Xi Liang,Jingnan Hu,Jianming He

Department of Radiotherapy,Hebei Province Hospital of Chinese Medicine,Hebei University of Chinese Medicine,Shijiazhuang 050011,China

Key words:animal model;colorectal cancer;inflammation;protocol

Abstract Since azoxymethane (AOM)-dextran sodium sulfate (DSS) induced tumorigenesis was used to explore inflammation-associated carcinogenesis of sporadic colorectal cancer (CRC),different administration modes of AOM or DSS have been reported.In this article we optimized the protocol of the AOM-DSS modeling using C57BL/6 mice for study on sporadic CRC by intraperitoneal injecting AOM solution at a proper concentration with a 100 μl sterile syringe once,feeding with DSS solution for 7 days in a roll and change DSS solution every day.More than 100 C57BL/6 mice had been treated with the optimized protocol,and all mice were demonstrated suffering from colorectal tumors when sacrificed in 8 to 20 weeks after AOM injection.These tumors mainly occurred in distal segment of colorectum with an increase in tumor density,which was similar to CRC in human beings.Tumor per mouse was high,and variation of tumor number per mouse was low.The histology of tumor developed through the defined stage ranged from precursor lesions,adenomatous lesions,adenomas to adenocarcinomas.The modified protocol of AOM-DSS model is easy,cheap,with high tumor formation rate of colorectal tumors.

INTRODUCTION

Background

Colorectal cancer (CRC) is the third most common cancer in males and the second in females.1The incidence is increasing in most of developing countries,which is partly attributed to lipid metabolism.1-3According to its incidence,CRC is categorized in three forms:familial,hereditary,and sporadic (about 80% of CRC).4,5

Manyin vitromodels andin vivomodels have been developed and used in understanding CRC.2,6In vivomodels include inoculated CRC models and tumors induced by“diet”(high fat,low calcium,etc.),by chemical,by gene mutation or genetic modification(APC lines,Kras,Msh2,P53,Rb,etc.).7-9Subcutaneous inoculation models are widely used to study responses of CRC to chemotherapy,radiotherapy and other types of therapy.10,11Inoculated CRC models (the orthotopic model,the intrasplenic inoculation model,the intraportal inoculation model,etc.) are options to study metastasis of CRC.9,12“Diet”(high fat,low calcium,etc.)alone can hardly induce tumors and they are commonly employed as a factor of promoting tumors.13Gene mutant or genetically modified colorectal tumor models are primarily used for studies addressing hereditary aspects of CRC.8,9These models are confounded by development of tumors outside colorectum.8,9,13Furthermore,these models rarely develop malignant tumors,let alone metastases.8,9,13

Chemically induced colorectal tumor models are optimal to study sporadic CRC.2,7,9,14Carcinogens used in these models include dimethyhydrazine(DMH),azoxymethane (AOM,a downstream metabolite of DMH),dextran sodium sulfate (DSS),2-amino-1-methyl-6-phenyl imidazol (4,5-b) pyridine (PhIP),N-methyl-N'-nitro-N-nitrosoguanidine (MNNG),methylnitrosourea (MNU),3,2'-dimethyl-4-aminobiphenyl(DMBA).8,9,13,14AOM can initiate cancer by alkylation of DNA.2,7,14DSS is a pro-inflammatory reagent and using DSS alone can hardly induce colorectal tumor.2,7,14However,the combination of AOM and DSS (the AOMDSS model) has been proved to be an easy,cheap,rapid and reproducible model with high incidence of colorectal tumors.2,7,14AOM-DSS induces much higher tumor incidence,more tumors per mouse at earlier time point than most of other carcinogens.2,8,13All tumors locate in colorectum,increase in density at the distal end,which is similar to CRC in human beings.2,14Tumors develop through defined stages ranging from precursor lesions (abberant crypt foci formation or mucin-depleted foci),adenomatous lesions,adenoma to adenocarcinomas.2,14Tumors induced by AOM-DSS resemble human CRC at the molecular level.Mutations of β-catenin play a key role in CRC carcinogenesis,progression,chemoresistance.AOM-DSS induced colorectal tumors often have gene mutations at codons 32 to 34 in mice and this is similar to human CRC.9,15The protein level of β-catenin increases in almost all tumors in the AOM-DSS model and in most of human CRC.2,7Other genes and proteins,for example,Kras,cyclin D1,also share the same feature in both the AOM-DSS model and human CRC.7Consequently,the AOM-DSS model is well accepted as an excellent model to analyze sporadic and inflammation-driven human CRC.

