Alpha diversity in feces of AOM/DSS and APCMin upon MS-444 treatment and indicator OTUs in AOM/DSS mouse feces.

CD8+ cytotoxic T-lymphocytes, NKp46+ natural killer cells and F4/80+ macrophages in tumors of AOM/DSS and APCMin mice. Analysis of cytokines secreted by small intestinal organoids upon MS-444 treatment.

Figure legends for the supplementary figures 1-4.

Crypt-villus length and HuR subcellular distribution as analyzed by immunohistochemistry and Western blot of nuclear and cytoplasmic fractions from isolated tumors in MS-444 treated and control mice.

Antibody list, organoid culture medium reagents and primer sequences

Phylum and family levels of APCMin and AOM/DSS mouse feces upon MS-444 treatment.

CRISPR/Cas9 HuR ko in RAW 264.7 macrophage cells confirmed by Sanger sequencing and Western blot analysis.

MS-444 restores splenic lymphoid follicles and decreases spleen weight in APCMin mice.

HuR nucleocytoplasmic shuttling is increased with increasing MS-444 concentrationsin an acute colitis model.

Supplementary Figure 1. PAK1 regulates β-catenin expression throughout the intestine. Supplementary Figure 2. PAK1 regulates β-catenin signaling in APCmin adenomas. Supplementary Figure 3 (A-E). Effect of PAK1 deletion on tumorigenesis in an AOM/DSS model. Supplementary Figure 4 (A-E). Inhibition of PAK1 impedes AKT/ β-catenin signaling in AOM/DSS mice.

Weight curves of AOM/DSS and APCMin mice, DSS water consummtion and large bowel tumorigenesis in APCMin mice

Additional information on mouse experiments, colonoscopy, histology, immunohistochemical analysis and amplicon sequencing for microbiota analysis

Abstract p21-activated kinase 1 (PAK1) is a serine/threonine kinase that is overexpressed in colorectal cancer. PAK1 is a target of mesalamine [5-aminosylicylic acid (5-ASA)], a common drug for the treatment of ulcerative colitis with prospective chemopreventive properties. Here, we investigated whether PAK1 deletion impedes tumorigenesis in murine intestinal cancer models. Ten-week-old APCmin or APCmin/PAK1−/− mice were monitored for 8 weeks, euthanized, and assessed for tumor number and size. Six- to 8-week-old PAK1−/− and wild-type (WT) mice received one 10 mg/kg intraperitoneal injection of azoxymethane (AOM) and four cycles of 1.7% dextran sodium sulfate (DSS) for 4 days followed by 14 days of regular water. Mice also received 5-ASA via diet. Tumor incidence and size was assessed via colonoscopy and pathology. Molecular targets of PAK1 and 5-ASA were evaluated via immunohistochemistry (IHC) in both models. PAK1 deletion reduced tumor multiplicity and tumor burden but did not alter average tumor size in APCmin mice. IHC revealed that PAK1 deletion reduced p-AKT, β-catenin, and c-Myc expression in APCmin adenomas. Colonoscopy and pathologic analysis revealed that PAK1 deletion reduced tumor multiplicity without affecting tumor size in AOM/DSS-treated mice. 5-ASA treatment and PAK1 deletion impeded tumor multiplicity and dysplastic lesions in AOM/DSS mice. IHC further revealed that 5-ASA blocked β-catenin signaling via inhibition of PAK1/p-AKT. These data indicate that PAK1 contributes to initiation of intestinal carcinogenesis. Cancer Prev Res; 8(11); 1093–101. ©2015 AACR.

Inflammatory bowel disease is a group of conditions with rising incidence caused by genetic and environmental factors including diet. The chelator ethylenediaminetetraacetate (EDTA) is widely used by the food and pharmaceutical industry among numerous other applications, leading to a considerable environmental exposure. Numerous safety studies in healthy animals have revealed no relevant toxicity by EDTA. Here we show that, in the presence of intestinal inflammation, EDTA is surprisingly capable of massively exacerbating inflammation and even inducing colorectal carcinogenesis at doses that are presumed to be safe. This toxicity is evident in two biologically different mouse models of inflammatory bowel disease, the AOM/DSS and the IL10-/- model. The mechanism of this effect may be attributed to disruption of intercellular contacts as demonstrated by in vivo confocal endomicroscopy, electron microscopy and cell culture studies. Our findings add EDTA to the list of food additives that might be detrimental in the presence of intestinal inflammation, but the toxicity of which may have been missed by regulatory safety testing procedures that utilize only healthy models. We conclude that the current use of EDTA especially in food and pharmaceuticals should be reconsidered. Moreover, we suggest that intestinal inflammatory models should be implemented in the testing of food additives to account for the exposure of this primary organ to environmental and dietary stress.

