp;lt;0,01) upregulated in the adenoma and tumor samples compared to the healthy colonic tissue controls that can potentially influence the expression of genes without remarkable DNA methylation alteration (e.g. BCL2, MAL, PTGS2). Decreasing protein levels of PTGDR and SFRP1 could be observed along the adenoma-carcinoma sequence. Conclusion: Genome-wide gene expression-based screening was found to be a suitable approach for the identification of genes, that can be potentially downregulated by DNA hypermethylation or miRNA upregulation. Hypermethylation of the selected markers (COL1A2, SFRP2, SOCS3) or miR21 upregulation might result in reduced expression and may contribute to the formation of colorectal cancer. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Peter Hollosi, Zsofia Brigitta Nagy, Barnabas Wichmann, Ilona Kovalszky, Zsolt Tulassay. DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2014-422

Orally administered liposomal formulations for colon targeted drug delivery.

Colon targeted drug delivery is an active area of research for diseases affecting the colon, as it shows promise in improving the efficacy of therapeutics and reducing systemic toxicity. Improved oral drug delivery design has unequivocally improved the bioavailability of drugs to the colon. The oral route of administration is the most common method of drug delivery. It is the preferred route of administration because of increased patient convenience and reduced invasiveness. Rectal formulations may seem ideal for colon targeted drug delivery; however in addition to the issues surrounding patient administration, the efficacy of these formulations is limited to conditions affecting the distal colon and rectum. They are not effective for the treatment of more widespread inflammation of the colon, which occurs in conditions such as Inflammatory Bowel Disease (IBD). Oral formulations can be designed to achieve either a local or systemic delivery of therapeutics. Local treatment requires that the drug will be delivered to the site of action within the gastrointestinal (GI) tract to have a localized effect, but will not be absorbed or only poorly absorbed. Systemic delivery for orally administered drugs requires systemic absorption occurring within the intestines prior to distribution of drug around the body. For conditions involving localized inflammation to the tissues of the colon, the ideal formulation would be one that (i) is administered orally, (ii) is able to reach the colon, (iii) delivers high concentrations of drug at the site of inflammation, and (iv) is not systemically absorbed. Current formulations have limited effect on specificity of targeting to diseased colon tissue vs. healthy colon tissue. In addition, despite coverage to the surface of the colon (including diseased tissue), there is no guarantee that the drug is effectively taken up into the tissue and cells at the site of inflammation. Therefore, the use of nanotechnology in formulation design has been investigated as a way to further improve the efficacy of therapeutics by allowing specific targeting and uptake into inflamed tissue within the colon (Jani et al., 1989, 1990). Nanoformulations have been developed for the targeted treatment of a number of pathological conditions, including IBD, cancers, and infections (Vingerhoeds et al., 1994; Singh, 1999; Maruyama, 2002). For the purpose of GI tract targeting, liposomes can be manipulated by the inclusion of polymer coatings on the liposomal surface. These coatings allow oral liposomal formulations to resist degradation in the hostile environment of the GI tract, which include bile salts and enzymes (e.g., pancreatic lipases) that would normally dissolve the lipid bilayer (Iwanaga et al., 1999; Takeuchi et al., 2003; Karn et al., 2011). This article will briefly discuss strategies which have been investigated for targeting drug-encapsulated liposomes to the colon.

Tu1707 Endogenous Opioids From Human IBD Supernatants Modulate TRPV1 on DRG Neurons During Chronic Colitis