Several AOM-DSS models (1 AOM followed by 1-5 cycles of DSS) have been reported.7,14,16,17The 1 AOM followed by 3 cycles of DSS model and the 1 AOM followed by 1 cycle of DSS model are widely used.2,14The prior model mimics inflammatory bowel disease (IBD) course of human beings,including Crohn's disease and ulcerative colitis.IBD is strongly associated with inherited factors and is undoubtedly a precancerous disease.7,8It develops both chronic colitis and colitis activity,and deep inflammatory lesions,active colitis,Ul-III ulcer and even more serious ulcer commonly coexists.18,19Therefore,it is widely used to study the role of inflammation in carcinogenesis of CRC.Most of sporadic CRC are not,at least not strongly,correlated with inflammation.To study sporadic CRC,the 1 AOM followed by 1 cycle of DSS model may be more ideal because it develops neither chronic colitis nor flares of colitis activity,at the same time,it has lower mortality rate (below 10%) than the 1 AOM followed by 3 cycles of DSS model.2,7,9,14In the 1 AOM followed by 1 cycle of DSS model,inflammation in colorectum gradually decrease after DSS treatment, and Ul-II ulcer or serious ulcer are not found in the colorectum in 6 week.20Several researchers mentioned that 1 AOM followed by 1 cycle of DSS induces tumors slower than 1 AOM followed by 3 cycles of DSS,but,to our knowledge,no head-tohead study has been reported.7Here,we studied the 1 AOM followed by 1 cycle of DSS model,addressing spontaneous colorectal tumor.

APPLICATION AND VALUE

We modified a few technicalities of the 1 AOM followed by 1 cycle of DSS models (Figure 1) and described the whole procedure of modeling in detail in this article.

Unprecise AOM intraperitoneal injection will definitely cause high variability.Some researchers injected intraperitoneally AOM working solution of 1 mg/ml,10 μl/g body weight using a 1 ml syringe.As the body weight of a C57BL/6 mouse is about 20 g to 25 g,usually 200 μl to 250 μl AOM solution is injected,that is a volumn not easy to be injected accurately with a 1 ml syringe.We proposed to inject intraperitoneally AOM working solution at a concentration of 2.5 mg/ml,volume of 4 μl/g body weight using a 100 μl sterile syringe for C57BL/6 mice.Under this condition,a mouse will be injected 80 μl to 100 μl AOM solution and the injection is easy to be performed accurately.

Different modes of DSS administration were reported.2,7Because DSS is not stable in solution at room temperature in light,we replaced DSS-containing water every day to avoid the influence of DSS degradation.

This protocol with technicality modifications is expected to provide researchers an easy,cheap,rapid and reproducible model with high incidence of CRC for studies on carcinogenesis,progression,prevention,biomarkers,identification and pre-clinical validation of therapeutic targets.2,7However,as an AOM-DSS model,it still shares the same limitations such as coexistence of benign tumors and malignant tumors in an experimental mouse,more low-grade cancers than high-grade cancer,as well as rare metastases.2,8

EXPERIMENTAL DESIGN

Mice selection

The study was approved by the Ethics Committee of Hebei Province Hospital of Chinese Medicine.This protocol has been reported in previous study with mice and rats.2,21Different mouse strains show different susceptibility to AOM or DSS.2,7,9,14Researchers may adjust concentration and volume of AOM solution,concentration and duration of DSS according to the employed strain.We have successfully used this protocol without any adjustment on six-to seven-week-old male C57BL/6 mice in our previous study.2

As in human beings,lots of factors increase CRC risk,such as heredity,gender,age,obesity,smoking,consumption of red meat,alcohol,gut microbiota,1,2,13in mice,heredity,gender,age,body weight,gut microbiota,diet,etc.,also influence colorectal tumorigenesis.2,7,22Therefore,taking these factors into account,we suggest that both the control mice and the experimental mice be maintained in one cage to exclude potential effects from uncertain factors,such as gut microbiota,diet,etc.

AOM treatment

Figure 1.The schematic of the AOM-DSS model protocol.AOM,azoxymethane;DSS,dextran sodium sulfate.

Different mouse strains show different susceptibility to AOM.Some mouse strains,such as Crj,A/J,SWR/J or FVB/N are considered to have high to moderate susceptibility to AOM,while C57BL/6,Balb/C,ICR,AKR/J,129SvJ and DBA/2J are considered to have low susceptibility to AOM.2,7,9,14,18A dose of 10 ± 2.5 mg AOM per kg body weight was reported in studies of different mouse sublines.7,23Some researchers injected intraperitoneally AOM working solution of 1 mg/ml,10 μl/g body weight.For C57BL/6 mice,we proposed to inject intraperitoneally AOM working solution of 2.5 mg/ml,4 μl/g body weight with a 100 μl sterile syringe once.It is easy to handle and easy to inject accurately the volumn as needed.If used in other stain of mice,adjusting concentration of AOM working solution may be needed.

