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

* These authors contributed equally
Cancer Research

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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.

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Vähätupa, M., Pemmari, T., Junttila, I., Pesu, M., Järvinen, T. A. H. Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA). J. Vis. Exp. (154), e60445, doi:10.3791/60445 (2019).

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 model1,2. 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 carcinogenesis model, the back skin of mice is treated with two different chemicals that together induce tumor formation. The model is initiated with a low dose of the mutagen, DMBA, followed by prolonged exposure to the tumor promoter, TPA3 (Figure 1). DMBA mutates DNA randomly by forming covalent adducts with the DNA of epidermal cells and primary keratinocyte stem cells4,5,6,7. Some of these random mutations take place in a proto-oncogene, such as Hras1 (mutations in Kras and Nras are also detected) and the conversion of proto-oncogenes to oncogenes drives the tumor formation under proper stimuli. TPA, in turn, is the most commonly used tumor growth-promoting agent. Its molecular target is protein kinase C (PKC)8. TPA also activates Wnt/β-catenin signaling that is crucial for tumor formation in the model9. Repeated and prolonged exposure to the promoting agent leads to enhanced cell signaling, increased production of growth factors, and a local inflammatory reaction, which are evident due to increased DNA synthesis and inflammatory cell infiltration in the treated skin.

The key inflammatory mediators in the DMBA-TPA model have been identified10. Interleukin-17A (IL-17A) is known to be particularly tumorigenic in the DMBA-TPA model11,12. It works in synergy with interleukin 6 (IL-6) and participates in macrophage and neutrophil recruitment13,14. In addition, CD4+ T cells and neutrophils have been shown to be tumorigenic in the DMBA-TPA model. Finally, macrophages can also promote tumorigenesis in the model15,16,17.

During the promotion phase, the cell proliferation of the mutated cells is enhanced and a sustained hyperplasia of the epidermis is maintained1. This leads to papilloma development in the skin in 10–20 weeks, after which the papillomas start to convert to malignant tumors, squamous cell carcinomas (SCCs)2. However, less than 10% of the papillomas progress to malignancy, although this percentage also depends on the genetic background of the mice2,18. For decades it was not known what type of cells were initially mutated in the tumors leading to malignancy, even though some studies had reported clearly distinct features in the malignant tumors when compared to benign papillomas19,20. However, recent studies have greatly increased our understanding on the clonal origin of tumor formation in the DMBA-TPA model21.22.23. It was demonstrated that both bone marrow-derived epithelial cells and hair follicle stem cells contribute to the tumor formation22. Stage-specific lineage tracing studies have unveiled that benign papillomas are of monoclonal origin, but they recruit new epithelial cell populations21,23. However, only one of the cell clones functions as a driver for carcinogenesis; it contains an Hras mutation23. The progression to carcinoma formation is associated with a clonal sweep23.

The carcinogen DMBA initiates the papilloma formation and TPA promotes tumor growth. Hence, the tumor initiation can be studied separately from the promotion by interrupting the experiment before the TPA treatment period. As the tumor progression is studied weekly it offers a great opportunity for detailed tumor growth analysis throughout the study. Because the tumors are generated by external chemicals, an oncogenic mutation in the germline is unnecessary. Thus, studying the effects of a genetic background (e.g., knockout/transgene vs. wild type) on tumorigenesis is straightforward2. In sum, the DMBA/TPA skin cancer model is a particularly useful approach for studying the role of the immune system in tumor progression as well as for the evaluation of tumor initiation and promotion steps independently or interdependently.

Figure 1
Figure 1: DMBA-TPA-induced skin carcinogenesis model outline. The carcinogen DMBA is topically applied to induce DNA mutations in the initiation phase of the model. The growth-promoting agent TPA is administered 2x a week to enhance cell proliferation during the promotion phase, leading to the development of papillomas in the skin. Animals are sacrificed after the papilloma response reaches a plateau, usually within weeks 15–20, depending on the genetic background of the mice. A small proportion of the papillomas can further develop into SCCs within 20–50 weeks. To study early events in the initiation and early promotion phase, samples can be collected (e.g., shortly after the second TPA application). A representative photograph and hematoxylin and eosin stained cross section of papillomas on a C57BL/6 mouse skin after 19 weeks of treatment are shown. Scale bar = 0.1 mm. Please click here to view a larger version of this figure.

