We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.
Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.
Colorectal cancer (CRC) is the 4th most common cause of malignancy worldwide1. Despite the significant progress in our understanding of the familial basis of this disease, genetic predisposition only contributes to ~20% of CRC cases2. The remainder are attributed to numerous extrinsic and environmental factors, including chronic inflammation. In humans, the link between chronic inflammation and colon cancer is evident in ulcerative colitis (UC) patients, who have a greater risk of developing colitis-associated colon cancer (CAC), depending on the duration, extent and severity of the inflammatory disease3-5. Accordingly, novel therapies are in development to control the immune response and the associated production of growth promoting factors by the inflammatory tumor microenvironment6-8. There is an increasing requirement for appropriate pre-clinical animal models to characterize the therapeutic efficacy of these drugs against the development and progression of disease.
Mouse models have unequivocally demonstrated that the inflammatory microenvironment contributes to CRC progression, even in the absence of overt inflammation9,10. These models include the use of the polysaccharide dextran sulfate sodium (DSS), provided in the drinking water of mice, to model epithelial injury and acute and chronic inflammatory bowel disease (IBD)11,12. Although the mechanism by which DSS induces mucosal damage and colitis is not completely understood, some studies suggest that DSS inhibits cellular reverse transcriptase and ribonuclease activities within cells, or promotes the formation of nano-lipid complexes that fuse with the colonic membrane leading to epithelial damage13,14. Modifications to the standard DSS model have also provided significant insight into the mechanisms by which colonic epithelial cells maintain tissue homeostasis and regulate mucosal immune responses15.
Intraperitoneal administration of Azoxymethane (AOM) alone, or in combination with DSS, provides a model for examining the interplay between somatic mutations in the epithelial mucosa and the inflammatory and stromal microenvironment16,17. AOM is a metabolite of the carcinogen 1,2-dimethylhydrazine (DMH) that does not directly result in DNA mutations. Instead, AOM is hydrolyzed to methylazoxymethanol (MAM) by the cytochrome isoform CYP2E1 in the liver, where MAM is conjugated with glucuronic acid and then transported to the intestine through bile secretions18. It is thought that the bacterial β-glucuronidase contributes to the degradation of MAM resulting in DNA alkylation and the accumulation of mutations in epithelial cells19. Most AOM-induced colonic tumors harbor missense mutations in the gene encoding β-catenin, rendering the protein resistant to proteasomal degradation, which results in aberrant activation of the canonical Wnt-signaling pathway20. When the activity of AOM is combined with the mucosal damage elicited by DSS, the ensuing wound healing response creates a microenvironment that is conducive to the growth and expansion of the mutagenized epithelium. In a variation of this model, repetitive administration of AOM alone over a period of several weeks can be used to model sporadic colorectal cancer, in the absence of DSS-induced colitis10,17. These two complimentary models provide experimental settings to study CAC and sporadic CRC respectively, both of which are associated with a pro-inflammatory tumor microenvironment10.
The use of serial endoscopy in mice was pioneered by Becker and colleagues21, and enables longitudinal monitoring of colitis and tumor progression. Here we provide three pre-clinical protocols based on DSS-induced mucosal damage and/or AOM-mediated tumor induction to reproducibly induce specific colonic pathologies. The first protocol describes inducing acute mucosal damage in response to DSS administration to elicit many of the histopathological features associated with IBD. The second protocol is based on three consecutive cycles of DSS administration to mimic the flares of inflammation commonly observed in IBD patients, and can be carried out in conjunction with AOM-induced mutations. The final protocol is based on AOM-induced sporadic epithelial mutations. For each of these protocols, we expand on the relevant standard procedures to include prophylactic and therapeutic intervention methods that we have developed to monitor the efficacy of new drugs.
The three protocols that are described outline methods of reliable and reproducible induction of colonic disease pathology in mice. When combined with routine endoscopic monitoring and the intervention strategies outlined here, these protocols will provide powerful pre-clinical insight into the efficacy of therapeutics. Our laboratories routinely use all of these protocols to monitor the success of novel therapeutics10,23,24.
