Colitis-Associated Colorectal Cancer in Mice: In vivo Disease Imaging and Insight into the AOM/DSS Model

Detta är en Master-uppsats från Lunds universitet/Tillämpad biokemi

Sammanfattning: Inflammatory bowel diseases as ulcerative colitis and Cohn’s disease represent significant health problems in the western world. Colon cancer is a common consequence of those chron- ic inflammatory disorders, thereby it is essential to improve knowledge and research level of this area. AOM/DSS is a known and well-established chemically induced mice model of coli- tis associated cancer. In order to make research based on this model more efficient and relia- ble, improvements are desired. In vivo imaging with the use of specific fluorescent probes is a very convenient and recently strongly developed method worth exploiting in monitoring tumors development. Furthermore, introduction of knockout model enabling in depth exploration of colon cancer mechanisms shades light on the behavior of the disease. Particularly, due to the importance of insulin re- ceptor, as a key regulator of homeostasis, also involved in cancer pathology, it would be of high interest to explore it in terms of colon cancer. On the other hand, exploring physiology of the healthy colon is also essential in fighting this serious medical issue and culturing colon epithelium ex vivo gives a broad range of opportunities for this matter. 3D in vitro culture of colon crypts enables development of spherical organoids, imitating intestinal epithelium envi- ronment. In combination with mice of different genetic background, growing colon crypts raises a vast spectrum of exploration. The used methodology was based on a combination of Azoxymethane (AOM) with dextran sulfate sodium (DSS) that allowed induction of colitis associated colon cancer mice model. Tumor development was confirmed with weights monitoring, colonoscopy and tumor counts. In vivo imaging system with the use of fluorescent probes was tested for imaging of tumors in AOM/DSS model. Two types of probes potent for emitting signal in near-infrared light were used: active IntegriSense 680 and activatable ProSense 750. Insulin receptor knock out mice were genotyped to confirm loss-of-function mutation. Colon crypts were taken from healthy mice donors and 3D matrigel-based culture was set up in order to induce organoids develop- ment and survival in vitro. As a result, AOM/DSS model was successfully obtained which was confirmed with numerous tumors developed in distal part of the colon. In vivo imaging with IntegriSens 680 did not give significantly strong signal either in vivo or ex vivo. Activatable probe ProSense 750 how- ever failed in vivo imaging of induced tumors, provided some specific distinguishable signal ex vivo. Colon crypts were successfully isolated from mouse donors. Culture was maintained for 9 days during which colonoids grown and developed into variously shaped spherical struc- tures. AOM/DSS model resembles human colitis associated colon cancer. Although utilized fluorescent probes did not give expected results in vivo, ProSense 750 due to positive ex vivo measurement sheds a light on potential use and future optimization of that method.

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