AOM/DSS Model of Colitis-Associated Cancer

Summary

Our understanding of colitis-associated carcinoma (CAC) has benefited substantially from mouse models that faithfully recapitulate human CAC. Chemical models, in particular, have enabled fast and efficient analysis of genetic and environmental modulators of CAC without the added requirement of time-intensive genetic crossings. Here we describe the Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) mouse model of inflammatory colorectal cancer.

1. Introduction

Colorectal cancer (CRC) is the fourth most common cancer in the world (1). It is well established that colitis predisposes individuals to colorectal tumorigenesis (2–4). Patients with inflammatory bowel disease, for example, are at an elevated risk for developing colon cancer, although the magnitude of this risk has recently come under debate (5–11). While the molecular pathogenesis of colitis-associated cancer (CAC) remains incompletely understood, significant advances have been made from studying murine models of CAC. Here we outline the application of the Azoxymethane (AOM)/Dextran sodium sulfate (DSS) model of CAC. The AOM/DSS model is a powerful, reproducible, and relatively inexpensive initiation-promotion model that utilizes chemical induction of DNA damage followed by repeated cycles of colitis (12–15).

AOM (Methyl-methylimino-oxidoazanium, CH₃N=N(→O)CH₃) is a procarcinogen that is metabolized by cytochrome p450, isoform CYP2E1, converting it into methylazocymethanol (MAM), a highly reactive alkylating species that induces O⁶ methylguanine adducts in DNA resulting in G→A transitions (16). After excretion into the bile, it is taken up by colonic epithelium and induces mutagenesis. DSS is a heparin-like polysaccharide that is dissolved in the drinking water and inflicts colonic epithelial damage, inducing colitis mimicking some of the features of IBD (17). Combining AOM and DSS provides a two-step tumor model of CAC.

Key features of the AOM/DSS model include its relatively short timeline and accurate modeling of CAC. Tumor development can occur in as short as 10 weeks (12). Moreover, the histopathology of AOM/DSS-induced tumors recapitulates key facets of human CAC such as distally-located tumors and invasive adenocarcinomas (13). Application of the AOM/DSS model has been critical in unraveling the pathogenesis of CAC: from the role of signaling pathways (e.g. Toll-like receptor 4, IKKβ, and IL-6 (18–20)) and antioxidant machinery (e.g. glutathione peroxidase (21)) to the influence of the microbiota (22) and transcriptional corepressors (e.g. Myeloid translocation genes (23)). Thus, the AOM/DSS model is a powerful platform to employ when studying the pathogenesis of inflammatory colorectal cancer.

2. Materials

1. 1 mg/ml Azoxyemethane solution: Dissolve 10 milligrams of AOM (Sigma-Aldrich, CAS 25843-45-2, Cat# A4586) in10 ml of sterile Phosphate-buffered saline (PBS). Filter the solution using a 0.45 µm cellulose acetate filter and aliquot into 1 ml sterile eppendorf tubes. Aliquots can be stored at −20 °C for up to a year.

2. 0.5 ml Tuberculin Syringe 28^1/2 G (Monoinject, Cat # 8881500105)

3. 3% (w/v) Dextran Sodium Sulfate: Weigh 30 grams of DSS (Affymetrix Cat# 14489, MW 40–50 kDa) and dissolve into 1 liter of water. Once the DSS is dissolved, filter sterilize the solution using 0.45 µm cellulose acetate filter.

4. Scale for weighing mice (Ohaus Scout Balance, Model SP402)

5. 10% Buffered Formalin (Fisher Scientific, Cat# SF100-4)

6. 70% Ethanol: Dilute 190 proof ethanol to 70% ethanol with sterile, deionized water.

