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This article has been retracted at the request of the Editor-in-Chief due to concerns regarding the legitimacy of images and data presented in the paper.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.

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.

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.

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.

Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease.

OBJECTIVES: Loss of intestinal barrier function plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Shedding of intestinal epithelial cells is a potential cause of barrier loss during inflammation. The objectives of the study were (1) to determine whether cell shedding and barrier loss in humans can be detected by confocal endomicroscopy and (2) whether these parameters predict relapse of IBD. METHODS: Confocal endomicroscopy was performed in IBD and control patients using intravenous fluorescein to determine the relationship between cell shedding and local barrier dysfunction. A grading system based on appearances at confocal endomicroscopy in humans was devised and used to predict relapse in a prospective pilot study of 47 patients with ulcerative colitis and 11 patients with Crohn's disease. RESULTS: Confocal endomicroscopy in humans detected shedding epithelial cells and local barrier defects as plumes of fluorescein effluxing through the epithelium. Mouse experiments demonstrated inward flow through some leakage-associated shedding events, which was increased when luminal osmolarity was decreased. In IBD patients in clinical remission, increased cell shedding with fluorescein leakage was associated with subsequent relapse within 12 months after endomicroscopic examination (p<0.001). The sensitivity, specificity and accuracy for the grading system to predict a flare were 62.5% (95% CI 40.8% to 80.4%), 91.2% (95% CI 75.2 to 97.7) and 79% (95% CI 57.7 to 95.5), respectively. CONCLUSIONS: Cell shedding and barrier loss detected by confocal endomicroscopy predicts relapse of IBD and has potential as a diagnostic tool for the management of the disease.

Radiation-induced gastric epithelial apoptosis occurs in the proliferative zone and is regulated by p53, bak, bax, and bcl-2.

Unlike the small intestine and colon where gamma-radiation-induced apoptosis has previously been well characterized, the response of murine gastric epithelium to gamma-radiation has not been investigated in detail. Apoptosis was therefore assessed on a cell positional basis in gastric antral and corpus glands from adult male mice following gamma-radiation. Maximum numbers of apoptotic cells were observed in both antrum and corpus at 48 h and at radiation doses greater than 12 Gy. However, the number of apoptotic cells observed in the gastric epithelium was much lower than observed in the small intestine or colon after similar doses of radiation. Hematoxylin and eosin, caspase 3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling detected similar numbers and cell positional distributions of apoptotic cells, hence hematoxylin and eosin was used for subsequent studies. The highest numbers of apoptotic cells were observed at cell positions 5-6 in the antrum and cell positions 15-18 in the corpus. These distributions coincided with the distributions of PCNA-labeled proliferating cells, but not with the distributions of H(+)-K(+)-ATPase-labeled parietal cells or TFF2-labeled mucous neck cells. Decreased numbers of apoptotic gastric epithelial cells were observed in p53-null, bak-null, and bax-null mice compared with wild-type counterparts 6 and 48 h after 12 Gy gamma-radiation. Significantly increased numbers of apoptotic gastric epithelial cells were observed in bcl-2-null mice compared with wild-type littermates 6 h after 12 Gy gamma-radiation. Radiation therefore induces apoptosis in the proliferative zone of mouse gastric epithelium. This response is regulated by the expression of p53, bak, bax, and bcl-2.