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 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&#8211;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&#39; welfare.</li>
	<li>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 &#181;g of DMBA in 200 &#181;L 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 &#181;g in 200 &#181;L 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 &#176;C, preferably protected from light.</li>
	<li>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.</li>
</ol>
2. Skin Papilloma Induction and Promotion
<ol>
	<li>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.</li>
	<li>Apply 50 &#181;g of DMBA in 200 &#181;L 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.</li>
	<li>After 7 days, give the first TPA dose. Apply 5 &#181;g of TPA in 200 &#181;L of acetone topically with a pipette 2x a week, preferably Monday and Thursday or Tuesday and Friday.</li>
	<li>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.</li>
</ol>
3. Animal Sacrifice and Sample Collection
<ol>
	<li>Continue the treatment until the tumor response reaches a plateau. Generally, the papilloma burden is expected to increase 10&#8211;20 weeks after the initiation, depending on the mouse strain used. The plateau is expected in 15&#8211;20 weeks. A small proportion of the papillomas (under 3%) can develop into SCCs within 20&#8211;50 weeks2.</li>
	<li>Sacrifice the animals 24 h after the last TPA application. Use carbon dioxide narcosis with cervical dislocation or another suitable method.</li>
	<li>Depending on the research question, gather appropriate sample material from the animals30,31.
	<ol>
		<li>For example, take blood samples before sacrifice and separate the plasma.</li>
		<li>Cut pieces of the skin for immunohistochemistry (IHC) staining (e.g., hematoxylin and eosin, proliferating or inflammatory cells).</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Statistics
<ol>
	<li>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.</li>
</ol>

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 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.

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.remotexs.ntu.edu.sg/files/ftp_upload/60445/60445fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><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 &amp;amp; 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 between the groups at that stage. Terminating the experiment too early may hide a statistically significant difference.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60445/60445fig2b.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60445/60445fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60445/60445fig3a.jpg" /> 
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 &#181;m. (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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60445/60445fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

chemical-induced-skin-carcinogenesis-model-using

Nanyang Technological University

Research

JoVE Journal

Cancer Research

14.1K Views.  Tampere University and Tampere University Hospital. 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.

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

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Bioengineering

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

Biochemistry

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Medicine

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER+ and R26-SmoM2YFP+ mice in this protocol); 2) inducing SmoM2YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Biology

Establishing a Compound Acne Model in Rats Using Oleic Acid and C. acnes

A Rat Model of Compound Acne

Acne vulgaris is a prevalent chronic skin condition characterized by the presence of comedones, papules, and pustules on the face, neck, and chest. To simulate the inflammation of acne vulgaris, this protocol details an approach to establish a compound acne rodent model by inducing acne inflammation in rat ears using oleic acid and Cutibacterium acnes (C. acnes).&#160;Rats were randomly divided into four groups: the normal control group (NC), ears treated with oleic acid group (OA), ears treated with C. acnes group (C. acnes), ears treated with oleic acid and C. acnes (OA + C. acnes). To mimic excessive sebum production, oleic acid was smeared on the ears of rats in OA and OA + C. acnes groups for 25 days.
From days 21 to 25, C. acnes suspension was injected intradermally into the ears of rats in the C. acnes and OA + C. acnes groups to aggravate the acne inflammation. Ear thickness was measured weekly as a gauge of inflammation severity. Gross observation, hematoxylin and eosin staining, and immunohistochemistry (IHC) were conducted and the results showed that the ears of the OA group and the OA + C. acnes group were thickened and indurated, accompanied by erythema and the presence of comedones. Additionally, papules were observed in C. acnes and OA + C. acnes groups. The histopathology exhibited hyperkeratinization and expanded infundibulum of the hair follicles in OA and OA + C. acnes groups. Infiltration of inflammatory cells and abscesses were found in the dermis of C. acnes and OA + C. acnes groups. The IHC results confirmed increased levels of tumor necrosis factor (TNF)-&#945; in the dermis of C. acnes and OA + C. acnes groups. All the above results collectively indicated the successful establishment of the compound acne rodent model.

Author Spotlight: Development and Evaluation of a Compound Acne Rodent Model Using C. acnes and Oleic Acid

This protocol outlines the procedure for inducing acne inflammation in rat skin with oleic acid and Cutibacterium acnes.

Acne vulgaris is a prevalent chronic skin condition characterized by the presence of comedones, papules, and pustules on the face, neck, and chest. To ...

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

Source: Chang, J. E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a>&#160;J. Vis. Exp.&#160;(2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.&#160;J. Vis. Exp.&#160;(2016).
...

JoVE Encyclopedia of Experiments

Lung Cancer

In vitro study

Chemical-induced Two-stage Skin Carcinogenesis Model: An Experimental In Vivo Mouse Model of Skin Cancer

Source: V&#228;h&#228;tupa, M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60445"> Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA)</a>. J. Vis. Exp. (2019)
This video describes the procedure to induce two-stage skin carcinogenesis to study tumor initiation by topical application of chemicals. In the first step, a mutagen, DMBA, is applied onto the skin of a mouse model which initiates tumor formation by causing mutations in the stem cells, followed by the application of a growth stimulator, TPA, which accelerates skin papilloma formation.

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

Summary

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