Murine models of colorectal cancer: the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of colitis-associated cancer

Spontaneous models

Spontaneous tumors in the intestine of laboratory mice and rats develop rarely. For example, C57BL/6J mice developed colon cancer at 1% of cases. In colon cancer-prone rats substrain WF-Osaka the incidence of colon carcinoma varied from 30 to 40%. Spontaneous models are rarely used due to unpredictability and low reproducibility (Nascimento-Gonçalves et al., 2021).

Genetically engineered murine models (GEMMs)

Genetically engineered models of colorectal cancer make it possible to study the genetic predisposition to the development of colorectal cancer, interaction with environmental and modifying factors, tumor microenvironment, as well as systemic immune responses (Bürtin, Mullins & Linnebacher, 2020; Nascimento-Gonçalves et al., 2021).

According to Ye et al. (2020) KRAS, KIT, PIK3CA, MET and EGFR are among the most frequently mutated oncogenes, and TP53, APC, CDKN2A, STK11 and FBXW7 are the most frequently mutated tumor suppressor genes in patients with CRC. In GEMMs mutations in Apc, p53, K-ras genes and DNA mismatch repair genes (MMR) are the most oftenly used, but other genetic mutations are also taken (De-Souza & Costa-Casagrande, 2018; Stastna et al., 2019; Oliveira et al., 2020; Bürtin, Mullins & Linnebacher, 2020; Nascimento-Gonçalves et al., 2021; Li et al., 2022a; Neto et al., 2023).

Apc The first genetic model for CRC was described in 1990. Min (Multiple intestinal neoplasia) is an ethylnitrosourea (Enu)-induced mutation in the murine Apc (adenomatous polyposis coli) gene (Moser, Pitot & Dove, 1990). The line of mice was named Apc^Min. The acronym “min” means multiple intestinal neoplasms, and this is an autosomal dominant mutation, which in homozygous conditions is lethal to animals. Animals that are heterozygous for the mutation develop important anemic conditions at 60 days of life and develop tumors in the large and small intestine. As in familial adenomatous polyposis cases, Apc^Min animals also develop colorectal adenomas, but they die at 120 days of life (De-Souza & Costa-Casagrande, 2018). APC encodes a key negative regulator of the Wnt-signaling pathway and it represents the most frequently mutated gene in CRC. Approximately 90% of sporadic colorectal tumors carry a mutation in APC. The APC locus was discovered through studying a rare hereditary syndrome, familial adenomatous polyposis (FAP). As the sequence identity of the human and mouse APC proteins exceed 89%, the mouse represents a suitable model to study the involvement of APC truncations in intestinal cancer (Stastna et al., 2019). The Apc^Min mouse model is the only animal model of cancer that contains a single genetic alteration capable of producing a fully penetrating, consistent, and organ-specific tumor phenotype (Nascimento-Gonçalves et al., 2021). Throughout aging, there was a high mortality of Apc^Minanimals because of intestinal obstruction and anemia. A significant part of animals died before the tumor progresses to invasive carcinoma (Bürtin, Mullins & Linnebacher, 2020). Moreover, the polyps predominantly develop in the small intestine, and to a way lesser extent in the colon, at the same time it is very rare for people to develop tumors of the small intestine (Stastna et al., 2019).

Mouse strains with other targeted genetic modifications at different locations of the Apc gene, such as Apc^Min/850, Apc^Δ716, Apc^1638N, Apc1^638T, Apc^Δ468 and Apc^Δ474, were developed to allow reproduction of disease models closer to CRC in humans and study the role of certain regions of the APC gene in the development of cancer. Transgenic mice with different mutations of the APC gene differ in the number of tumors and their morphology (Stastna et al., 2019; Bürtin, Mullins & Linnebacher, 2020; Nascimento-Gonçalves et al., 2021). Also rat model with Apc mutation created: Pirc rats. They developed adenomas similar to those found in humans, demonstrated the same progression to invasive carcinomas, and dependence on gender was observed, with males more prone to develop tumors in the intestinal tract than female rats (Nascimento-Gonçalves et al., 2021).