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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 typical experimental group size varies between 8–20 animals per group24,25,26,27.
    NOTE: Power calculations based on the biological variance seen in earlier studies help to choose a sufficiently large group size. The strains used as examples in this article include Balb/c and C57BL/6. However, many other mouse strains such as SENCAR and FVB have been used with the DMBA-TPA-model, as well as Wistar and Sprague-Dawley rats2,28,29. A license from the national or local committee of animal work is needed before the initiation of the study. In addition to general welfare considerations, the model-specific endpoints are typically squamous cell carcinoma (SCC) and an infection of the skin. Scratches in the skin due to itching after application of the tumorigenic chemicals in acetone is typical but otherwise the animals should show no signs of discomfort. Weighing regularly (e.g., 2x a month) helps evaluate the animals' welfare.
  3. Use the DMBA and TPA, both diluted in acetone. The working concentration of DMBA is 250 g/L. A dose for one animal is 50 µg of DMBA in 200 µL of acetone. The stock concentration of TPA is 125 g/L and the treatment concentration 25 g/L. A TPA dose for one animal is 5 µg in 200 µL of acetone.
    CAUTION: DMBA is harmful if swallowed and may cause cancer. Acetone evaporates rapidly and is flammable. It may cause dizziness and irritate eyes. Use a breathing mask and/or work under a vacuum flow. Change gloves after handling any of these chemicals. Individually ventilated cages prevent the spread of the chemicals during housing of the mice. After application, gather the pipette tips used for handling the DMBA and dispose as dangerous waste.
    NOTE: The DMBA must be protected from light. The diluted TPA is stored in -20 °C, preferably protected from light.
  4. Procure the following equipment: a scale, an ordinary shaver for the fur, pipettes and tips of an appropriate size, an ordinary ruler, a digital camera, a notepad and a pen or a computer for recording the papillomas, an inhaled anesthesia system or a mouse restrainer with an opening on the back top, and a carbon dioxide narcosis system for sacrificing mice.

2. Skin Papilloma Induction and Promotion

  1. Shave the back skin and weigh the animal. Later, shave the skin whenever needed but not at the time of the chemical exposure.
    NOTE: Be careful when shaving around the papillomas and avoid making any cuts on the skin. Weigh each animal every 2 weeks to notice any potential weight loss.
  2. Apply 50 µg of DMBA in 200 µL of acetone topically on the shaved area using a pipette 48 h after shaving the fur. If needed, restrain the animal using light inhalation anesthesia or a mouse restrainer.
  3. After 7 days, give the first TPA dose. Apply 5 µg of TPA in 200 µL of acetone topically with a pipette 2x a week, preferably Monday and Thursday or Tuesday and Friday.
  4. Count, record, and photograph the papillomas every week. A palpable mass greater than 1 mm in diameter is considered a papilloma if it stays longer than 1 week. Mark each individual papilloma on a map and list its size every week. Store digital photographs.

3. Animal Sacrifice and Sample Collection

  1. Continue the treatment until the tumor response reaches a plateau. Generally, the papilloma burden is expected to increase 10–20 weeks after the initiation, depending on the mouse strain used. The plateau is expected in 15–20 weeks. A small proportion of the papillomas (under 3%) can develop into SCCs within 20–50 weeks2.
  2. Sacrifice the animals 24 h after the last TPA application. Use carbon dioxide narcosis with cervical dislocation or another suitable method.
  3. Depending on the research question, gather appropriate sample material from the animals30,31.
    1. For example, take blood samples before sacrifice and separate the plasma.
    2. Cut pieces of the skin for immunohistochemistry (IHC) staining (e.g., hematoxylin and eosin, proliferating or inflammatory cells).
    3. Use biopsy punches to collect skin pieces with either papilloma tissue or non-papilloma (treated) skin for gene expression (e.g., qPCR) and/or protein analyses (e.g., Western blot or ELISA).
    4. Collect a piece of the spleen and skin-draining lymph nodes for flow cytometry analysis if more detailed analysis of the immune cell populations is desired. You may also separate epidermal and dermal layers for further analyses.

4. Statistics

  1. Draw a Kaplan-Meier survival curve of the papilloma-free time and use the Mantel-Cox log-rank test for the survival. Draw a linear curve of the number of papillomas per week. Because this is count data, use a nonlinear regression model. Consult a statistician if needed.

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Representative 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 between the groups at that stage. Terminating the experiment too early may hide a statistically significant difference.

Figure 2
Figure 2: The main outcomes of the DMBA-TPA-induced skin carcinogenesis model. Representative images of papillomas on mouse skin and the two main outcomes. Data were combined from four separate experiments. NOTE: Experiments with Balb/C and C57BL/6 mice were done separately. (A) A survival plot of the papilloma free time. Dashed line = Balb/c and solid line = C57BL/6. (B) The average number of papillomas per mouse. Dashed line = Balb/c and solid line = C57BL/6. Error bars = SEM. (C) A Balb/c mouse with papillomas at 11 weeks after DMBA treatment. (D) A C57BL/6 mouse with papillomas at 11 weeks after DMBA treatment. Please click here to view a larger version of this figure.

