Murine Colonoscopy Research Articles

Abstract 721: Epigenetic intervention in the prevention of Lynch Syndrome Colorectal Cancer

Abstract Introduction: Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC) affecting >1 million Americans. LS is caused by germline mutations in one of four DNA mismatch repair (MMR) genes, generating several hundreds of mutations in microsatellite coding sequences. Although defects in the MMR machinery are the principal mechanism driving LS carcinogenesis, the role of the epigenome through histone and DNA methylation remains unexplored. Hypermethylation of MLH1 is detected in 15% of sporadic MMR-deficient CRC and also in LSCRC, indicating a pivotal role of DNA methylation in LSCRC. In this study, we investigated the landscape of DNA methylation and EZH2-mediated histone H3 lysine 27 methylation in LS tumor samples, and tested the preventive effects of GSK-503, on tumor formation using a novel LS mouse model. Methods: We profiled the somatic mutational, global DNA methylation landscape, and performed ChIP-seq in LS precancers and tumors. Patient-derived organoids (PDO) from colonic tissues treated with EZH2 and 5-AZA were used for gene expression analysis. LS mice (Villin-Cre;Msh2fl/fl;TgfβRII-KI) were treated for 9 weeks with EZH2 inhibitor GSK503 to assess the effect on polyp formation in vivo monitored through murine colonoscopy. Results: Our mutational profiling via whole-exome-seq and gene expression survey of 10 LSCRC and 33 pre-cancers showed accumulation of somatic mutations and aberrant expression patterns in key epigenetic regulators including EZH2 and DNMT3A, which suggests dysregulation of epigenetic control in LS. Global DNA methylation using RRBS in tumors of 3 LS patients showed that gene promoters were more hypermethylated when compared to matched normal samples. Immunohistochemistry data showed higher expression of EZH2 in tumors when compared to precancerous and normal colonic mucosa. ChIPseq analysis demonstrated high levels of H3K27me3 present in gene promoters of stem cell marker genes (WNT9A, LGR5, and ASCL2), and immune-related genes (CD4, CD8a, and HLA-E). Interestingly, 577 out of 2756 DNA hypermethylated genes from RRBS analysis displayed high levels of H3K27me3, thus indicating a central role of EZH2 in epigenetic programming of LS neoplasms. RNAseq data from PDOs treated with 5-AZA (DNMT inhibitor) and GSK503 (EZH2 inhibitor) showed that both epigenetic inhibitors led to the reactivation of silenced genes in colorectal carcinogenesis such as CDKN2A, RUNX3, IGFBP3, and immune genes, including CD40, CD4, SEMA3B, CXRC6/6, and ICAM1. Finally, a 9-week chemo-preventive treatment of a cohort of LS mice with GSK503 significantly decreased polyp formation when compared to control, and polyp reduction was associated with increased infiltration of cytotoxic CD8a+ Tcells and NK in the large intestine mucosa. Conclusions: These results demonstrate the chemopreventive potential of EZH2 inhibitors in prevention of LS-associated tumors through an immune modulation approach. Citation Format: Laura Reyes Uribe, Charles M Bowen, Kyera Evans, Hong Wu, Fahriye Duzagac, Krishna Sinha, Eduardo Vilar. Epigenetic intervention in the prevention of Lynch Syndrome Colorectal Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 721.

Establishment of an Endoscopy-Guided Minimally Invasive Orthotopic Mouse Model of Colorectal Cancer.

Simple SummaryOpen orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these limitations, this study aimed to evaluate the establishment of an endoscopy-guided minimally invasive model without laparotomy. Different concentrations of the murine CRC cell lines CT26 and MC38 were endoscopically injected into the colorectal wall of BALB/C and C57BL/6J mice, respectively. Consistent tumor growth with the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. Analysis of the learning curve demonstrated that this model is easy to learn and quick to establish. It enables intra-individual follow-up endoscopies, and features tumors to study mechanisms of metastasis and the interaction with the immune system. The application of specific cell lines and concentrations enables a controlled local tumor growth and metastatic formation within short observation periods.Open orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these drawbacks, this study aimed to evaluate the establishment of a minimally invasive model using murine colonoscopy. CT26 and MC38 CRC cells of different concentrations were injected into BALB/C and C57BL/6J mice, respectively. Follow-up endoscopies were performed to assign an endoscopic score to tumor growth. Gross autopsy, histologic and immuno-histochemical evaluation, and immune scoring were performed. To describe the learning curve of the procedures, a performance score was given. Local tumor growth with colorectal wall infiltration, luminal ulceration, the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. The tumors showed cytoplasmic immuno-staining for CK20. Compared to the MC38/C57BL/6J model, tumorigenicity and immunogenicity of the CT26/BALB/C model were higher. Tumor volume correlated with the endoscopic score. This endoscopy-guided orthotopic mouse model is easy to learn and quick to establish. It features early metastasis and enables the study of interactions with the immune system. When specific cell concentrations and cell lines are applied, controlled local tumor growth and metastasis can be achieved within short observation periods.