DSS treatment

Different mouse strains show different susceptibility to DSS.C57BL/6 and ICR showed moderate to high susceptibility to DSS,while FVB/N mice were low to moderately susceptible to DSS.2,7,9Variant dose (1%-4%weight/volume),apply time (5-7 days) and administration method of DSS have been reported.7,9,24Dr.Neufert suggested to feed mice with DSS-containing water at day 7 and replaced the water at day 10 and day 12.7Because DSS is not stable enough in solution at room temperature in light,degradation will accumulate with time.To avoid the influence from DSS degeneration,we empty and refill DSS-containing water every day to ensure its effectiveness.

Waiting time period

The pro gress of tumor also depends on the duration of induction.AOM/DSS treatment in mice offers a powerful model ranged from precursor lesions to adenocarcinoma.2,9,25Precursor lesions represent early end points and in this case,they should be scored several weeks after DSS treatment.According to literature,3 to 13 weeks of waiting period were adopted.7,26,27To score spontaneous tumor progression,mice are commonly sacrificed at 10 to 20 weeks after DSS treatment,rarely at 29 weeks after AOM treatment.2,7,25

Monitoring body weight

Since both AOM and DSS are toxic,the body weight of mice should be monitored.AOM rarely causes decrease in body weight but almost all mice decrease their body weights at the beginning or after DSS treatment because of enteritis caused by DSS.If body weight of a mouse decrease by more than 20% within 1 week during this period,euthanasia is suggested.The mortality of mice is usually kept under 5%;if the mortality reaches 10% or higher,0.5% to 1% of concentration reduction is required.Colorectal tumors cause weight loss too,but this is expected to happen in a few months after DSS treatment.Prolapse of rectum and hematochezia are common symptoms at 10 weeks after DSS treatment.

MATERIALS

Regents

AOM (Sigma-aldrich;Cat.:A5486)

DSS (MP Biomedicals;Cat.:0218055880-100g)

PBS (beyotime;Cat.:C0221A)

Equipments

N95 mask (Medicom;Cat.:MED0002321-0080020),

100 μl syringes (Hamilton Company;Cat.:80601)

0.22 μm filters (Millipore;Cat.:SLGP033RB)

60 ml plastic water bottles with a steel ball in the steel tube (Alex;Cat.:AL142)

Biological safety cabinets (Thermo Scientific;Cat.:51025411)

PROCEDURES

Mouse preparations for AOM-DSS induced colorectal tumorigenesis

1.Gender-and age-matched C57BL/6 mice are housed in cages in a specific pathogen-free animal facility under standard conditions of 22°C±2°C with a daylight cycle from 6 AM to 6 PM.

2.Label mice.Divide mice into groups,maintain both control mice and experimental mice in one cage if the experiment has a control group and a experimental group.Allow no less than one week of acclimation before any treatment.

Note:a) Use animals of the same gender,male mice are preferred to avoid influence from menstrual periods.b) We suggest that the age gap in one experiment be less than 1 week.c) Perform the step 2 at mouse age of three-to five-week old to reduce fighting among mice,and the earlier the better.d) We suggest that mice in control group and experimental group are littermates.This could reduce effects by differences in origin/genetic backgrounds.e) If possible,separate mice in different cages and one cage contains both control mice and experimental mice.

AOM stock solution preparation

3.When mice reach six-to eight-week-old,weigh them.Calculate the amount of AOM needed (10 mg/kg body weight).Dissolve AOM in saline at the concentration of 10 mg/ml,and filtrate AOM solution with a sterile 0.22 μm filter.

4.Aliquot and store at -20°C in sterile glass tubes.

Note:a) AOM is more stable in saline than in PBS.b) AOM solution should be stored in glassware rather than in plastic tubes.c) Aliquot to avoid multiple freeze/thaw cycles of stock solution.d) Mice with body weight below 18 g are not used.

AOM treatment

5.Thaw stock solution in amount as needed;dilute the AOM stock solution 1:4 in sterile saline to obtain working solution at a concentration of 2.5 mg/ml.

6.Weigh mouse and calculate the volume of AOM working solution needed by weight (4 μl/g body weight).

7.Inject AOM working solution intraperitoneally with a 100 μl sterile syringe.

8.After AOM injection,weight mice and record for day 0.

Note:a) When treated with AOM,the body weight gap of mice in one experiment should be less than 5 g.b) As AOM is a strong carcinogen and flammable,AOM stock solution and working solution should be prepared in a laminar airflow biosafety cabinetry,and intraperitoneal injection in a fume hood.c) We suggest the administration of AOM working solution be a one-time intraperitoneal injection at a proper concentration using a 100 μl sterile syringe.d) In case that body weigh of a mouse is over 25 g,the concentration of AOM working solution should be calculated to meet the maximum volume of a 100 μl sterie syringe.