Histology, mRNA, and cellular and protein analysis can be conducted to explore the mechanisms underlying the observed differences. For example, histological analyses are suitable for studying the structure of the papillomas, morphology of the skin (epidermal and dermal thickness), different biological processes (proliferation, apoptosis, angiogenesis), or the infiltration of immune cells32 (Figure 3). Skin is ideal for reliable quantification of morphological and IHC parameters by state-of-art image analysis systems30,31,32,33. Gene and protein expressions from the (papilloma) skin samples can be easily studied with standard methods30. Flow cytometry is a useful tool for the analysis of different immune cell populations of the spleen and skin-draining lymph nodes, as previously described30. The choice of analytical methods depends on the research question and needs to be considered before initiating the experiment.

Figure 3
Figure 3: Histological analysis of inflammatory cell infiltration in the DMBA-TPA model. Representative histological cross sections from PFA-fixed paraffin-embedded skin in the DMBA-TPA model. Skin sections were stained with a macrophage marker F4/80 (brown). Scale bars = 200 µm. (A) Only a few F4/80 positive macrophages were seen in the normal, untreated skin. (B) Macrophage infiltration in the dermis was evident at the early stage of cancer promotion, at 2 weeks and 36 h after the second TPA administration. Also, epidermal hyperproliferation was seen at 2 weeks, as shown by the thickening of the epidermis. (C) Non-papilloma skin at 19 weeks still showed a remarkable infiltration of macrophages. (D) A strong accumulation of macrophages was seen under papillomas in the dermis of the DMBA-TPA treated animals. Please click here to view a larger version of this figure.

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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 progression in genetically modified animals. When applying the model for any given strain, it is advisable to study the existing literature to achieve an idea of how the strain reacts in the model (e.g., to find out how quickly the first papillomas will appear). When using genetically modified mice, the use of wild type littermates is always recommended. The use of potential additional controls, such as replacing either DMBA or TPA or both by acetone, is highly debatable, because it is known that removing either DMBA or TPA or inverting their administration order is highly unlikely to result in the induction of papilloma growth34. When it is essential to study tumor initiation (i.e., by administration of DMBA alone), it can be achieved by sacrificing the animals after the DMBA dose. When studying the effect of an individual gene in a transgenic or knockout setting, it is recommended to take samples from untreated animals to detect possible differences that might exist before tumor formation between genetically engineered and wild type strains30,31.

Although the DMBA-TPA model is relatively simple to conduct technically, accurate timing is essential for success. The application of DMBA and the regular application of TPA are both crucial for the tumor development34. A careful weekly examination of the papillomas is essential to gather all data. It needs to be noted that certain treatments or genetic backgrounds may cause papillomas to grow and later shrink or even vanish30. Sometimes, other noncancerous lesions and SCCs arise. In this case, histological examination of the lesion is recommended. Another critical step is to ensure that TPA is the only tumor promotor. Skin trauma induces the production of cytokines that may affect tumor promotion2,34. Thus, the iatrogenic wounding of the skin during shaving needs to be avoided, and aggressive, fighting mice must be housed separately. In addition to fighting, other aspects of animal welfare are also important. Weight loss, dull and tangled fur, and apathetic behavior are signs of discomfort. The discomfort is not only an ethical issue, because negative energy balance inhibits the tumor growth34.

The DMBA-TPA model provides an opportunity for many modifications. Because some strains are known to be more sensitive than others, a careful selection enables scheduling the experiment and adjusting the amount and frequency of TPA application18,35. The model is mainly used for developing skin tumors, but local application to the gastrointestinal tract is an option36. In addition, a modified version of the model has been used to study tumor development in the trachea37. Because the tumor development can be regulated with DMBA and TPA applications, the model enables studying the outcomes after modifying environmental aspects such as diet and circadian rhythm38. By separating tumor initiation (DMBA) and promotion (TPA), the model enables studying those two processes independently. For instance, sacrificing the mice after the DMBA treatment permits studying the mechanism of the treatment or the effect of a gene of interest in tumor initiation2,34. To study the early events in the tumor initiation and promotion, animals can be sacrificed at an early time point. For example, samples can be collected shortly after the second TPA administration (for example 3 h, 12 h, 36 h, or 48 h after)30,31. It is also worth noting that the classical DMBA-TPA two-stage carcinogenesis model can be modified by using different chemicals or by using the "third" chemical. These are reviewed by Abel et al2.