DNA damage response genes mark the early transition from colitis to neoplasia in colitis-associated colon cancer

Chronic intestinal inflammation predisposes patients with Inflammatory Bowel Disease (IBD) to Colitis-Associated Cancer (CAC). In the setting of chronic inflammation, microsatellite instability (MSI) results from early loss of DNA damage response (DDR) genes, ultimately leading to tumor formation. Despite continued efforts to improve early detection of high risk, pre-dysplastic regions in IBD patients, current macroscopic and genetic surveillance modalities remain limited. Therefore, understanding the regulation of key DDR genes in the progression from colitis to cancer may improve molecular surveillance of CAC. To evaluate DDR gene regulation in the transition from colitis to tumorigenesis, we utilized the well-established Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) pre-clinical murine model of CAC in C57BL/6 mice. In order to assess colonic tumor burden in the setting of mutagen and intestinal irritation, tumors were visualized and graded in real time through high-resolution murine colonoscopy. Upon sacrifice, colons were opened and assessed for macroscopic tumor via high magnification surgical lenses (HMSL). Tissues were then sectioned and separated into groups based on the presence or absence of macroscopically visible tumor. Critical DDR genes were evaluated by semi-quantitative RT-PCR. Interestingly, colon tissue with macroscopically visible tumor (MVT) and colon tissue prior to observable tumor (the non-macroscopically visible tumor-developing group, NMVT) were identical in reduced mRNA expression of mlh1, anapc1, and ercc4 relative to colitic mice without mutagen, or those receiving mutagen alone. Colitis alone was sufficient to reduce colonic ercc4 expression when compared to NMVT mice. Therefore, reduced ercc4 expression may mark the early transition to CAC in a pre-clinical model, with expression reduced prior to the onset of observable tumor. Moreover, the expression of select DDR genes inversely correlated with chronicity of inflammatory disease. These data suggest ercc4 expression may define early stages in the progression to CAC.

Abstract 618: Dual PI3K/mTOR inhibition as a treatment strategy for PIK3CA and APC mutant colorectal cancers

Abstract BACKGROUND: Colorectal cancer (CRC) is understood as a diverse group of cancers calling for the personalization of therapeutics to individual subtypes of CRC. Mutations in PIK3CA, which result in a constitutively active phosphoinositide-3-kinase protein (PI3K), are present in 20-30% of CRCs. Aberrations in the Adenomatous Polyposis Coli (APC) gene are found in 86% of PIK3CA mutant CRCs. The potential for treatment of tumors with this specific profile is of great interest. METHODS: Mice expressing a constitutively active PI3K and loss of APC were generated and spheroid cultures were derived from the resulting cancers. PIK3CA and APC mutant spheroids were treated with GDC0941 (GDC), a pan PI3K inhibitor, NVP-BYL719 (BYL), a PI3K alpha isomer specific inhibitor, or NVP-BEZ235 (BEZ), a dual mTOR/PI3K inhibitor. Proliferation was measured by changes in spheroid diameter over time. BEZ treatment of mice with PIK3CA and APC mutant CRCs was performed over 14 days. Tumor response was measured with serial murine colonoscopy and dual hybrid 18F-FDG PET/CT imaging. RESULTS: Persistent PIK3CA and APC mutant CRC spheroid growth was observed with BYL and GDC treatment, while BEZ treatment resulted in a significant reduction in spheroid size. Immunoblotting determined that this correlated with significant suppression of phosphorylated AKT, RPS6 and 4EBP1 in BEZ-treated spheroids and incomplete PI3K pathway inhibition following BYL and GDC treatment. BEZ treatment was then investigated in mice with PIK3CA and APC mutant CRCs. BEZ elicited a dramatic treatment response in these cancers on endoscopy, with a 53% decrease in median lumen occlusion compared to a 60% increase in controls over the 14 day treatment period. PET/CT imaging results confirmed these findings demonstrating a decrease in tumor size and avidity post BEZ treatment. BEZ resulted in inhibition of the PI3K pathway in these tumors. Reactivation of PI3K signaling was observed within 24 hours of the withdrawal of BEZ. BEZ was well-tolerated in these mice and associated with a decrease in the endoscopic anemia score. CONCLUSIONS: PIK3CA and APC mutant CRCs are a potentially targetable subtype of cancer. These cancers are resistant to proximal inhibition of the PI3K pathway, but are responsive to dual PI3K/mTOR inhibition. These results warrant further investigation in clinical studies. Citation Format: Susan Payne, Tyler Foley, Cheri Pasch, Alexander Yueh, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. Dual PI3K/mTOR inhibition as a treatment strategy for PIK3CA and APC mutant colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 618.

