Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications

Abstract

For more than 60 years, the chemical induction of tumors in mouse skin has been used to study mechanisms of epithelial carcinogenesis and evaluate modifying factors. In the traditional two-stage skin carcinogenesis model, the initiation phase is accomplished by the application of a sub-carcinogenic dose of a carcinogen. Subsequently, tumor development is elicited by repeated treatment with a tumor-promoting agent. The initiation protocol can be completed within 1-3 h depending on the number of mice used; whereas the promotion phase requires twice weekly treatments (1-2 h) and once weekly tumor palpation (1-2 h) for the duration of the study. Using the protocol described here, a highly reproducible papilloma burden is expected within 10-20 weeks with progression of a portion of the tumors to squamous cell carcinomas within 20-50 weeks. In contrast to complete skin carcinogenesis, the two-stage model allows for greater yield of premalignant lesions, as well as separation of the initiation and promotion phases.

Figure 1. Two-stage model of skin carcinogenesis in mice

During initiation, topical application of a sub-carcinogenic dose of a mutating agent induces mutations in target genes in keratinocyte stem cells. Repeated topical application of a promoting agent begins two weeks after initiation and continues for the duration of the study. Papillomas begin to arise after approximately 6–12 weeks of promotion and a fraction begin to convert to SCC after approximately 20 weeks. Representative H&E stained sections of normal skin, hyperplastic skin, a papilloma, and a SCC are presented. All mice were handled in accordance with institutional and national regulations. This figure is a modification of a previously published figure.

Figure 2. Expression of several marker proteins in each sequential stage of skin carcinogenesis in mice

Tumor tissue was harvested from FVB mice that had undergone two-stage skin carcinogenesis initiated by DMBA and promoted by TPA. The tumors as well as hyperplastic dorsal skin from between tumors and untreated ventral skin were fixed in formalin and embedded in paraffin for immunohistochemical analyses. Representative images of normal epidermis, hyperplastic epidermis, papilloma (PAP), differentiated SCC, poorly differentiated SCC, lymph node metastasis and spindle cell carcinoma are shown. The lymph node metastasis was harvested from the same mouse bearing the poorly differentiated SCC. Immunostaining using the following antibodies was performed at the Histology Core at M.D. Anderson Cancer Center Science Park-Research Division as previously described: Loricrin (Covance, Princeton, NJ), K1 (Covance), K8 (Origene, Rockville, MD), K15 (Covance), E-cadherin (Santa Cruz), and GTT (Abcam, Cambridge, MA). All mice were handled in accordance with institutional and national regulations.

Figure 3

Representative data from a previously-published two-stage skin carcinogenesis study in FVB mice. This study was designed to test the effect of Bcl-x_L deficiency on tumor development in the two-stage skin carcinogenesis model. BK5.Cre × Bcl-x_L mice (lack Bcl-x_L expression in the basal layer of the epidermis) and wild-type mice (n=11 per group) were initiated with 25 nmol DMBA and promoted twice weekly with 6.8 nmol TPA. Tumor incidence (A) and multiplicity (B) were monitored until the maximum papilloma response was achieved (21 weeks). In panel B, the average number (mean ±SEM) of papillomas per mouse is presented. Bcl-x_L deficiency significantly reduced tumor multiplicity (P < 0.05 by Mann-Whitney U test). All mice were handled in accordance with institutional and national regulations.