Mouse organoids as an in vitro tool to study the in vivo intestinal response to cytotoxicants.

Cross-species comparison of drug responses at the organoid level could help to determine the human relevance of findings from animal studies. To this end, we first need to evaluate the in vitro to in vivo translatability of preclinical organoids. Here, we used 5-fluorouracil (5-FU) as an exemplar drug to test whether the in vivo gut response to this cytotoxicant was preserved in murine intestinal organoids. Mice treated with 5-FU at 20 or 50 mg/kg IV (low and high dose, respectively) displayed diarrhea at clinically relevant exposures. 5-FU also induced intestinal lesions, increased epithelial apoptosis, and decreased proliferation in a dose-dependent manner. To enable comparison between the in vitro and in vivo response, top nominal in vitro drug concentrations that caused significant cytotoxicity were chosen (dose range 1-1000 µM). The inferred intracellular concentration in organoids at 1000 µM was within the tissue exposure range related to intestinal toxicity in vivo. 5-FU at ≥ 100 µM decreased ATP levels and increased Caspase-3 activity in intestinal organoids. In keeping with the in vivo findings, 5-FU increased the percentage of Caspase-3-positive cells and reduced Ki67 staining. At the transcriptome level, there was an overlap in the activity of pathways related to 5-FU's mode of action, lipid and cholesterol metabolism and integrin signaling across in vivo gut and organoids. The predicted activity state of upstream regulators was generally well preserved between setups. Collectively, our results suggest that despite their inherent limitations, organoids represent an adequate tool to explore the intestinal response to cytotoxicants.

RETRACTED: IP1867B suppresses the insulin-like growth factor 1 receptor (IGF1R) ablating epidermal growth factor receptor inhibitor resistance in adult high grade gliomas.

This article has been retracted at the request of the Editor-in-Chief due to concerns regarding the legitimacy of images and data presented in the paper. Though a corrigendum (Can. Lett. Vol. 469, 2020, pages 524-535) was previously published to address some of these concerns, this corrigendum has also been found to contain errors and therefore cannot stand. Specific concerns are listed below. The Editor and Publisher received a letter from the University of Portsmouth alerting us to an investigation into alleged research misconduct. The University concluded their investigation with external experts and determined that misconduct did take place in relation to the research involved in this paper. Upon our separate investigation, it has been determined that the paper headline relies on showing that there was considerable reduction of IGF1R, IL6R and EGFR post treatment in all cell lines. During review, it was determined that this cannot be concluded from the presented data. For example, in SEBTA-003 the EGFR levels go up and there is no difference in IGFR1. It is apparent from Fig 4d that in the SEBTA-003 cell line the EGFR level does not go down, which is stated in the Results section on page 32, it is rather going up. The data for IGFR1 are inconclusive and there are concerns regarding the blot. The general implications would be that the effects of the drug IP1867B does not seem to be the same for all tested cell lines, and this should have been discussed in detail by the authors. Additionally, in subsequent experiments (Fig. 4g and h) the SEBTA-003 cell line (no reduction of EGFR, rather increased expression) and the other 3 cell lines (reduction of EGFR) show similar responses. This is particularly evident in Fig. 4g: Two cell lines are compared, SEBTA-003 (increased EGFR expression) and UP-029 (decreased EGFR expression), both behave similarly after exposure to drugs. The corrigendum (https://doi.org/10.1016/j.canlet.2019.10.002) issue is with respect to the Supplemental Figure 6i EGFR, particularly panel IP1867B. The Corrigendum states that the left part is a cut out of the very right part. If so, the bands for IP1867B should show the same staining pattern - but they do not. Also, in the Corrigendum, there are incorrect mentions between day 14 in the Figure and day 19 in the Figure legend. All authors were informed of the retraction in advance. Drs. Pritchard and Duckworth agreed to the retraction. The corresponding author, Dr Hill, did not agree to the retraction. No response had been received from Drs. Mihajluk, Simms, Reay, Madureira, Howarth, Murray, Nasser and Pilkinton at the time of the retraction being published.

Author Correction: Mice lacking NF-κB1 exhibit marked DNA damage responses and more severe gastric pathology in response to intraperitoneal tamoxifen administration.

Following the publication of this article [1], it was noted that the author list was incomplete and was missing the following author.

A cohort study of local excision followed by adjuvant therapy incorporating a contact X-ray brachytherapy boost instead of radical resection in 180 patients with rectal cancer.