DNA MMR The most common form of hereditary CRC in humans is hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome. It is caused by autosomal dominant mutations in the DNA mismatch repair genes such as MLH1, MSH2, MSH6, and PMS2, that leads to the development of various cancers, including colorectal cancer. Developed tumors are not specific to the colon-rectum; they occur in other organs such as skin, lungs, lymphatic system, stomach, and small intestine (Nascimento-Gonçalves et al., 2021). Inactivation of DNA MMR genes leads to the development of microsatellite instability and hypermutative tumor phenotype. Microsatellite instability is also detected in some cases of spontaneous CRC (Bürtin, Mullins & Linnebacher, 2020). Therefore, a number of mouse models were developed with knockouts MMR genes such as Mlh1, Mlh3, Msh2, Msh6, and Pms2, which is in details described in the Lee, Tosti & Edelmann (2016).

p53 Mutations in the p53 gene are found in 60% of human CRC (Neto et al., 2023). Animals with p53 knockout rarely develop or don’t develop colorectal tumors (De-Souza & Costa-Casagrande, 2018). Surprisingly, given the proposed role for loss of function mutations of the P53 gene in the development of human colorectal cancer, Clarke, Cummings & Harrison (1995) found no evidence for either an increase in the rate of adenoma formation in Apc^+/- p53^-/- animals, or an increased rate of progression to malignancy compared with Apc^+/- p53^+/+mice. The role of mutations in p53 in the development of colon cancer is being actively studied, and the suppression of the mutant p53 function via the inhibition of nuclear accumulation is expected to be an effective strategy against malignant progression of colorectal cancer (Nakayama & Oshima, 2019).

KRAS KRAS belonging to the RAS family, is one of the most prominent proto-oncogenes, and associated with various oncogenic pathways including PI3K/AKT/mTOR signaling to promote proliferation and to suppress apoptosis of tumor cells. KRAS mutation found in more than 40% of patients with CRC (Li et al., 2022a). Mice with mutations in codon 12 of the K-ras gene (K-ras^G12D) have regions of hyperplasia in the colon as well as aberrant crypt and ring cells. Associating the K-ras^G12D and Apc^Min mutations lead to increase in the number of lesions in the colon, as well as the presence of completely undifferentiated cells. Another mutation, K-ras^v12, alone is not capable of inducing tumorigenesis, but once it is associated with mutations in Msh2 gene, it promotes a greater number of tumors in the small and in the large intestine, than Msh2^-/- animals (De-Souza & Costa-Casagrande, 2018).

Thus, genetically engineered models made a huge contribution to understanding the molecular mechanisms of CRC initiation and progression. However, these models include a number of limitations. Firstly, cancer development is a step-by-step process with initial driver mutation and subsequent acquisition of further mutations, and therefore this process can only be partially captured in mouse tumor models by combination of mutation. Secondly, the number of combined mutations in mice is limited, as the resulting phenotype often demonstrates a dramatic lifespan reduction. In addition, creating genetically engineered models is time consuming and expensive, as breeding transgenic mice often takes several generations and requires careful breeding to produce the desired changes. With regard to animal welfare, it should be noted that during the breeding process, many “rejected” mice appear, which are not used either for further breeding or for the research purposes. Furthermore, the constructing technique for a transgene or viral vector requires special skills. Although genetically engineered models are a valuable tool for fundamental research, their use for preclinical studies is limited due to the lack of genetic heterogeneity, on the one hand, and inconsistencies with the mechanisms of human tumor development, on the other (Bürtin, Mullins & Linnebacher, 2020).

Transplant models

Transplant models are performed by transplanting a tumor from one living organism to another. Transplant models are classified according to three main parameters:

(1) Type of transplant. Either tumor cell suspensions or tumor tissue fragments are used. It is easier to work with a cell suspension, cheaper, and the tumor engraftment rate is higher. Cell lines of tumors are phenotypically and genetically quite homogeneous and do not reproduce the heterogeneity of real tumors; in addition, tumor cells due to in vitro selection usually tend to be poorly differentiated and developing tumors are more aggressive in comparison to human CRC. Tumor tissue fragments make it possible to preserve the initial tumor microenvironment, genetic and molecular heterogeneity of tumor cells, and are more adequate model, but their engraftment rate is lower.

(2) Source of the graft. Syngeneic tumor transplantation is characterized by the engraftment of tumor tissue or a tumor cell line within the same animal line; xenogeneic transplants are obtained from other animal lines or human donors. Patient-derived xenografts retain the pathological and molecular characteristics of individual patient CRC and are therefore the most reliable for preclinical drug development. However, in xenograft models, tumor cells or tumor fragments are implanted in immunocompromised animals, which makes it impossible to study the role of immune responses in carcinogenesis.