7. Isoflurane, USP (Phoenix Pharmaceuticals, Inc., Cat# NDC 57319-474-06)

8. Tissue Pathology Macrosette Cassettes (Fisher Scientific, Cat# 15182706)

9. 20G Straight feeding needle (VWR International, Cat# 2068-612)

10. Dissection Scissors Sharp/Blunt Tip (VWR International, Cat# 82027-588)

11. Waugh Forceps (VWR International, Cat# 82027-428)

12. 27^1/2 G Precision Glide Needle (BD Biosciences, Cat# 30519)

13. Whatman Blotting Paper (VWR International, Cat# 28298-020)

14. Carbon Fiber Composites Digital Caliper (Fisher Scientific, Cat# 15-007-958)

15. Nalgene Surfactant-Free Cellulose Acetate (SFCA) Filter (Cole-Palmer, Cat# EW-06731-2)

16. RNAlater (Invitrogen, Cat# AM7020)

17. RIPA buffer (Thermo Scientific, Cat# 89900)

18. Sterile Phosphate-Buffered Saline (PBS)

3. Methods

3.1 Treating mice with AOM/DSS (see Figure 1 for an example of a typical experimental timeline)

Figure 1.

Schematic timeline for AOM/DSS induced inflammatory carcinogenesis.

Day 1: Injecting Azoxymethane

• 1.Ensure experimental groups are age and gender-matched with control mice (see Note 1). Weigh 8–12 week old C57BL/6 (see Note 2) mice and record weights. Accurate weights are required in order to ensure uniform dosing of AOM (see Note 3). We recommend weighing each mouse three times to increase precision. Calculate the volume of AOM (1 mg/ml) to inject to achieve a dose of 12 mg/kg. For example: a 25 gram mouse would receive a 300 µl injection of 1 mg/ml AOM solution. It may be necessary to reduce the dosage if substantial toxicity is observed (see Note 4).
• 2.Once you have recorded weights and injection volumes, anesthetize mice using isoflurane in accordance with your institution’s IACUC protocols. Using a 28^1/2 G tuberculin syringe, inject each mouse intraperitoneally with the appropriate volume of AOM.
• 3.Place the mice back in their cages. Weigh and monitor them over the next 48 hours.
• 4.If your mouse facility provides lixit drinking valves or other automatic watering systems, be sure to cap or disengage this water supply to ensure each cage only has one water supply. It is important for mice to become accustomed to drinking only from a water bottle, as this will be the source of DSS (see Note 5). We recommend disengaging automatic watering systems for the duration of the experiment (see Note 6).

Day 3: Start DSS cycle 1

• 5.Replace drinking water in cages with 3% DSS formula.

Day 3–8: Monitoring Animals: DSS cycle 1

• 6.Weigh mice daily to evaluate response to DSS-induced colitis.
• 7.During treatment with DSS, mice can lose significant body weight depending on strain and genotype (see Figure 2). If mice lose substantial body weight (between 10% and 20% weight loss relative to the day prior to DSS administration), it is advisable to administer up to 1 ml of sterile saline via IP injection or provide wet food (see Note 7).
• 8.If mice lose greater than 20% body weight, demonstrate hunched posture, or move in a limited fashion, then it may be necessary to euthanize the animal. Be sure to follow all appropriate IACUC protocols.

Figure 2.

Example of weight loss during repeat cycle DSS treatment.

Day 8: End DSS Cycle 1

• 9.Replace 3% DSS with sterile drinking water.

Day 9–12: Initial Recovery

• 10.It is important to continue to monitor the mice, especially in the 3–5 days after replacing the 3% DSS with water. It is not unusual for mice to continue to lose weight several days after 3% DSS administration.

Day 13–24: Recovery

• 11.Weigh mice every 2–3 days.

Day 25: Start DSS cycle 2

• 12.Replace water with 3% DSS and weigh mice daily.

Day 25–30: Monitoring Animals: DSS cycle 2

• 13.Monitor and weigh mice as exactly as detailed in Day 3–8.

Day 30: End DSS cycle 2

• 14.Replace 3% DSS with water.
• 15.Tumor burden can be safely monitored via endoscopy (Figure 3A) throughout the duration of the experiment. We recommend using endoscopy 1 week after completion of the second cycle of DSS (see Note 8).

