This work was funded by the Academy of Finland (grants 25013080481 and 25013142041 (I.J.), 286377 and 295814 (M.P.), 287907 (T.J.)), P&#228;ivikki 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.).

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
<ol>
	<li>Use age and sex-matched mice. Start the study at 7&#8211;9 weeks of age, because the skin in most mice is in telogen (the resting phase) around that age2.</li>
	<li>Observe the behavior of the animals during the study and if they fight, which often happens with males, house them ...

The authors have nothing to disclose.

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...

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/&#946;-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&#8211;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60445/60445fig1a.jpg" /> 
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&#8211;20, depending on the genetic background of the mice. A small proportion of the papillomas can further develop into SCCs within 20&#8211;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. <a href="https://www.jove.com/files/ftp_upload/60445/60445fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 ul RPT XL Graduated Filter Tip (Sterile), Refill</td>
			<td>Starlab</td>
			<td>S1182-1730-C</td>
			<td></td>
		</tr>
		<tr>
			<td>300 ul RTP Graduated Filter Tip (sterile), Refill</td>
			<td>Starlab</td>
			<td>S1180-9710-C</td>
			<td></td>
		</tr>
		<tr>
			<td>7,12-Dimethylbenz[a]anthracene (DMBA)</td>
			<td>Sigma</td>
			<td>D3254-100MG</td>
			<td>Harmful if swallowed and may cause cancer. Store protected from light.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>1000141011</td>
			<td>Evaporates rapidly and is inflammable.</td>
		</tr>
		<tr>
			<td>Attane vet 1000 mg/g</td>
			<td>Piramal Critical Care Limited</td>
			<td></td>
			<td>Liquid isoflurane for inhalation</td>
		</tr>
		<tr>
			<td>Battery-Operated Clipper Isis</td>
			<td>Albert Kerlb GmbH</td>
			<td>GT421</td>
			<td>For shaving the fur</td>
		</tr>
		<tr>
			<td>CONTRAfluran-Restgasfilter</td>
			<td>ZeoSys GmbH</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Linex Nature N1030 Ruler 30 cm</td>
			<td>Staples Business Advantage</td>
			<td>60383</td>
			<td>For measuring papillomas</td>
		</tr>
		<tr>
			<td>Medium CO2 Chamber 300 x 200 x 200mm - Red</td>
			<td>VetTech Solutions Ltd</td>
			<td>AN045AR</td>
			<td>For sacrifice</td>
		</tr>
		<tr>
			<td>Mekasoft</td>
			<td>Mekalasi</td>
			<td>23008</td>
			<td>Table cover</td>
		</tr>
		<tr>
			<td>Mice (Balb/c JRj)</td>
			<td>Janvier labs</td>
			<td></td>
			<td>Other strains also possible</td>
		</tr>
		<tr>
			<td>Mice (C57BL/6JRj)</td>
			<td>Janvier labs</td>
			<td></td>
			<td>Other strains also possible</td>
		</tr>
		<tr>
			<td>Panasonic Lumix DMC-FS5 Digital Camera</td>
			<td>Panasonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>30525-89-4</td>
			<td>For histology samples</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate aka 12-Otetradecanoylphorbol-13-acetate (TPA)</td>
			<td>Enzo</td>
			<td>BML-PE160-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance PLJ-C/PLJ-G</td>
			<td>KERN &#38; SOHN GmbH</td>
			<td>PLJ 600-3CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Set CO2 System-2 Chamber-S/S Housing</td>
			<td>VetTech Solutions Ltd</td>
			<td>AN044BX</td>
			<td>For sacrifice</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>For nucleic acid samples</td>
		</tr>
		<tr>
			<td>Tacta pipette 100-1000 ul</td>
			<td>Sartorius</td>
			<td>LH-729070</td>
			<td></td>
		</tr>
		<tr>
			<td>Tacta pipette 20-200 ul</td>
			<td>Sartorius</td>
			<td>LH-729060</td>
			<td></td>
		</tr>
		<tr>
			<td>UNO Anaesthetic Key Filler</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO Face Mask for Mouse</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO FM2200 Flowmeter</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO Gas Exhaust Unit</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO Induction Box</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>UNO200VAP Vaporizer</td>
			<td>Scintica instrumentation inc.</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
	</tbody>
</table>

chemical-induced skin carcinogenesis model using dimethylbenz[a]anthracene and 12-o-tetradecanoyl phorbol-13-acetate (dmba-tpa)

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.

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...

Watch this Scientific Journal Video about Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate DMBA-TPA at JoVE.com

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

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.

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

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different ...

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.

chemical-induced-skin-carcinogenesis-model-using

Research

JoVE Journal

Cancer Research

13.7K visualizações.  Tampere University and Tampere University Hospital. Este modelo de câncer de pele de duas etapas permite o estudo do papel da inflamação no câncer de pele e permite ao estudo a iniciação do tumor como um processo separado da progressão do tumor. A formação de tumores neste modelo é quimicamente induzida e permite o uso de cepas de nocaute para estudar os efeitos de fatores ambientais ou candidatos terapêuticos. O modelo é adequado para estudar o papel do sistema imunológico e da angiogênese no câncer.Demonstra uma patolog...