Our previous mouse studies have shown that endogenous opioid signaling is enhanced during the evolution of colonic inflammation, altering pain signaling. The mechanisms underlying this action are unknown but given that TRPV1 channels are important in mechano and chemosensitive nociceptive signaling, and that TRPV1 is co-expressed with opioid receptors on dorsal root ganglia (DRG) neurons, we postulated that TRPV1 signaling may be involved. Therefore, in the present work we sought to investigate if the endogenous opioids released during the chronic inflammation model regulate TRPV1 activity. The 2% chronic DSS (cDSS) colitis model was employed in C57/BL6 mice. Human colonic biopsies from patients with chronic ulcerative colitis, crohn's desease or healthy controls and colonic tissue from cDSS colitis were used to generate supernatants (sup). DRG neurons were incubated overnight with mouse or human supernatants or μ and δ opioid agonists DAMGO (10 nM) and DADLE (1 μM) respectively and with or without the opioid receptor antagonist naloxone (10 μM). For Ca2+ imaging 1 μM Fura-2 AM and capsaicin (250 nM) was used as a TRPV1 agonist. T-test or one way ANOVA were used to analyze the data obtained, n= number of neurons. The capsaicin-evoked Ca2+ signal was inhibited 66% by cDSS sup compared to control sup (F340/380: Ctrl=2.08±0.33 n=19, cDSS=0.70±0.14 n=16, P= <0.01). This effect was blocked by naloxone 10 μM (F340/380: cDSS+NLX= 2.32±0.65, n=6, P= <0.05), demonstrating that endogenous opioids were involved. Moreover, the supernatant effect was mimicked by the use of low concentration DAMGO (10 nM) or DADLE (1 μM) alone. (F340/380: Ctrl=5.66±0.52 n=26, DAMGO=2.42±0.55 n=19, DADLE=3.45±50, P= <0.001), DRG neurons incubated with the human UC sup showed a similar effect with a 58% reduction of the Ca2+ signal (F340/380: Ctrl=5.51±0.51 n=18, UC sup=2.30±0.67 n= 9, P= 0.0011). In contrast, in a symptomatic IBD patient where inflammation had resolved (post-inflammatory IBS) there was no difference in the Ca2+ signal compared to control, thus suggesting the endogenous opioid effect was lost (F340/380: Ctrl=4.59±0.58 n=15, Crohn's sup=3.95±0.79 n=11). These results suggest that endogenous opioids released during chronic inflammation have an important role in inhibiting TRPV1 channels in nociceptive neurons. Following resolution of the inflammation in some patients however, the pain persists but the endogenous opioid system appears to be inactive.

Cortactin regulates intestinal epithelial permeability by stabilizing tight junctions (650.6)

Chronic inflammatory diseases of the intestines such as inflammatory bowel disease (IBD) are characterized by disturbed epithelial contacts and increased intestinal permeability. The actin cytoskeleton and its regulators are critical for these features. Analyzing cortactin‐KO mice, we found that the actin‐binding molecule cortactin (CTTN) controls vascular permeability. However, it is unknown if CTTN also regulates intestinal epithelial permeability. Thus, we generated a stable CTTN‐depleted Caco‐2 cell line that showed reduced transepithelial resistence (TER) and increased paracellular flux of 4 kDa FITC‐dextran. In these cells, we also observed a less developed cortical actin ring and internalization of occludin and ZO‐1. However, CTTN depletion did not cause increased apoptosis. CTTN‐KO mice also showed increased basal permeability in the colon. In healthy human colon tissue biopsies, we found that CTTN colocalized with actin and the tight junction protein ZO‐1. By contrast, in IBD patients, we observed strong internalization of CTTN resulting in a loss of colocalization with actin and ZO‐1. Our data imply that CTTN regulates intestinal permeability by controlling actin dynamics at cell contacts to stabilize epithelial junctions.Grant Funding Source: CONACYT 179895

A 3-D Artificial Colon Tissue Mimic for the Evaluation of Nanoparticle–Based Drug Delivery System

Functional engineered nanoparticles are promising drug delivery carriers. As the construction of a functional nanocarrier always needs the optimization of multiple technical variables, efficient in vitro high-throughput evaluation methods would help to shorten the development cycle. In the present study, we generated a tissue mimic of the colon of inflammatory bowel disease (IBD) patients. Generally, Caco-2 cells and THP-1 cells were grown in a 3-D matrix with different number, spatial distribution and specific extracellular cell matrix (ECM) composition according to real healthy and inflamed animal colon tissues. After interlerukin-1β/lipopolysaccharide (LPS) stimulation, the artificial model closely resembled the pathological features of IBD patient's colon, including massive cytokines and mucus production, epithelium defect and leukocytic infiltration. The tissue and cellular uptake of three different nanoparticles in the artificial model was similar to that in 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitic mice. Most importantly, our artificial tissue can be placed into 96-well plates for high-throughput screening of drug delivery carriers for the treatment of IBD. Our study suggested a readily achievable way to improve current methodologies for the development of colon targeted drug delivery systems.