AIM: Recent data have suggested near-equivalent oncological results when treating early rectal cancer by local excision followed by radio- ± chemotherapy rather than salvage radical surgery. The aim of this retrospective study was to assess the use of contact X-ray brachytherapy within this paradigm. METHOD: All patients had undergone local excision and were referred to our radiotherapy centre for treatment with contact X-ray brachytherapy. Postoperative (chemo)radiotherapy was also given in their local hospital in most cases. Variables assessed were local excision method, postoperative therapy received, follow-up duration, disease-free survival, salvage surgery and stoma-free survival. RESULTS: In total, 180 patients with a median age of 70 (range 36-99) years were assessed. Following local excision, pT stages were pT1 = 131 (72%), pT2 = 44 (26%), pT3 = 5 (2%). All patients received contact X-ray brachytherapy boosting at our centre and, in addition, 110 received chemoradiotherapy and 60 received radiotherapy alone. After a median follow-up of 36 months (range 6-48), 169 patients (94%) remained free of local recurrence. Of the 11 patients with local recurrence (three isolated nodal), five underwent salvage abdominoperineal excision. Eight patients developed distant disease, of whom five underwent metastasis surgery. At last included follow-up 173 (96%) patients were free of all disease and 170 (94%) were stoma free. CONCLUSIONS: Contact therapy can be offered in addition to external beam radio (±chemo) therapy instead of radical surgery as follow-on treatment after local excision of early rectal cancer. This combination can provide equivalent outcomes to radical surgery. The added value of contact therapy should be formally assessed in a clinical trial.

Contact X-ray Brachytherapy as an Adjunct to a Watch and Wait Approach is an Affordable Alternative to Standard Surgical Management of Rectal Cancer for Patients with a Partial Clinical Response to Chemoradiotherapy.

AIMS: Emerging evidence suggests that contact X-ray brachytherapy (CXB) may increase the clinical complete response rate and durability when administered after standard chemoradiotherapy in patients with rectal cancer. The addition of CXB in partial responders is therefore probably cost-effective. The affordability of widening access to CXB in the UK, however, has not been evaluated. MATERIALS AND METHODS: Decision analytical modelling with Monte Carlo simulation was used to evaluate long-term costs for the management of patients with rectal cancers who were given a CXB boost when a clinical complete response was not initially achieved following chemoradiotherapy in order to facilitate a watch and wait approach. A third-party payer (National Health Service) perspective was adopted, probabilistic sensitivity analysis was carried out and a scenario analysis was performed to investigate the effect of the number of referral centres and number of patients treated with CXB. RESULTS: We estimate that 818 (95% confidence interval 628-1021) patients per year are eligible for CXB as an adjunct to a watch and wait approach in England and Wales. As this management is less costly than surgical management for each individual patient, the more patients treated, the more affordable the technology. Even if as few as 125 patients are treated nationally in 15 centres, the cost of implementing this technology would be less than £4 million. If the average number of patients treated in each centre is 30, this technology would be cost saving within 5 years. CONCLUSIONS: The cost of CXB is not prohibitive according to the National Institute for Health and Care Excellence threshold for implementation of new technology and may even be cost saving within 5 years compared with standard surgical management, depending on the uptake of the technology and the number of referral centres.

The stomach in health and disease.

The stomach is traditionally regarded as a hollow muscular sac that initiates the second phase of digestion. Yet this simple view ignores the fact that it is the most sophisticated endocrine organ with unique physiology, biochemistry, immunology and microbiology. All ingested materials, including our nutrition, have to negotiate this organ first, and as such, the stomach is arguably the most important segment within the GI tract. The unique biological function of gastric acid secretion not only initiates the digestive process but also acts as a first line of defence against food-borne microbes. Normal gastric physiology and morphology may be disrupted by Helicobacter pylori infection, the most common chronic bacterial infection in the world and the aetiological agent for most peptic ulcers and gastric cancer. In this state-of-the-art review, the most relevant new aspects of the stomach in health and disease are addressed. Topics include gastric physiology and the role of gastric dysmotility in dyspepsia and gastroparesis; the stomach in appetite control and obesity; there is an update on the immunology of the stomach and the emerging field of the gastric microbiome. H. pylori-induced gastritis and its associated diseases including peptic ulcers and gastric cancer are addressed together with advances in diagnosis. The conclusions provide a future approach to gastric diseases underpinned by the concept that a healthy stomach is the gateway to a healthy and balanced host. This philosophy should reinforce any public health efforts designed to eradicate major gastric diseases, including stomach cancer.

bak deletion stimulates gastric epithelial proliferation and enhances Helicobacter felis-induced gastric atrophy and dysplasia in mice.