Primary Myofibroblasts Maintain Short-Term Viability following Submucosal Injection in Syngeneic, Immune-Competent Mice Utilizing Murine Colonoscopy.

The myofibroblast is an important stromal cell of the gastrointestinal tract. Current in vitro and in vivo models either do not accurately recreate stromal-epithelial interactions or are not specific to myofibroblasts. We sought to create an animal model that would allow the study of myofibroblast-epithelial interactions. We isolated and cultured colonic myofibroblasts from FVB mice. Cells were α-SMA and vimentin positive but desmin negative on immunoblot analysis. We injected the myofibroblasts into the colonic submucosa of syngeneic adult mice (n = 8) via a miniendoscopic system. We then isolated green fluorescent protein (GFP) positive colonic myofibroblasts from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and injected them into the colonic lamina propria of C57BL/6J mice at 1x105 (n = 14), 1x106 (n = 9), or 5x106 cells/mL (n = 4). A subset of mice were injected with serum-free media and ink without cells (n = 3). Mice underwent repeat endoscopy and euthanasia one or 7 days after injection. Colons were isolated and either fixed in 10% formalin or the inked sites were individually excised and lysed for DNA. We assessed the injection sites via histology and immunohistochemical stains for α-SMA and GFP. We used qPCR to quantify GFP DNA transcripts at the lamina propria injection sites. Submucosal injection of myofibroblasts resulted in the formation of a subepithelial wheal on endoscopy, which persisted to day 7. Myofibroblasts injected either into the submucosa or lamina propria maintained viability on post-injection day 7 as evidenced by positive α-SMA staining. qPCR of lamina propria injections showed a dose-dependent increase in GFP DNA transcripts on post-injection day 1, whereas the number of transcripts on day 7 was equivalent for the concentrations injected. We demonstrate short-term survival of primary cultured colonic myofibroblasts in syngeneic mice. This may prove to be a valuable model for studying the role of myofibroblasts in states of health and disease.

Abstract 1009: Tumor-specific expression of fatty acid synthase promotes angiogenesis in colorectal cancer

Abstract Up regulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation of metastasis by FASN are elusive. The induction of angiogenesis is crucial for metastasis. Therefore, the purpose of the present study was (i) to determine the role of FASN in regulation of secretion of angiogenic factors by CRC cells, (ii) to evaluate the effect of tumor-specific expression of FASN on endothelial cells, and (iii) to assess the role of FASN in regulation of tumor vasculature. METHODS: The effect of FASN inhibition on secretion of angiogenic factors was analyzed using the Human Angiogenesis Antibody Array 1 (RayBiotech). Co-culture of CRC and HMVEC-L cells was used to assess the effect of FASN expression on growth, migration, and tube formation of endothelial cells. To evaluate the effect of FASN on vasculature, we utilized murine colonoscopy, the orthotopic colon cancer model, the chick chorioallantoic membrane (CAM) model, and the Matrigel plug assay. RESULTS: We found that inhibition of FASN is associated with an evident change in the profile of secreted angiogenic factors by CRC cells and this change is associated with a significant decrease in microvessel density as determined by analysis of CD31 staining (Aperio Image Scope) in orthotopic colon tumors. A decrease in expression of FASN is also associated with “normalization” of tumor vasculature in the CAM model. The mechanisms of inhibition of angiogenesis by knockdown of FASN included down regulation of VEGF189, an isoform associated with aggressive CRC, up regulation of anti-angiogenic isoform VEGF165b, and a decrease in expression and activity of MMP-9. Furthermore, medium conditioned on FASN-knockdown CRC cells inhibited proliferation, migration, and tube formation of endothelial cells via inhibition of VEGFR-2 activation and its downstream signaling as compared to control medium. CONCLUSIONS: This study suggests that FASN promotes angiogenesis via alteration of the profile of secreted angiogenic factors by CRC cell and stimulation of endothelial cell proliferation. Furthermore, the regulation of angiogenesis by FASN, at least in part, is mediated by up-regulation of VEGF189, a VEGF-A isoform that tightly correlates with poor prognosis in CRC, and by an increase in activity of MMP-9. Together, these findings suggest that inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC. Citation Format: Yekaterina Y. Zaytseva, Victoria Elliott, Piotr Rychahou, William Mustain, Ji Tae Kim, Joseph Valentino, Tianyan Gao, Kathleen O'Connor, Janna Neltner, Heidi Weiss, B. Mark Evers. Tumor-specific expression of fatty acid synthase promotes angiogenesis in colorectal 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 1009. doi:10.1158/1538-7445.AM2014-1009