The DMBA-TPA model has been used for decades for different purposes34. It has been used for studying the effects of different therapeutics24,25,26 and genes31,32,39,40 but also for studying nutrition27, the circadian rhythm38, and the development of diagnostic tools41. As the tumors form from the skin, the model provides a unique opportunity to collect tumor tissue samples at different stages of the progression21. When introducing new chemopreventive agents or immunomodulators, it is advisable to first gather data on how the strain has reacted in the DMBA-TPA model in previous studies. If not available, a prestudy without the potential therapeutic agent is recommended to determine the timing and number of papillomas as well as the variance in these parameters. Existing data from published studies may also help to determine the dosage of the tested compound24,27, but if unavailable, using a serial dilution of the tested compound enables evaluating the dose response of the agent studied25,26.

Inflammation is essential for human tumorigenesis, as it is involved in all phases of tumor progression: the initiation, the promotion, and the conversion to malignancy42,43. The chemically induced skin carcinogenesis model is an inflammation-driven carcinogenesis model. Pro-inflammatory cytokine response and the infiltration of inflammatory cells to the treated skin are already evident in the early phase of the model. The first infiltrating cells are neutrophils, followed by accumulation of macrophages, T cells, etc.30,31. These cells create an inflammatory microenvironment in the skin, and a cross talk between inflammatory and epidermal cells is initiated, leading to the production of cytokines, chemokines, and prostaglandins that drive the tumor initiation and growth locally. As the DMBA-TPA model is an inflammation-driven model, it does not include some other aspects of clinical cancer. Although it is known that the SCCs eventually may cause metastasis in the DMBA-TPA model, only a small portion of papillomas develop into SCCs2,23. Consequently, metastasis is a rare feature in the DMBA-TPA model, although some sensitive outbred stocks such as CD-1 or SENCAR may have an increased likelihood of developing metastasis44. The appearance of SCC is often considered an animal welfare issue and thus an endpoint criterium. Therefore, despite the existence of metastasis in the model, some other models such as transplantation or genetically engineered models are more suitable for studying metastasis45. Another limitation of the model is that it requires a surface to apply the chemicals. Thus, studying cancer progression in a specific internal organ is out of the scope of the model.

Another common skin cancer model is the UV radiation model46. The model is comparable to the DMBA-TPA model because neither of these two models needs cell transplantation or genetic modification to cause cancer. The fundamental difference between the models is that the UV radiation model induces aggressive SSC formation, whereas the tumor outcome in the DMBA-TPA model is generally benign papillomas. If UV radiation is combined with the application of chemical carcinogens or in mouse strains with a genetic background that makes the strain susceptible for cancer development, even melanoma formation can take place. The most common mutations in the UV radiation model inactivate the p53 gene, a crucial tumor suppressor gene in humans46, whereas the most common mutations in the DMBA-TPA model activate the Hras-gene23. The UV radiation causes also immunosuppression46,47, which may be a confounding factor when studying the aspects of the immune system. However, these two models also work in combination. UV radiation can be used as a promoting agent with DMBA or as an initiating agent with TPA46.

A reasonable question is whether the DMBA-TPA model mimics human cancer. A small proportion of papillomas develop to SCCs. Nevertheless, the premalignant stage of human SCC is actinic keratosis, and it differs from the papilloma formation seen in the model. Still, papilloma formation similar to the model is detected in human individuals treated for melanoma with vemurafenib. This suggests that the Hras-mutated stem cells that are essential for carcinogenesis in the DMBA-TPA model also exist on human skin34. Furthermore, it is reported that the uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) papillomas and microinvasive SCCs is higher than in the surrounding skin but not as high as in a fully invasive SCC, reflecting the similarity of the premalignant stages41. If using the DMBA-TPA model for toxicity evaluation, one must remember that the repetition of exposure has a great effect on tumor promotion34. At least some of the molecular mechanisms are common to the model and human skin.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

This work was funded by the Academy of Finland (grants 25013080481 and 25013142041 (I.J.), 286377 and 295814 (M.P.), 287907 (T.J.)), Päivikki and Sakari Sohlberg Foundation (M.P., T.J.), Finnish Medical Foundation (T.P.), The Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital (grant 9V049 and 9X044 (M.P.), 9X011 and 9V010 (T.J.)), The Competitive State Research Financing of the Expert Responsibility Area of Fimlab Laboratories (grant X51409 (I.J.)), Tays Support Foundation (I.J., M.P., T.J.), Tampere Tuberculosis Foundation (I.J., M.P., T.J.), the Finnish Cultural Foundation (M.V.), the Paulo Foundation (T.P.), Cancer Society of Finland (M.P.), and the Emil Aaltonen Foundation (T.P.).

Materials

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

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References

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