Assessment of Tumor Development and Wound Healing Using Endoscopic Techniques in Mice

doi:10.1053/j.gastro.2010.10.007

Gastroenterology

Volume 139, Issue 6, December 2010, Pages 1837-1843.e1

https://doi.org/10.1053/j.gastro.2010.10.007 Get rights and content

Mouse models of intestinal inflammation and colon cancer are valuable tools to gain insights into the pathogenesis of the corresponding human diseases. Recently, in vivo mouse endoscopy has been developed, allowing not only the high-resolution monitoring and scoring of experimental disease development, but also enables the investigator to perform manipulations, including local injection of reagents or the taking of biopsies for molecular and histopathologic analyses. Chromoendoscopic staining with methylene blue enables visualization of the crypt structure and allows discrimination between inflammatory and neoplastic changes. The development of endoscopic techniques in live mice opened new options for the investigation of disease mechanisms in the gut and for the preclinical testing of potential therapeutic effects of drug candidates. Finally, mouse endoscopy can help to reduce animal numbers needed to gain significant experimental data.

Section snippets

Endoscopic Procedure

The high-resolution mouse endoscopic system contains of a miniature endoscope with an outer diameter of 1.9 mm. Furthermore, the endoscopic setup is composed of a xenon light source, a digital camera device, and an air pump to provide a continuous air flow necessary for the inflation of the mouse colon (Figure 1A). The camera device can be connected to a personal computer to allow recording of digital videos. Instruments, including a biopsy forceps or an injection needle, can be introduced

Longitudinal Tumor Development Using Mouse Endoscopy

Despite numerous studies on the etiology of CRC, the mechanisms driving tumor initiation, promotion, and progression remain incompletely understood. Mouse models of CRC have proven to be an indispensable tool for basic research on colon cancer. Furthermore, mouse models allowed the preclinical evaluation of potential drug candidates and treatment regimens. Numerous mouse models of colon cancer have been developed in recent years, including chemically induced CRC models, mutant mouse models, and

High-Resolution Endoscopy for Monitoring of Colitis and Wound Healing In Vivo

The described endoscopic procedure is an adequate method for the investigation of colitis and wound healing as well. For the analysis of mucosal damage in the gut, investigators have either used in vitro assay systems using colon epithelial cell lines or used DSS colitis as a model for intestinal wound healing. DSS is a polysaccharide that leads to the destruction of the epithelial layer of the colon. Endoscopically, healthy mice show a normal blood vessel structure and a smooth and translucent

Acknowledgments

M.F.N. and N. W. share first authorship.

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Cited by (34)