Helicobacter infection causes a chronic superficial gastritis that in some cases progresses via atrophic gastritis to adenocarcinoma. Proapoptotic bak has been shown to regulate radiation-induced apoptosis in the stomach and colon and also susceptibility to colorectal carcinogenesis in vivo. Therefore we investigated the gastric mucosal pathology following H. felis infection in bak-null mice at 6 or 48 wk postinfection. Primary gastric gland culture from bak-null mice was also used to assess the effects of bak deletion on IFN-γ-, TNF-α-, or IL-1ß-induced apoptosis. bak-null gastric corpus glands were longer, had increased epithelial Ki-67 expression, and contained fewer parietal and enteroendocrine cells compared with the wild type (wt). In wt mice, bak was expressed at the luminal surface of gastric corpus glands, and this increased 2 wk post-H. felis infection. Apoptotic cell numbers were decreased in bak-null corpus 6 and 48 wk following infection and in primary gland cultures following cytokine administration. Increased gastric epithelial Ki-67 labeling index was observed in C57BL/6 mice after H. felis infection, whereas no such increase was detected in bak-null mice. More severe gastric atrophy was observed in bak-null compared with C57BL/6 mice 6 and 48 wk postinfection, and 76% of bak-null compared with 25% of C57BL/6 mice showed evidence of gastric dysplasia following long-term infection. Collectively, bak therefore regulates gastric epithelial cell apoptosis, proliferation, differentiation, mucosal thickness, and susceptibility to gastric atrophy and dysplasia following H. felis infection.

Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip.

The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed.

Serial surveillance of carcinoid heart disease: factors associated with echocardiographic progression and mortality.

BACKGROUND: Carcinoid heart disease is a complication of metastatic neuroendocrine tumours (NETs). We sought to identify factors associated with echocardiographic progression of carcinoid heart disease and death in patients with metastatic NETs. METHODS: Patients with advanced non-pancreatic NETs and documented liver metastases and/or carcinoid syndrome underwent prospective serial clinical, biochemical, echocardiographic and radiological assessment. Patients were categorised as carcinoid heart disease progressors, non-progressors or deceased. Multinomial regression was used to assess the univariate association between variables and carcinoid heart disease progression. RESULTS: One hundred and thirty-seven patients were included. Thirteen patients (9%) were progressors, 95 (69%) non-progressors and 29 (21%) patients deceased. Baseline median levels of serum N-terminal pro-brain natriuretic peptide (NT-proBNP) and plasma 5-hydroxyindoleacetic acid (5-HIAA) were significantly higher in the progressors. Every 100 nmol l(-1) increase in 5-HIAA yielded a 5% greater odds of disease progression (OR 1.05, 95% CI: 1.01, 1.09; P=0.012) and a 7% greater odds of death (OR 1.07, 95% CI: 1.03, 1.10; P=0.001). A 100 ng l(-1) increase in NT-proBNP did not increase the risk of progression, but did increase the risk of death by 11%. CONCLUSIONS: The biochemical burden of disease, in particular baseline plasma 5-HIAA concentration, is independently associated with carcinoid heart disease progression and death. Clinical and radiological factors are less useful prognostic indicators of carcinoid heart disease progression and/or death.

The clinical presentation and management of carcinoid heart disease.

Carcinoid heart disease is a major cause of morbidity and mortality in patients with metastatic neuroendocrine tumours (NETs). Although cases of carcinoid syndrome and severe carcinoid heart disease requiring urgent intervention are well described, many patients with significant carcinoid heart disease may have insidious symptoms or even be asymptomatic. As haemodynamically significant carcinoid heart disease may be clinically silent, specific and individualised considerations must be made as to the most appropriate clinical criteria and time point at which surgical valve replacement should be undertaken in patients with carcinoid heart disease.

Circulating galectins -2, -4 and -8 in cancer patients make important contributions to the increased circulation of several cytokines and chemokines that promote angiogenesis and metastasis.