Oral iron promotes intestinal tumourigenesis in animal models. In humans, expression of iron transport proteins are altered in colorectal cancer. This study examined whether the route of iron therapy alters iron transport and tumour growth. Colorectal adenocarcinoma patients with pre-operative iron deficiency anaemia received oral ferrous sulphate (n = 15), or intravenous ferric carboxymaltose (n = 15). Paired (normal and tumour tissues) samples were compared for expression of iron loading, iron transporters, proliferation, apoptosis and Wnt signalling using immunohistochemistry and RT-PCR. Iron loading was increased in tumour and distributed to the stroma in intravenous treatment and to the epithelium in oral treatment. Protein and mRNA expression of proliferation and iron transporters were increased in tumours compared to normal tissues but there were no significant differences between the treatment groups. However, intravenous iron treatment reduced ferritin mRNA levels in tumours and replenished body iron stores. Iron distribution to non-epithelial cells in intravenous iron suggests that iron is less bioavailable to tumour cells. Therefore, intravenous iron may be a better option in the treatment of colorectal cancer patients with iron deficiency anaemia due to its efficiency in replenishing iron levels while its effect on proliferation and iron metabolism is similar to that of oral iron treatment.

BACKGROUND & AIMS:Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism. METHODS:The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning-based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays. RESULTS:Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P < .001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea. CONCLUSIONS:The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease.

Bioimpedance spectroscopy (BIS) is routinely used in peritoneal dialysis patients and might aid fluid status assessment in patients with liver cirrhosis, but the effect of ascites volume removal on BIS-readings is unknown. Here we determined changes in BIS-derived parameters and clinical signs of fluid overload from before to after abdominal paracentesis. Per our pre-specified sample size calculation, we studied 31 cirrhotic patients, analyzing demographics, labs and clinical parameters along with BIS results. Mean volume of the abdominal paracentesis was 7.8 ± 2.6 L. From pre-to post-paracentesis, extracellular volume (ECV) decreased (20.2 ± 5.2 L to 19.0 ± 4.8 L), total body volume decreased (39.8 ± 9.8 L to 37.8 ± 8.5 L) and adipose tissue mass decreased (38.4 ± 16.0 kg to 29.9 ± 12.9 kg; all p < 0.002). Correlation of BIS-derived parameters from pre to post-paracentesis ranged from R² = 0.26 for body cell mass to R² = 0.99 for ECV. Edema did not correlate with BIS-derived fluid overload (FO ≥ 15% ECV), which occurred in 16 patients (51.6%). In conclusion, BIS-derived information on fluid status did not coincide with clinical judgement. The changes in adipose tissue mass support the BIS-model assumption that fluid in the peritoneal cavity is not detectable, suggesting that ascites (or peritoneal dialysis fluid) mass should be subtracted from adipose tissue if BIS is used in patients with a full peritoneal cavity.

Iron deficiency (ID) is globally prevalent, and apart from anemia is associated with thrombocytosis. While considered benign, studies linking thrombotic events with prior ID anemia suggest otherwise. Herein we used animal models to assess the influence of ID on thrombotic tendency. Sprague-Dawley rats were fed control or iron deficient diets and ferric carboxymaltose was used to reverse ID. Thrombosis was induced via stenosis of the inferior vena cava or damage to the right carotid artery using ferric chloride. Thrombi were evaluated histologically and via high frequency ultrasound in the venous model. ID consistently induced thrombocytosis alongside anemia. Venous thrombus growth and final dimensions in both arterial and venous thrombi were largest in ID. In both models, platelet numbers correlated with the final thrombus size, with ID thrombi having the largest platelet areas. Platelet function was also evaluated in surgically naive rats. Coagulability on thromboelastography and hemostasis on tail transection were augmented in ID. Platelet and plasma P-selectin expression were both higher in ID. Platelet adhesion and aggregation in ID was impaired under shear flow but was intact on static assays. Iron replacement therapy reversed all ID-related changes in hematological parameters, thrombus dimensions, and platelet assays. In summary, ID alone increases thrombotic tendency. Iron replacement therapy reverses these changes, making it a viable strategy for prevention of ID-related thrombotic disease. This may be of importance in patients with chronic illnesses which may already be at increased risk for thrombosis such as inflammatory bowel disease, chronic kidney disease, or cancer.

Background: Oral iron promotes intestinal tumourigenesis in animal models. In humans, expression of iron transport proteins are altered in colorectal cancer. This study examines whether the route of iron therapy alters iron transport and tumour growth. Methods: Colorectal adenocarcinoma patients with pre-operative iron deficiency anaemia received oral ferrous sulphate (n=15), or intravenous ferric carboxymaltose (n=15). Paired (normal and tumour tissues) samples were compared for expression of iron loading, iron transporters, proliferation, apoptosis, Wnt signalling pathway and microsatellite instability using immunohistochemistry and RT-PCR. Results: Iron loading was increased in tumour and distributed to the stroma in intravenous treatment and to the epithelium in oral treatment. The protein and mRNA expression of iron transporters were increased in tumours compared to normal tissues but there were no significant differences between the treatment groups. However, intravenous iron treatment reduced ferritin mRNA levels in tumours (p<0.001) and successfully replenished body iron stores without increasing tumour growth, DNA damage markers, proliferation or apoptosis compared with oral iron treatment. Conclusion: Iron distribution to non-epithelial cells in intravenous iron treatment suggests that iron is less bioavailable to tumour cells. Therefore, intravenous iron supplementation could be a safer option in the treatment of colorectal cancer patients with iron deficiency anaemia due to the differential compartmentalisation of iron within the intestinal mucosa and its efficiency in replenishing body iron levels without increasing the risk of tumour growth.Trial registration: The study was registered with the Medicines and Healthcare Regulatory Agency, clinical trials.gov (NCT01927328) and EudraCT (2013-000209-22).