Sa1711 Primary Myofibroblasts Maintain Short-Term Viability Following Submucosal Injection in Syngeneic, Immune-Competent Mice Utilizing Murine Colonoscopy

Sa1711 Primary Myofibroblasts Maintain Short-Term Viability Following Submucosal Injection in Syngeneic, Immune-Competent Mice Utilizing Murine Colonoscopy

Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer

BackgroundColorectal-cancer (CRC) research has greatly benefited from the availability of small animal tumor models. Spontaneous and chemically-induced CRC models are widely used yet limited in their resemblance to human disease and are often prolonged, not accurately repetitive, and associated with inflammatory side effects. In-situ murine or human tumor implantation in the gastrointestinal tract of mice is extremely challenging, and limited by inter-animal variability and procedure-related complications and mortality. As a result, in frequent studies CRC is implanted in distal sites, most commonly the subcutaneous region, an approach that is greatly limited by the absence of normal gastrointestinal tumor milieu and has substantial effects on tumor development.AimsIn this study we aimed to develop a well-tolerated repetitive tool to study CRC in small animals by adapting the murine colonoscopy system to serve as a platform for colonic sub-mucosal orthotopic implantation of human and murine CRC tumor cells.ResultsWe report the establishment of a novel small-animal CRC model that is minimally invasive, rapid, well-tolerated, highly reproducible, and confers precise control of tumor number, location and growth rate. Moreover, we show that this model uniquely allows the side-by-side induction of distinct genetically manipulated tumors, enabling the mechanistic study of tumor interaction and cross-talk within the native intestinal microenvironment.ConclusionsEmployment of this new approach may represent a major technical advance for the in-vivo study of CRC.

Simultaneous fluorescence imaging of protease expression and vascularity during murine colonoscopy for colonic lesion characterization

Molecularly targeted fluorescent probes are currently being developed to improve the endoscopic detection of intestinal pathologic conditions. We report on the development and testing of a novel multichannel microendoscope capable of quantitatively reporting such probes simultaneously at different wavelengths in real time. We assessed the feasibility of detecting and quantifying beacons that can be activated by protease and correlating imaging with disease state. The microendoscope consisted of a 20-gauge fiberoptic catheter and dichroic beam splitters that simultaneously display visible light, 700 nm and 800 nm near infrared (NIR) fluorescent light. NIR interchannel separation was tested on in vitro phantoms. Two mouse models were used (Apcmin(+/-) mice for colonic adenomas and CT26 murine colon cancer). A perfusion probe and one activated by protease at a separate wavelength were injected before endoscopic evaluation. The microendoscope fluorochrome detection limit was approximately 10 fmol; ratio imaging in the NIR was accurate (+/-8% of true probe concentration between 0.3 to 100 microg/ml of a protease sensor). Both colonic adenomas and adenocarcinomas were clearly visible in the NIR channel on protease probe administration in live mice. Ratio imaging of protease activity/perfusion increased from healthy colon to adenomas to adenocarcinomas. Evaluation across additional spontaneous tumor models may provide more data on the translation of these findings. Our data show the feasibility of multichannel microendoscopic imaging of molecular targets in vivo and that ratio imaging may provide a novel means for characterizing colonic lesions. When scaled up clinically, this could aid in increasing lesion detection and quantitative assessment of distinct molecular markers.

Colonoscopy in mice.

Current investigational models of murine colitis and colon cancer necessitate sacrifice of animals in order to obtain colonic tissue. The purpose of this study was to develop a safe method of murine colonoscopy that would allow serial evaluation and mucosal biopsies of the same animal. Nine mice (two C3H, two C57/BL6, and five IL-10 deficient) were studied a total of four times each over 4 weeks. Three mice [APC (Min +/-)] were examined three times each. Mice were gavaged with 1 cc of a polyethylene glycol solution on the day prior to colonoscopy. Solid chow was withheld and the mice were maintained on Pedialyte. Mice were anesthetized with ketamine and xylazine. A flexible pediatric cystoscope (2.1-mm diameter) with a single biopsy channel was introduced per anum, and the colon was gently insufflated with air to a mean pressure of less than 5 mmHg. Saline irrigation was used when necessary. A single biopsy was obtained from the rectosigmoid colon during each examination. A total of 46 examinations were carried out. One mouse died after being anesthesized for the fourth examination, and two mice [one IL-10 knockout and one APC (Min+/-)] died one day after the 3rd examination. No other complications were noted. The average length of insertion was 3 cm. Transillumination allowed for localization of the endoscope tip. Biopsies, although quite small, were sufficient for pathologic evaluation and diagnosis. Murine colonoscopy is a safe and feasible technique. It permits consecutive visual and histopathological examinations, and it allows the investigator to monitor the response of the murine colon to experimental interventions.