Figure 3.

A) Above: Endoscopy image of normal colon. Below: Endoscopy of colon after AOM injection followed by two cycles of DSS. White arrows indicate tumors. B) Above: Colons harvested and oriented with the distal end toward the dissector. Below: Example of gross tumor burden. Images are reproduced from prior report (23).

Day 30–44: Recovery

• 16.Weigh mice every 2–3 days.

Day 45: Start DSS cycle 3

• 17.Replace drinking water in cages with 3% DSS formula.

Day 45–50: Monitoring Animals: DSS cycle 3

• 18.Monitor and weigh mice as exactly as detailed in Day 3–8.

Day 50: End DSS Cycle 3

• 19.Replace 3% DSS with sterile drinking water.

3.2 Sacrificing Mice

1. Weigh mice before euthanizing. Euthanize mice by a combination of inhalational isoflurane overdose and cervical dislocation or other institutionally, IACUC-approved protocols. Expose the ventral side of the mouse by placing the mouse on a surgical dissection table with its abdomen facing up. Secure legs for unobstructed access to the abdomen. Cover the abdomen with 70% ethanol to prevent fur from interfering with dissection.

2. Using forceps pinch and pull the abdomen up at the midline (thus forming a “tent”). Using scissors incise the pinched abdominal tissue to access the peritoneum. Then extend the incision to the xyphoid process at the midline (away from the dissector) and to the costal margins bilaterally (toward the dissector). Gently push peritoneal fat and small intestine to the side and locate the cecum (see Note 9)

3. Once the cecum is identified, cut immediately distal to isolate proximal colon. Follow the colon using forceps and gently dissect away the mesentery. Cut through the pelvis to allow removal of the distal colon including the anus (see Note 10). Because DSS-induced colitis damages the distal colon, it is critical to remove the entire colon to accurately assess tumor burden.

4. Flush the colon with PBS using a 10 ml syringe. Place the colon lengthwise on Whatman paper (see Figure 3B) with the distal end (anus end) nearest the dissector and the proximal (cecum end) furthest away. Cut the colon longitudinally along the proximal-distal axis so that the colon is splayed open length-wise and the distal most portion of the colon is located nearest the dissector.

5. Assess and record tumor burden grossly. Tumor size can be measured using digital calipers. If desired, isolate tumor tissue or adjacent tissue for RNA or protein analysis using a scalpel. Place tissue for RNA analysis directly into RNAlater; for protein analysis, place tissue directly in lysis buffer with protease and phosphatase inhibitors. We recommend doubling the normal concentration of protease and phosphatase inhibitors to preserve protein integrity (see Note 11).

6. Using two fine-tipped forceps held in two hands, grasp both lateral sides of the distal edge of the colon and roll the colon. The end product should be a rolled colon resembling a Swiss roll and the distal colon will be in the center and the proximal colon will be the outermost layer (see Figure 4 and Note 12). Once the colon is rolled, place a 27^1/2 gauge needle through the roll to secure it. Place the Swiss-rolled colon into a labeled tissue cassette.

7. Immerse the cassette into 10% buffered formalin for 24 hours.

8. Process samples by for histological analysis according to your lab’s preferred method (see Note 13).

Figure 4.

Hematoxylin and eosin stain of Swiss-rolled colon. The most distal portion of the colon is located in the innermost segment of the roll. A large polyp can be seen in the distal colon surrounded by inflammatory infiltrate.

Figure 5.

Representative histology of a distal colonic tumor isolated from a mouse treated with the AOM/DSS protocol. Pathology features indicating injury include inflammatory infiltrates are observed at low power (top panel) and intratumoral crypt abscesses (middle panel). Features of neoplasia such as hyperchromatic nuclei and increased nuclei/cytoplasmic ratios are identified at higher magnification (middle and bottom panel).