There are a number of considerations when choosing a pre-clinical animal model to test new therapeutics. These include relevance of the model to the human disease, and the contribution of the tumor microenvironment to the proposed action of the therapeutic target. Here we provide three protocols for therapeutic intervention in established intestinal disease models. These models are reproducible and the delivery of reagents to induce disease is easy to manage. Importantly, the models are highly relevant to multiple facets and stages of colitis onset, and of tumor initiation and progression. Researchers should take into consideration the genetic background of the mouse strains used when designing experiments, as the susceptibility to disease induced by DSS and/or AOM can vary considerably25. In addition, different microbial communities may have different metabolic capacities in the context of AOM, which is metabolized by bacteria. We caution against using different cohorts of mice that were born into different animal facilities (including commercial vendors) in a single experiment. Similarly, the different microflora in mice used from different facilities may elicit different host responses to DSS-induced epithelial barrier damage11. Moreover, the appropriate analysis of tissue (for example RNA purification) should also be considered, since the ability of DSS to inhibit reverse transcriptase will impact on subsequent molecular analysis26,27.
Mouse endoscopy is a cutting edge technique to repeatedly monitor disease onset and progression in an individual mouse. The ability to record videos and extract still images permits easy monitoring of multiple disease parameters and tumors. In addition to improving animal welfare, endoscopic monitoring also reduces the need for multiple cohorts of experimental mice, which traditionally were culled at different time-points to track disease outcome. The MEICS scoring system is not a substitute for histopathological analysis, but provides an alternative means to monitor animal health and mucosal damage in live mice. Mouse endoscopy is a specialized laboratory technique, and all procedures should be performed by trained personnel to ensure appropriate manipulation and handling of the mice, as well as to provide consistent quality in the images used for disease scoring. In the hands of qualified personal, we have found that endoscopy induces little or no damage to the tumors that would cause intra-tumoral bleeding. For the therapeutic protocols outlined, we consider endoscopy highly advantageous, since it provides a way to determine the initial tumor burden, and allows us to group cohorts of animals with similar tumor burdens prior to the administration of a therapeutic drug. Sequential monitoring of the mice enables researchers to determine the efficacy of novel therapies early on, with the option of terminating unsuccessful experiments in a timely manner.
As our understanding of inflammatory bowel disease and colorectal cancers advance, new targets for therapy will be identified. Appropriate animal models will be integral to ensuring that the most promising new therapies are moved towards clinical trials.
The authors have nothing to disclose.
We would like to thank CSL Ltd. for supporting the purchase of the endoscopy equipment. The research in the laboratory of ME is supported by the Ludwig Institute for Cancer Research, and the laboratories of TP and ME are supported by the Victorian State Government Operational Infrastructure Support and the National Health and Medical Research Council of Australia. ME is an NHMRC Senior Research Fellow.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Dextran Sulfate Sodium (MW 36,000-50,000) | MP Biochemicals | 160110 | Requires batch testing. |
Azoxymethane | Sigma | A5486-100MG | Requires batch testing. |
Vanilla Protein Shake | N/A | N/A | Available from hospital pharmacies. |
Isoflourane | PPC | M60303 | This is a restricted reagent, which should be stored under lock and key. |
70% Ethanol | N/A | N/A | Standard lab reagent. |
Coloview miniendoscopic system | |||
Endovision Tricam | Karl Storz | 20212001-020 | |
Xenon 175 light source with anti-fog pump | Karl Storz | 20134001 | |
HOPKINS straight Forward Telescope | Karl Storz | 64301AA | |
Endoscopic Sheath (total diameter 3 mm) | Kalr Stroz | 61029C | |
Fubre Optic Light Cable | Kalr Stroz | 69495ND | |
Computer and media player software | MAC, imovies | ||
Scale | Any | Any scale suitable for weighing mice. |