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

Melanoma is widely diagnosed worldwide, with increasing incidence. These protocols allow the early stages of melanoma initiation to be carefully controlled, facilitating the study of its development. By controlling the time and location of melanocyte stem cell activation, we have the ability to discern the changes within the stem cell population and its new strain melanoma initiation.

Demonstrating the procedure will be Dahihm Kim and Luye An, graduate students from the laboratory. Two to three days before beginning the procedure, use an electric trimmer to shave the dorsal skin from each anesthetized seven-week-old mouse. On the day of the experiment, use a cotton swab to apply a thin layer of depilatory cream to the exposed skin.

Once the cream has been evenly applied, use clean cotton swabs to remove the cream, followed by gentle wiping with a damp cloth to remove any residual cream. Then return each mouse to its home cage with monitoring until full recovery. For UV-B irradiation, cover the dorsal skin with UV-B protective material so that only the desired region will be exposed, and cover the UV chamber with a UV-resistant lid.

Then turn on the UV-B lamp to irradiate the mouse for the appropriate experimental time period before returning the mouse to an empty cage with monitoring until full recovery. To isolate the skin samples, use an electric trimmer to remove any hair from the dorsal skin area of the euthanized irradiated mice, and lightly brush the exposed skin with a lint-free tissue to clear the area. Make a small incision with sharp scissors just above the base of the tail and insert large, blunt scissors into the incision to separate the subdermal connective tissues.

Using sharp scissors, carefully cut along the edge of the skin region of interest to isolate the tissue sample and place the excised skin tissue onto a clean paper towel. Then use dull forceps to stretch the skin so that it adheres to the towel and is taut. When all of the samples have been harvested, place a skin sample and paper towel backing into a piece of folded filter paper immediately adjacent to the crease, taking care that the sample is smooth and not folded or crumpled.

Close the sides of the filter paper with staples, and trim the paper. Then fully submerge the samples in 10%neutral buffered formalin for three to five hours at room temperature before washing the tissues with two five-minute washes in deionized water. After the second wash, carefully remove any residual material from the skin tissues, and trim the edges of the samples so that they are not jagged.

Next, make three sagittal cuts in each sample so that the width of the strips is no more than five millimeters, and make transverse cuts so that the samples are no more than 20 millimeters wide. Position a cryo mold containing optimum cutting temperature or OCT medium in a portrait orientation, and place four to five pieces of skin on top of the OCT. When all of the pieces are in place, use two pairs of fine forceps to bring the long edge of each piece of skin to the bottom of the cryo mold so that the samples are vertical within the OCT and perpendicular to the base of the mold.

Then fill the mold with additional OCT to the second lip and place the mold onto a flat surface of a piece of dry ice, using forceps to adjust any skin pieces that may have shifted during the transfer as the block begins to freeze as necessary. When mice are seven weeks post-natal, their dorsal skin is in telogen. During telogen, hair follicle and melanocyte stem cells are known to be in a quiescent resting state, and the skin should show no significant hair growth after shaving.

Chemical depilation, however, can induce hair follicle stem cell activation, inducing significant hair growth within the region of interest. As chemical depilation can significantly induce the activation of both hair follicle and melanocyte stem cells, melanoma-prone melanocyte stem cells in an active state can form tumors and microscopic melanoma initiation can be observed within two weeks of chemical depilation. UV-B irradiation can also induce tumor initiation from quiescent, tumor-prone melanocyte stem cells.

Importantly, UV-B induces the direct translocation of melanocyte stem cells from their follicular stem cell niche to the inter-follicular epidermis. Furthermore, UV-B can induce melanocyte stem cell-originating cutaneous melanoma formation throughout the interfollicular epidermis. Similar to dorsal skin, UV-B-induced melanoma initiation can be notably observed in other skin areas, such as ear skin.

Indeed, compared to control ear skin, covered by UV-resistant cloth, ear skin exposed to UV-B demonstrates a higher pigmentation due to a higher burden of melanoma initiation. Different doses of UV-B may have different effects on mouse skin and melanocyte activity. Therefore, it is important to determine and optimize the irradiation dose prior to starting your experiment.