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 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://jove.unicartagenaproxy.elogim.com/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 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 ...

chemical-induced-skin-carcinogenesis-model-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

14.2K 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

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 ...

Medicine

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 ...

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Immunology and Infection

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

Biology

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://jove.unicartagenaproxy.elogim.com/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).
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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://jove.unicartagenaproxy.elogim.com/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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