BACKGROUND: Circulating concentrations of the cytokines interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) and chemokines monocyte chemotatic protein 1 (MCP-1)/CCL2 and growth-regulator oncogene α (GROα)/chemokine C-X-C motif ligand 1 are commonly increased in cancer patients and they are increasingly recognised as important promoters, via divergent mechanisms, of cancer progression and metastasis. METHODS: The effect of galectins-2, -4 and -8, whose circulating levels are highly increased in cancer patients, on endothelial secretion of cytokines was assessed in vitro by cytokine array and in mice. The relationship between serum levels of galectins and cytokines was analysed in colon and breast cancer patients. RESULTS: Galectins-2, -4 and -8 at pathological concentrations induce secretion of G-CSF, IL-6, MCP-1 and GROα from the blood vascular endothelial cells in vitro and in mice. Multiple regression analysis indicates that increased circulation of these galectins accounts for 41∼83% of the variance of these cytokines in the sera of colon and breast cancer patients. The galectin-induced secretion of these cytokines/chemokines is shown to enhance the expression of endothelial cell surface adhesion molecules, causing increased cancer-endothelial adhesion and increased endothelial tubule formation. CONCLUSION: The increased circulation of galectins -2, -4 and -8 in cancer patients contributes substantially to the increased circulation of G-CSF, IL-6 and MCP-1 by interaction with the blood vascular endothelium. These cytokines and chemokines in turn enhance endothelial cell activities in angiogenesis and metastasis.

Signaling mediated by the NF-κB sub-units NF-κB1, NF-κB2 and c-Rel differentially regulate Helicobacter felis-induced gastric carcinogenesis in C57BL/6 mice.

The classical nuclear factor-kappaB (NF-κB) signaling pathway has been shown to be important in a number of models of inflammation-associated cancer. In a mouse model of Helicobacter-induced gastric cancer, impairment of classical NF-κB signaling in the gastric epithelium led to the development of increased preneoplastic pathology, however the role of specific NF-κB proteins in Helicobacter-associated gastric cancer development remains poorly understood. To investigate this C57BL/6, Nfkb1(-/-), Nfkb2(-/-) and c-Rel(-/-) mice were infected with Helicobacter felis for 6 weeks or 12 months. Bacterial colonization, gastric atrophy and preneoplastic changes were assessed histologically and cytokine expression was assessed by qPCR. Nfkb1(-/-) mice developed spontaneous gastric atrophy when maintained for 12 months in conventional animal house conditions. They also developed more pronounced gastric atrophy after short-term H. felis colonization with a similar extent of preneoplasia to wild-type (WT) mice after 12 months. c-Rel(-/-) mice developed a similar degree of gastric atrophy to WT mice; 3 of 6 of these animals also developed lymphoproliferative lesions after 12 months of infection. Nfkb2(-/-) mice developed minimal gastric epithelial pathology even 12 months after H. felis infection. These findings demonstrate that NF-κB1- and NF-κB2-mediated signaling pathways differentially regulate the epithelial consequences of H. felis infection in the stomach, while c-Rel-mediated signaling also appears to modulate the risk of lymphomagenesis in gastric mucosa-associated lymphoid tissue.

Peptide receptor radionuclide therapy with (90)Y-DOTATATE/(90)Y-DOTATOC in patients with progressive metastatic neuroendocrine tumours: assessment of response, survival and toxicity.

BACKGROUND: Peptide receptor radionuclide therapy (PRRT) is an established treatment for patients with metastatic neuroendocrine tumours (NETs), although which factors are associated with an improved overall survival (OS) remains unclear. The primary aim of this study is to determine to what extent a radiological response to (90)Y-DOTATOC/(90)Y-DOTATATE PRRT is associated with an improved OS. The association of biochemical and clinical response to OS were assessed as secondary outcome measures. METHODS: A retrospective analysis was conducted on 57 patients: radiological response was classified using RECIST criteria, biochemical response was classified using WHO criteria and clinical response was assessed subjectively. Responses were recorded as positive response (PR), stable disease (SD) or progressive disease (PD), and survival analysed. RESULTS: Radiological response was achieved in 71.5% (24.5% PR, 47% SD) and was associated with a greater OS (51 and 56 months, respectively), compared with PD (18 months). A biochemical or clinical response post PRRT were not associated with a statistically significant improvement in OS. However, when combined with radiological response a survival benefit was observed according to the number of outcomes (radiological, biochemical, clinical), in which a response was observed. Mild haematological toxicity was common, renal toxicity was rare. CONCLUSION: In patients with progressive metastatic NETs receiving (90)Y-DOTATOC/(90)Y-DOTATATE PRRT, a radiological response with either a PR or a SD post therapy confers a significant OS benefit.

Acute WNT signalling activation perturbs differentiation within the adult stomach and rapidly leads to tumour formation.

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active ß-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear ß-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach.

CYP2C19*17 gain-of-function polymorphism is associated with peptic ulcer disease.

