Cancer Research
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Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA)
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Summary December 19th, 2019
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.
Transcript
This two step skin cancer model enables the study of the role of inflammation in skin cancer and allows the study the tumor initiation as a separate process from tumor progression. The tumor formation in this model is chemically induced and allows the use of knockout strains to study the effects of environmental factors or therapeutic candidates. The model is well suited for studying the role of immune system and angiogenesis in cancer.
It demonstrates a similar disease pathology to squamous cell carcinoma in human patients. Although this method is relatively easy to conduct, accurate timing is essential. It's also important to take appropriate safety measures when handling DMBA, which is a carcinogen.
Before beginning the experiment, house any aggressive seven to nine week old mice in separate cages to avoid fighting and skin lesions. For skin papilloma induction, first shave the back skins of the experimental animals and weigh the mice individually. 48 hours after shaving, use a pipette to apply 50 micrograms of DMBA in 200 microliters of acetone onto the shaved area of each anesthetized mouse.
For skin papilloma promotion, seven days after DMBA application treat the skin with five micrograms of TPA in 200 microliters of acetone two times a week through the end of the experiment. Examine the animals for papillomas and photograph and record the size of each individual papilloma on a map every week. A palpable mass greater than one millimeter in diameter is considered a papilloma if it remains longer than one week.
When the tumor response reaches a plateau, harvest the appropriate sample material from the animals 24 hours after the last TPA application and collection full thickness pieces of skin for immunohistochemical analysis. Use a biopsy punch to collect skin pieces from papilloma and non-papilloma skin samples for gene expression and or protein anaylses and use a scalpel to collect pieces from papilloma and non-papilloma skin for immunohistochemical analysis. Then collect the skin draining lymph nodes and a piece of the spleen for flow cytometric analysis as desired.
A statistically significant difference in the papilloma free time and in the number of papillomas between the experimental groups is typically observed in the DMBA TPA tumor model. Histological analyses are suitable for studying the structure of the papillomas, the morphology of the skin, or the infiltration of immune cells of interest. It is essential to apply the DMBA and TPA evenly on the skin and to apply TPA in consistent intervals.
It also important to count the papillomas regularly. The mechanisms that affect the number and time of tumor formation can be studied using different methods, including immunohistochemistry, flow cytometry, quantitative PCR, and protein analysis. This conventional technique can be easily adapted and applied to study the effects of different genes, environmental factors, or therapeutics of interest on tumor initiation and progression.
Caution is required when performing the technique since DMBA is carcinogenic and acetone evaporates quickly. A suitable breathing mask and the use of flow cabinet are recommended.
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