(3) Site of the transplantation. The implantation of tumor could be various: directly into the colon or rectum (orthotopic models) or subcutaneously, into the renal capsule, spleen, bloodstream (heterotopic models). The most widely used method in case to initiate reproduction, provide access and fast tumor growth is subcutaneous inoculation, nonetheless, the tumor microenvironment is different from the colon and metastasis do not develop. In comparison to heterotopic models the orthotopic model, are technically more difficult to create, requiring the use of imaging techniques (e.g., ultrasound) to control cell implantation and animal anesthesia.

Transplant models make it possible to reproduce tumor invasion, cancer progression to advanced stages, and metastasis to other organs. But they are not suitable for studying the induction and early stages of carcinogenesis, as well as studying the role of the immune system and inflammation in the pathogenesis of CRC (De-Souza & Costa-Casagrande, 2018; Stastna et al., 2019; Oliveira et al., 2020; Bürtin, Mullins & Linnebacher, 2020; Nascimento-Gonçalves et al., 2021; Neto et al., 2023).

Chemically-induced CRC models (CIMs)

Carcinogen-induced CRC in mice is a rapid, relatively inexpensive, highly reproducible model that mimics the human adenoma-to-adenocarcinoma progression sequence.

At the moment, a wide range of chemically induced CRC models are available. The most commonly used groups of carcinogens are:

(1) heterocyclic amines (HCAs) such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP);

(2) aromatic amines such as 3,2-dimethyl-4-aminobiphenyl (DMAB);

(3) alkylnitrosamide compounds such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU);

(4) 1,2-dimethylhydrazine (DMH), azoxymethane (AOM), methylazoxymethanol (MAM).

The introduction of chemical carcinogens to animals is possible with the free access to water and food or through a gastric tube, enema, or intraperitoneal or subcutaneous injection (Stastna et al., 2019; Li et al., 2022a).

Heterocyclic amines

Heterocyclic amines are among the most common genotoxic mutagens present in the environment, and the human body is exposed to them in daily life. Heterocyclic amines are formed in food when amino acids and proteins are heated. Throughout frying meat and fish, various imidazoquinoline, imidazoquinoxaline and imidazopyridine compounds are formed, which have a strong mutagenic effect.

Among heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinolone (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are used to induce CRC in rodents. Rats given long-term IQ or PhIP develop tumors in the small and large intestine, mammary gland, and prostate. However, the incidence of colon tumors is low and ranges from 5% to 28% when these drugs are administered with food for up to 1 year. When adding 100 p.p.m. PhIP in rat diets takes approximately 2 years to reach a 50% incidence of colon cancer, and at 25 p.p.m. PhIP after 2 years no colon carcinomas were observed. It should be stated that the combination of PhIP with a high-fat diet leads to accelerated tumor formation.

Thus, the model of CRC induced by heterocyclic amines is applicable for the development of methods for preventing the development of tumors under the action of food carcinogens. The disadvantages of this model are the relatively low incidence of tumor development and too long time of their development (De-Souza & Costa-Casagrande, 2018; Stastna et al., 2019; Oliveira et al., 2020; Neto et al., 2023).

Aromatic amines

Lorenz & Stewart (1941) initially observed the chemical induction of intestinal tumors in mice treated with polyaromatic hydrocarbons—dibenzanthracene or methylcholanthrene. DMAB shares structural similarities with mutagens found in well-done meat. The carcinogenic effect of DMAB (3, 2′-dimethyl-4-aminobiphenyl) was first described in 1950th by Walpole, Williams & Roberts. The researchers established the induction of colon tumors in rats by DMAB subcutaneous administration.

Subcutaneous weekly administration of DMAB at 50 mg/kg body weight in male F344 rats provided multiple colon tumors in 27% animals fed low and 75% animals fed high fat diet, respectively (Doi et al., 2007). DMAB contributes to both adenomas and adenocarcinomas epithelial neoplasms with a multiplicity of 1.2–2.7 tumor nodes per animal.

One of the key shortcommings of this model is that it suggests multiple injections of DMAB to induce colon tumors. On a molar basis, DMAB is considered to be less effective than AOM or DMH in rodent models. Another shortcoming of this model is the initiation of neoplasms in other organs: mammary adenocarcinomas in female rats, salivary gland sarcomas, squamous cell carcinomas of the ear canal and skin, squamous cell papillomas of the fundus of the stomach (forestomach), sarcomas and lymphomas, and bladder urothelial carcinomas (Nascimento-Gonçalves et al., 2021; Neto et al., 2023).