Inhibiting Interleukin 36 Receptor Signaling Reduces Fibrosis in Mice With Chronic Intestinal Inflammation
2019, Gastroenterology
Intestinal fibrosis is a long-term complication in inflammatory bowel diseases (IBD) that frequently results in functional damage, bowel obstruction, and surgery. Interleukin (IL) 36 is a group of cytokines in the IL1 family with inflammatory effects. We studied the expression of IL36 and its receptor, interleukin 1 receptor like 2 (IL1RL2 or IL36R) in the development of intestinal fibrosis in human tissues and mice.
We obtained intestinal tissues from 92 patients with Crohn’s disease (CD), 48 patients with ulcerative colitis, and 26 patients without inflammatory bowel diseases (control individuals). Tissues were analyzed by histology to detect fibrosis and by immunohistochemistry to determine the distribution of fibroblasts and levels of IL36R ligands. Human and mouse fibroblasts were incubated with IL36 or control medium, and transcriptome-wide RNA sequences were analyzed. Mice were given neutralizing antibodies against IL36R, and we studied intestinal tissues from Il1rl2^–/– mice; colitis and fibrosis were induced in mice by repetitive administration of DSS or TNBS. Bone marrow cells were transplanted from Il1rl2^–/– to irradiated wild-type mice and intestinal tissues were analyzed. Antibodies against IL36R were applied to mice with established chronic colitis and fibrosis and intestinal tissues were studied.
Mucosal and submucosal tissue from patients with CD or ulcerative colitis had higher levels of collagens, including type VI collagen, compared with tissue from control individuals. In tissues from patients with fibrostenotic CD, significantly higher levels of IL36A were noted, which correlated with high numbers of activated fibroblasts that expressed α-smooth muscle actin. IL36R activation of mouse and human fibroblasts resulted in expression of genes that regulate fibrosis and tissue remodeling, as well as expression of collagen type VI. Il1rl2^–/– mice and mice given injections of an antibody against IL36R developed less severe colitis and fibrosis after administration of DSS or TNBS, but bone marrow cells from Il1rl2^–/– mice did not prevent induction of colitis and fibrosis. Injection of antibodies against IL36R significantly reduced established fibrosis in mice with chronic intestinal inflammation.
We found higher levels of IL36A in fibrotic intestinal tissues from patients with IBD compared with control individuals. IL36 induced expression of genes that regulate fibrogenesis in fibroblasts. Inhibition or knockout of the IL36R gene in mice reduces chronic colitis and intestinal fibrosis. Agents designed to block IL36R signaling could be developed for prevention and treatment of intestinal fibrosis in patients with IBD.
Colonoscopic-Guided Pinch Biopsies in Mice as a Useful Model for Evaluating the Roles of Host and Luminal Factors in Colonic Inflammation
2018, American Journal of Pathology
Citation Excerpt :
This work carefully examined the histologic changes and select inflammatory responses that occur after biopsy, in addition to developing the method to measure wound closure.39 These findings laid the groundwork for later studies interrogating how inflammatory mediators, stem cells, and oxidation-reduction molecules affect the wound healing process.39–46 Although mainly focused on wound healing, a previous study showed increased neutrophils and macrophages in and around the wound bed by immunostaining 6 hours after biopsy, as well as gene expression changes of type 1 and type 2 helper T-cell–related cytokines at both early and late stages of the injury response.39
Colonic inflammation, a hallmark of inflammatory bowel disease, can be influenced by host intrinsic and extrinsic factors. There continues to be a need for models of colonic inflammation that can both provide insights into disease pathogenesis and be used to investigate potential therapies. Herein, we tested the utility of colonoscopic-guided pinch biopsies in mice for studying colonic inflammation and its treatment. Gene expression profiling of colonic wound beds after injury showed marked changes, including increased expression of genes important for the inflammatory response. Interestingly, many of these gene expression changes mimicked those alterations found in inflammatory bowel disease patients. Biopsy-induced inflammation was associated with increases in neutrophils, macrophages, and natural killer cells. Injury also led to elevated levels of sphingosine-1-phosphate (S1P), a bioactive lipid that is an important mediator of inflammation mainly through its receptor, S1P₁. Genetic deletion of S1P₁ in the endothelium did not alter the inflammatory response but led to increased colonic bleeding. Bacteria invaded into the wound beds, raising the possibility that microbes contributed to the observed changes in mucosal gene expression. In support of this, reducing bacterial abundance markedly attenuated the inflammatory response to wounding. Taken together, this study demonstrates the utility of the pinch biopsy model of colonic injury to elucidate the molecular underpinnings of colonic inflammation and its treatment.
Activation of Epithelial Signal Transducer and Activator of Transcription 1 by Interleukin 28 Controls Mucosal Healing in Mice With Colitis and Is Increased in Mucosa of Patients With Inflammatory Bowel Disease
2017, Gastroenterology
Citation Excerpt :
Caco-2 cells cultured for 2 days with IL28 showed higher proliferation rates compared to untreated cells in another assay (Supplementary Figure 3B). We next addressed the effects of IL28/STAT1 signaling on epithelial restitution in vivo using an established experimental model of mucosal wound healing.39 Wound closure was significantly accelerated by IL28 administration (Figures 7A and B and Supplementary Figure 3D).
We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing.
Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra^−/−), and mice with disruption of the interferon regulatory factor 3 gene (Irf3^−/−), with or without disruption of Irf7 (Irf7^−/−). We used high-resolution mini-endoscopy and in vivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n = 62) and patients without colon inflammation (controls, n = 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging.
Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 in primary human and mouse IECs, increasing with dose. Il28ra^−/−, Irf3^−/−, Irf3^−/−Irf7^−/−, as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice, with reduced epithelial restitution. Il28ra^−/− and Stat1IEC-KO mice also developed more severe colitis in response to oxazolone than control mice. We found IL28 to induce phosphorylation (activation) of STAT1 in epithelial cells, leading to their proliferation in organoid culture. Administration of IL28 to mice with induced colonic wounds promoted mucosal healing.
IL28 controls proliferation of IECs in mice with colitis and accelerates mucosal healing by activating STAT1. IL28 might be developed as a therapeutic agent for patients with IBD.
VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis
2015, Gastroenterology
Citation Excerpt :
Endoscopy of VEGF2ΔIEC mice revealed no spontaneous phenotype in the lower gastrointestinal tract, which was confirmed by histology (Supplementary Figure 1B). To induce colitis-associated tumor development, VEGFR2ΔIEC and matched VEGFR2fl/fl control mice were exposed to AOM+DSS (Figure 1C).17,18 Colonoscopy was performed to evaluate different tumor parameters and the severity of intestinal inflammation.16
Senescence prevents cellular transformation. We investigated whether vascular endothelial growth factor (VEGF) signaling via its receptor, VEGFR2, regulates senescence and proliferation of tumor cells in mice with colitis-associated cancer (CAC).
CAC was induced in VEGFR2^ΔIEC mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2^fl/fl mice (controls) by administration of azoxymethane followed by dextran sodium sulfate. Tumor development and inflammation were determined by endoscopy. Colorectal tissues were collected for immunoblot, immunohistochemical, and quantitative polymerase chain reaction analyses. Findings from mouse tissues were confirmed in human HCT116 colorectal cancer cells. We analyzed colorectal tumor samples from patients before and after treatment with bevacizumab.
After colitis induction, VEGFR2^ΔIEC mice developed significantly fewer tumors than control mice. A greater number of intestinal tumor cells from VEGFR2^ΔIEC mice were in senescence than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Tumor cell senescence promoted an anti-tumor immune response by CD8⁺ T cells in mice. Patients whose tumor samples showed an increase in the proportion of senescent cells after treatment with bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment.
Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with bevacizumab.
C/EBP homologous protein inhibits tissue repair in response to gut injury and is inversely regulated with chronic inflammation
2014, Mucosal Immunology
Loss of intestinal epithelial cell (IEC) homeostasis and apoptosis negatively affect intestinal barrier function. Uncontrolled activation of the unfolded protein response (UPR) in IEC contributes to an impaired barrier and is implicated in the pathogenesis of inflammatory bowel diseases. However, the contribution of the UPR target gene C/EBP homologous protein (CHOP), an apoptosis-associated transcription factor, to inflammation-related disease susceptibility remains unclear. Consistent with observations in patients with ulcerative colitis, we show that despite UPR activation in the epithelium, CHOP expression was reduced in mouse models of T-cell-mediated and bacteria-driven colitis. To elucidate the molecular mechanisms of IEC-specific CHOP expression, we generated a conditional transgenic mouse model (Chop^{IEC Tg/Tg}). Chop overexpression increased the susceptibility toward dextran sodium sulfate (DSS)-induced intestinal inflammation and mucosal tissue injury. Furthermore, a delayed recovery from DSS-induced colitis and impaired closure of mechanically induced mucosal wounds was observed. Interestingly, these findings seemed to be independent of CHOP-mediated apoptosis. In vitro and in vivo cell cycle analyses rather indicated a role for CHOP in epithelial cell proliferation. In conclusion, these data show that IEC-specific overexpression impairs epithelial cell proliferation and mucosal tissue regeneration, suggesting an important role for CHOP beyond mediating apoptosis.
Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer
2014, Cancer Letters
Citation Excerpt :
No toxicity was observed during the treatment period. Tumor biopsies were taken before the first treatment and one hour after the final treatment using biopsy forceps passed through the working channel of the endoscope sheath (Karl Storz), then flash-frozen in liquid nitrogen for subsequent western blot analysis [22]. Mice were sacrificed immediately following final biopsy, one hour after final treatment dose.
PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer.

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: Funding The research leading to these results has received funding from the European Community's 7th Framework Programme (FP7/2007-2013) under grant agreement n° 202230, acronym GENINCA.

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Imaging and Advanced TechnologyAssessment of Tumor Development and Wound Healing Using Endoscopic Techniques in Mice

Section snippets

Endoscopic Procedure

Longitudinal Tumor Development Using Mouse Endoscopy

High-Resolution Endoscopy for Monitoring of Colitis and Wound Healing In Vivo

Acknowledgments

Eur J Cancer

Cancer facts and figures 2010

Imaging and Advanced Technology
Assessment of Tumor Development and Wound Healing Using Endoscopic Techniques in Mice