The treatment interval

9.House mice for 6 days and weight mice every day.

DSS treatment

10.On day 7,prepare 2% DSS solution of the amount as needed (6 ml per mouse per day for 7 days).

11.Fill the 60 ml plastic water bottle of the mouse cage with 2% DSS solution and record the volume (A mouse drinks less than 5 ml DSS solution per day).Store the left DSS solution at 4°C.

12.On the next day,record the volume of DSS solution left in the bottle.Then,empty and refill the water bottle with DSS solution stored.

13.Repeat step 11 and 12 every day from day 9 to day 13.

14.On the day 14,write down the left DSS solution in the bottle,replace the water bottle and fill it with water.

Note:a) DSS is toxic.Preparation of DSS solution should be in a laminar airflow biosafety cabinetry.For procedure 10-14,the operator should wear a N95 mask or a better one.b) Molecular weight of DSS should be 36,000-50,000 Da.c) We suggest DSS solution be replaced every day.

Waiting period

15.House mice for 5 to 20 weeks.

Colon collection

16.At the time point of choice,fast mice for 6 to 12 hours.

17.Euthanize mice.Immobilize the dead mouse in a supine position.Spray the abdomen with 70%ethanol.

18.Open the abdominal cavity using forceps and scissors.Cut the pelvic bone to dissect the distal part of the colon completely.

19.Remove the entire colon (including the rectum and the anus) using forceps and scissors.

20.Immediately sink the colon in a Petri dish filled with icy cold PBS.

21.Open the colon longitudinally,rinse the colon twice with ice cold PBS to clean up feces,and be ready for subsequent studies.

TIMING

Steps 1-2,mouse preparations:1 week.

Steps 3-4,AOM stock solution preparation:1-2 hours.

Steps 5-8,AOM treatment:1-4 hours (depending on the number of mice).

Step 9,time interval between AOM and DSS treatment:6 days.

Steps 10-14,DSS treatment:every day for 7 days.

Step 15,waiting period for collection:5 to 20 weeks (depending on the objective of the study).

Steps 16-21,colon collection:1 day.

ANTICIPATING RESULTS

The AOM-DSS model in this protocol is optimized to induce colorectal tumor for the study of sporadic CRC in mice.All C57BL/6 mice treated using this protocol had colorectal tumors when sacrificed 8 to 20 weeks after AOM injection.The incidence of tumors in C57BL/6 mice after AOM treatment was 100%,and the multiplicity at 8 weeks,10 weeks and 20 weeks were 5.17±0.95,6.83±0.79,and 11.83±1.58,respectively (Table 1).All tumors located in colorectum,with higher tumors density in distal end (Figure 2),which is similar to CRC in human beings.After AOM-DSS treatment,colorectal tumors increased in number and size along with time.Tumors developed through defined stages ranged from precursor lesions(aberrant crypt foci formation or mucin-depleted foci),adenomatous lesions,adenoma to adenocarcinomas(Figure 3).The progression of tumors also depends on the time duration,which means,colorectal tumors increased in number,size and ratio of malignancy along with time.

This protocol offers an easy,rapid,reproducible and pow erful model for studies on CRC.It produces high tumor incidence,more tumors per mouse,and low variation in tumor number per mouse.Meanwhile,although after DSS treatment,the body weighs of almost all mice decreased due to enteritis caused by DSS,most of these mice regained weight to the pretreatment level in two weeks after termination of DSS treatment (Figure 4).The body weight of mice decreased at the beginning of and early after DSS treatment (the second week),regained to the level before DSS treatment in two weeks after the termination of DSS treatment.The data shown are the mean ± standard error.

Table 1.The colorectal tumor in C57BL/6 mice induced by AOM-DSS model after AOM injection

TROUBLE SHOOTING

Figure 2.Gross specimen photographs of colorectums in mice with colorectal tumors induced by AOM-DSS.A.Mouse sacrificed 10 weeks after AOM injection;B.mouse sacrificed 20 weeks after AOM injection.Colon was excised(up) and longitudinally opened (down),showing tumors were mainly distributed at the distal end of colorectum,increased in number and size along with time.

Figure 3.Histopathological findings of colorectal adenoma (up) and colorectal adenocarcinoma (down) of mice induced by AOM-DSS model (sacrificed 20 weeks after AOM injection) (HE staining).

Figure 4.Body weight changes of the mice treated with AOM-DSS along with time (n=10).

Conflict of interest statement

The authors declared no potential conflict of interests.