Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA)

Summary

Two-stage skin carcinogenesis is induced by two topically applied chemicals. A mutagen 7,12-dimethylbenz[a]anthracene) causes mutations in the epidermal cells and a continuous application of general growth stimulator 12-O-tetradecanoyl phorbol-13-acetate accelerates skin papilloma formation.

Abstract

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different cell types and genes in promoting tumor progression and to provide a platform for testing the efficacy of different therapeutic approaches. One of the most commonly used experimental inflammatory cancer models is the DMBA-TPA two-stage skin carcinogenesis model. Tumor formation is induced in this model by the topical application of two different chemicals, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA), that together cause papilloma formation in the skin. As the primary outcome is papilloma formation in the skin, the model is an ideal, reliable, and reproducible way to address both tumor initiation (tumor-free survival) and tumor progression (number and size of visible tumors). The effects of the DMBA-TPA treatment are transmitted via an inflammatory mechanism, which makes this model especially suitable for studying the role of the immune system in tumor formation. However, this model is restricted to the skin and other surfaces where the chemicals can be applied on. A detailed protocol is provided in this article to use the model successfully.

Introduction

Cancer is one of the leading causes of death in the world. Therefore, there is a demand to develop reliable experimental disease models to obtain a better understanding of the disease as well as to explore potential therapeutic approaches. One of the most commonly used experimental in vivo models to study skin cancer development is the chemically induced two-stage skin carcinogenesis model^1,2. The model provides a tool to study tumor initiation, promotion, and progression in addition to specific events such as immune cell infiltration and angiogenesis.

To use the two-stage skin carc....

Protocol

The protocol described here has been approved by the National Animal Ethics Committee of Finland (protocol number ESAVI/23659/2018).

1. Experimental Animals, Reagents, and Equipment

1. Use age and sex-matched mice. Start the study at 7–9 weeks of age, because the skin in most mice is in telogen (the resting phase) around that age².
2. Observe the behavior of the animals during the study and if they fight, which often happens with males, house them separately. Fights may cause cuts in the skin, which promote tumor formation. Female mice are preferred due to their less aggressive behavior. The typ....

Results

The main outcome is the survival (i.e., papilloma free) time between the treatment or genotype groups. The secondary outcome is the number of papillomas per week in each group (Figure 2). The expected results are a statistically significant difference in the papilloma free time and in the number of papillomas between the experimental (two or more) groups. It is recommended to count the number of papillomas and draw a curve during the promotion (TPA) phase to get an idea of the differences be.......

Discussion

DMBA-TPA-induced skin cancer is one of the most commonly used cancer models because it is highly reproducible and provides information on tumor progression from initiation to malignancy. The key outcome measure, papilloma formation, is easily and reliably quantitative. The model addresses both tumor initiation (tumor-free survival) and progression (tumor numbers and sizes) simultaneously. The model is suitable for studying different compounds, such as potential therapeutics, and the effects of individual genes on tumor p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1000 ul RPT XL Graduated Filter Tip (Sterile), Refill	Starlab	S1182-1730-C
300 ul RTP Graduated Filter Tip (sterile), Refill	Starlab	S1180-9710-C
7,12-Dimethylbenz[a]anthracene (DMBA)	Sigma	D3254-100MG	Harmful if swallowed and may cause cancer. Store protected from light.
Acetone	Sigma	1000141011	Evaporates rapidly and is inflammable.
Attane vet 1000 mg/g	Piramal Critical Care Limited		Liquid isoflurane for inhalation
Battery-Operated Clipper Isis	Albert Kerlb GmbH	GT421	For shaving the fur
CONTRAfluran-Restgasfilter	ZeoSys GmbH		For anesthesia
Linex Nature N1030 Ruler 30 cm	Staples Business Advantage	60383	For measuring papillomas
Medium CO2 Chamber 300 x 200 x 200mm - Red	VetTech Solutions Ltd	AN045AR	For sacrifice
Mekasoft	Mekalasi	23008	Table cover
Mice (Balb/c JRj)	Janvier labs		Other strains also possible
Mice (C57BL/6JRj)	Janvier labs		Other strains also possible
Panasonic Lumix DMC-FS5 Digital Camera	Panasonic
Paraformaldehyde	Merck	30525-89-4	For histology samples
Phorbol 12-myristate 13-acetate aka 12-Otetradecanoylphorbol-13-acetate (TPA)	Enzo	BML-PE160-0001
Precision balance PLJ-C/PLJ-G	KERN & SOHN GmbH	PLJ 600-3CM
Pre-Set CO2 System-2 Chamber-S/S Housing	VetTech Solutions Ltd	AN044BX	For sacrifice
RNAlater	Qiagen	76104	For nucleic acid samples
Tacta pipette 100-1000 ul	Sartorius	LH-729070
Tacta pipette 20-200 ul	Sartorius	LH-729060
UNO Anaesthetic Key Filler	Scintica instrumentation inc.		For anesthesia
UNO Face Mask for Mouse	Scintica instrumentation inc.		For anesthesia
UNO FM2200 Flowmeter	Scintica instrumentation inc.		For anesthesia
UNO Gas Exhaust Unit	Scintica instrumentation inc.		For anesthesia
UNO Induction Box	Scintica instrumentation inc.		For anesthesia
UNO200VAP Vaporizer	Scintica instrumentation inc.		For anesthesia