Alkyl nitrosamide substances

Methylnitrosourea (MNU) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) are direct alkylating agents that do not require metabolic activation and thus are potent local carcinogens.

Intrarectal injection of MNU at 1–3 mg per rat weekly for 5 month provided colon tumors in 100% of male F344 rats, of which 43% of the tumors were adenocarcinomas and 57% were adenomas. All neoplasms were detected in rectum and distal colon, where MNU and MNNG were injected.

When using alkylnitrosamide substances in rats and mice, most of the induced colon tumors were sessile or polypoid lesions that were well differentiated and often invaded the submucosa. Metastases are usually not detected.

Since biochemical activation is not necessary, these carcinogens are perfect for initiation tumors of colon in animals and investigation the changing effects of xenobiotics without establishing the metabolism of the initiating carcinogen. MNU and MNNG administered intrarectally selectively initiate tumors in the rectum and distal colon, such models are widely spread.

The main disadvantage of this model is that intrarectal injection is a significant technical problem, the reproducibility of such experiments depends on the skills of the experimenter, and the quantification of carcinogens administered intrarectally is difficult (Bürtin, Mullins & Linnebacher, 2020; Nascimento-Gonçalves et al., 2021; Neto et al., 2023).

DMH, AOM, MAM

The most commonly used chemicals to induce CRC are 1,2-dimethylhydrazine (DMH) and its metabolite azoxymethane (AOM). They are potent carcinogens, causing a wide range of mutations in key genes that code for components of multiple intracellular signaling cascades (De-Souza & Costa-Casagrande, 2018; Stastna et al., 2019; Bürtin, Mullins & Linnebacher, 2020; Nascimento-Gonçalves et al., 2021; Li et al., 2022a; Neto et al., 2023).

DMH is oxidized in the liver to azomethane, which is then oxidized to AOM and then hydroxylated to methylazoxymethanol (MAM). MAM enters the intestine with bile or through the bloodstream in the form of glucuronides and glucosides.

MAM is cleaved by the effect of colon and liver enzymes to form formaldehyde and a strong alkylating agent,—the methyldiazonium ion. DNA alkylation results in base mismatch and mutagenesis. Thus, guanine is methylated in DNA at the N-7 position. Alkylated guanine combines with thymidine instead of cytosine. In the course of further replication, a nucleotide replacement occurs (Venkatachalam et al., 2020).

DMH is typically administered subcutaneously, but intraperitoneal and intrarectal administration were also stated in the literature. Dosing, number of injections and duration of the experiment vary significantly: 2–200 mg/kg body weight, 1–30 injections, 2–20 weeks (Venkatachalam et al., 2020). Protocol proposed by Gurley, Moser & Kemp (2015), suggests administering 15 µg of DMH per gram of body weight to mice subcutaneously once a week for 12 weeks.

AOM is administered, as a rule, once a week intraperitoneally at a dose of 10-15 mg/kg of animal body weight for 2-10 weeks, the development of tumors is assessed after 16-36 weeks from the start of the experiment (Waly et al., 2014; Whetstone et al., 2016; Velázquez et al., 2016; Uyar et al., 2022). According to Whetstone et al. (2016) in male Balb/c mice, which were intraperitoneally injected with 10 mg/kg of AOM once a week for 6 weeks, 30 weeks after the first injection of AOM, adenomas were observed in 63% of the animals, and adenocarcinomas—in 21%.

The introduction of DMH or AOM leads to the development of epithelial neoplasia, which begins with the developing of abnormal crypts in the colon—the so-called aberrant crypt foci (ACF); ACF further progresses to adenoma and then to malignant adenocarcinoma (Stastna et al., 2019).

Different mice strains vary according to the AOM sensitivity. A/J and SWR/J mice are highly sensitive to AOM with high incidence of colon tumors. C57B/L6 and Balb/c mice are moderately sensitive with a relatively lower incidence of colon tumors in comparison to A/J and SWR/J mice, while administration of AOM to AKR/J and 129/SV mice does not induce colon tumors (Rosenberg, Giardina & Tanaka, 2009).

The disadvantages of this model are the long tumor induction period, not very high reproducibility potential, and the need for multiple injections of carcinogen.