Single-nucleotide polymorphisms (SNPs) in the CYP2C gene cluster have been extensively investigated as predisposing factors for nonsteroidal anti-inflammatory drug (NSAID)-induced peptic ulcer disease (PUD) or upper gastrointestinal bleeding (UGIB). However, results have been inconclusive owing to different study designs, limited genotyping strategies, and small sample sizes. We investigated whether eight functional SNPs in the CYP2C family of genes--CYP2C8*3 (rs11572080 and rs10509681), CYP2C8*4, CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, and CYP2C19*17--are associated with PUD in 1,239 Caucasian patients. Logistic regression analysis showed that only CYP2C19*17 was associated with PUD (odds ratio additive model: 1.47 (95% confidence interval (CI) 1.12 to 1.92); P = 0.005; R(2) 16%), but not UGIB, independent of NSAID use or Helicobacter pylori infection. PUD distribution varied (P = 0.024) according to CYP2C19*17 genotype: *1/*1, 490 (64.3%); *1/*17, 304 (71.7%); and *17/*17, 31 (73.8%). CYP2C19*17, a gain-of-function polymorphism, is associated with PUD irrespective of etiology.

CD24 is expressed in gastric parietal cells and regulates apoptosis and the response to Helicobacter felis infection in the murine stomach.

CD24 is expressed in the putative stem cells within several tissues and is overexpressed in gastric and colonic adenocarcinomas. Perturbed CD24 expression may therefore alter the response of gastrointestinal epithelia to damage-inducing stimuli that induce cancer. We have investigated the effects of CD24 deletion on gastric responses to Helicobacter felis infection and γ-irradiation using CD24-null mice. Gastric CD24 expression was determined by immunohistochemistry in C57BL/6 mice. Female CD24-null and C57BL/6 mice were infected with H. felis for 6 wk, and inflammation, proliferation, apoptosis, and parietal cell numbers were assessed in gastric tissue sections. Apoptosis and proliferation were analyzed on a cell-positional basis in stomach, small intestine, and colon of CD24-null and C57BL/6 mice following γ-irradiation. Apoptosis was also assessed in HT29 cells following CD24 siRNA transfection. Of CD24-positive cells in the gastric corpus, 98% were H(+)-K(+)-ATPase-expressing parietal cells. CD24-null mice showed more prominent gastric H. felis colonization than C57BL/6 mice but displayed a marked reduction in corpus inflammation, reduced Ki67 labeling, and less gastric atrophy 6 wk following infection. Corpus apoptosis was elevated in CD24-null mice, but this did not increase further with H. felis infection as observed in C57BL/6 mice. More apoptotic cells were found following γ-irradiation in the stomach, small intestine, and colon of CD24-null mice and following CD24 knockdown in vitro. In conclusion, CD24 is expressed in gastric parietal cells, where it modulates gastric responses to H. felis and γ-radiation. CD24 also regulates susceptibility to apoptosis in the distal murine gastrointestinal tract.

The relative contribution of NSAIDs and Helicobacter pylori to the aetiology of endoscopically-diagnosed peptic ulcer disease: observations from a tertiary referral hospital in the UK between 2005 and 2010.

BACKGROUND: Recent data from Western countries indicate that the aetiology of peptic ulcer disease (PUD) is changing as the prevalence of Helicobacter pylori is decreasing while the use of low-dose aspirin (LDA, ≤325 mg/day) is increasing. AIM: To investigate the changing aetiology and demographics of PUD in a well-characterised patient cohort at a large tertiary hospital in the UK between July 2005 and June 2010. METHODS: Patients diagnosed with PUD following endoscopy were categorised as non-steroidal anti-inflammatory drug (NSAID)-users or non-users, and their H. pylori status determined. Comparisons between NSAID-users and non-users, and between non-aspirin NSAID-users and LDA-users were summarised using counts and corresponding percentages (for categorical variables) and means and standard deviations (for continuous variables). RESULTS: Overall, 386 patients were enrolled; 57% used NSAIDs (51% LDA only) and 43% were non-users. 57% of the whole cohort was H. pylori-positive (including 66% with duodenal ulcers and 47% with gastric ulcers). Compared with non-users, NSAID-users were older (mean age 68 vs. 61 years) and fewer were H. pylori-positive (52% vs. 63%). LDA-users were older (mean age 71 vs. 62 years) and more likely to be H. pylori-positive (61% vs. 41%) than those using non-aspirin NSAIDs. Twelve per cent of the patients were neither using NSAIDs nor were H. pylori-positive. CONCLUSIONS: The NSAIDs, particularly LDA, were most commonly associated with PUD in this cohort. Our findings are compatible with the decline in the prevalence of H. pylori-positive PUD and increase in non-NSAID, non-H. pylori PUD previously reported.