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

Summary

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.

Abstract

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' 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.

Introduction

Melanoma, the malignant form of melanocytic tumors, is the most aggressive cancer in the skin with cutaneous melanoma responsible for the majority of skin cancer deaths¹. In the United States, melanoma is commonly diagnosed; it is projected to be the 5^th to 6^th most common cancer type among the estimated new cancer cases in 2018². Furthermore, while overall cancer incidences have shown the trend of gradual reduction in recent decades, cutaneous melanoma incidence rates during the last few decades demonstrate a continuous and rapid increase in both genders².

Protocol

All animal procedures are performed in accordance with Cornell University Institutional Animal Care and Use Committee (IACUC).

1. Preparation

1. Collect tail clips from 12 day postnatal mice and digest in 400 µL of 0.05 N NaOH at 95 °C for 1 h. Vortex and add 32 µL of 1 M Tris-HCl, pH 7. Genotype mice according to PCR protocols provided through the Jackson Laboratory and identify mice with genotype of interest⁶.
   - Tyr-CreER – hemizygous (+/CreER)
   - LSL-Braf^V600E – heterozygous (+/LSL-V600E)
   - Pten – <....

Results

Cutaneous melanoma initiation induced by chemical depilation

The procedure of chemical depilation is depicted in Figure 2. When mice are 7-weeks postnatal, their dorsal skin is in telogen. During telogen, hair follicle stem cells and MCSCs are known to be in a quiescent, resting state. The skin should show no significant hair growth after shaving. On the other hand, chemical de.......

Discussion

Hallmark genetic alterations frequently found in cutaneous melanoma tumors have been well described¹³. The most dominant driver mutation is Braf^V600E, and a genetically engineered mouse model for Braf^V600E-mediated melanoma was generated by the Bosenberg group¹⁴ and deposited in the Jackson Laboratory. Using this mouse model, our recent study demonstrated the requirement of cellular activation of tumor-prone MCSCs for the significant .......

Materials

Name	Company	Catalog Number	Comments
Tamoxifen	Sigma	T5648-1G	For systemic injection
Tamoxifen	Cayman Chemical	13258	For systemic injection
Corn oil	Sigma	45-C8267-2.5L-EA
4OH-tamoxifen	Sigma	H7904-25MG	For topical treatment
26g 1/2" needles	various		Veterinary grade
1 mL syringe	various		Veterinary grade
Pet hair trimmer	Wahl	09990-502
Hair removal cream	Nair	n/a	Available at most drug stores
Cotton swabs	various
Ultraviolet light bulb	UVP	95-0042-08	model XX-15M midrange UV lamp
200 proof ethanol	various		pure ethanol
Histoplast PE	Fisher Scientific	22900700	paraffin pellets
Neutral Buffered Formalin, 10%	Sigma	HT501128-4L
Clear-Rite 3	Thermo Scientific	6901	xylene substitute
O.C.T. Compound	Thermo	23730571
Tissue Cassette	Sakura	89199-430	for FFPE processing
Cryomolds	Sakura	4557	25 x 20 mm
FFPE metal mold	Leica	3803082	24 x 24 mm
Isoflurane	various		Veterinary grade
Anesthesia inhalation system	various		Veterinary grade
Fine scissor	FST	14085-09	Straight, sharp/sharp
Fine scissor	FST	14558-09	Straight, sharp/sharp
Metzenbaum	FST	14018-13	Straight, blunt/blunt
Forcep	FST	11252-00	Dumont #5
Forcep	FST	11018-12	Micro-Adson
Tyr-CreER; LSL-BrafV600E; Pten-f/f	Jackson Labs	13590
LSL-tdTomato	Jackson Labs	007914	ai14
Cre-1		n/a	GCATTACCGGTCGATGCAACGAGTGATGAG
Cre-2		n/a	GAGTGAACGAACCTGGTCGAAATCAGTGCG
Braf-V600E-1		n/a	TGAGTATTTTTGTGGCAACTGC
Braf-V600E-2		n/a	CTCTGCTGGGAAAGCGGC
Kras-G12D-1		n/a	AGCTAGCCACCATGGCTTGAGTAAGTCTGCA
Kras-G12D-2		n/a	CCTTTACAAGCGCACGCAGACTGTAGA
Pten-1		n/a	ACTCAAGGCAGGGATGAGC
Pten-2		n/a	AATCTAGGGCCTCTTGTGCC
Pten-3		n/a	GCTTGATATCGAATTCCTGCAGC
tdTomato-1		n/a	AAGGGAGCTGCAGTGGAGTA
tdTomato-2		n/a	CCGAAAATCTGTGGGAAGTC
tdTomato-3		n/a	GGCATTAAAGCAGCGTATCC
tdTomato-4		n/a	CTGTTCCTGTACGGCATGG