General strategy to analyse melanoma in mice

Abstract

Many studies on melanocyte biology have gathered a great deal of knowledge on the genetic and developmental mechanisms driving skin and hair pigmentation. The first coat colour genetics studies date from the early 1900s when mouse pigmentation mutants were used to test Mendelian theories and to describe genetic linkage analyses in vertebrates (Castle, 1903; Haldane et al., 1915). In addition to the information on pigmentation genetics, interest in melanocytes was amplified by their transformation and formation of melanoma, an aggressive and often lethal type of cancer with an increasing incidence in Caucasian populations (Diepgen and Mahler, 2002; Whiteman et al., 2011). The genetic and biochemical mechanisms responsible for the melanocyte development, function, and malignancy are complex and not fully understood. Nowadays, research in melanocyte pigmentation and transformation continues to highlight new genes governing these processes. Mice represent an exceptional animal model for identifying genes behind phenotypic traits, because of the wealth of information on mouse genetics and to the similarity of mouse and human genetics; the mouse genome sequence was the first to be completed after the human genome was sequenced. The histology and organization of mouse and human skin are generally similar (in the epidermis, either interfollicular or follicular, and in the dermis), with the main distinction that melanocyte distribution varies across species, as the melanocyte ratio in different locations is different. In humans, melanocytes are mostly located in the basal layer of the epidermis, where they make dendritic connections with the surrounding keratinocytes, whereas in mice, they are found mostly in hair follicles hidden in the dermis. Mouse melanocytes can also be found in the epidermis of non-hairy skin (tail, ear, nose and footpad), where they are located in the basal layer or in the hair follicles, and in the dermis of the pinna of the ears. Dermal melanocytes are mostly surrounded by fibroblasts, whereas the cells surrounding epidermal melanocytes are keratinocytes. As a result, mouse melanomas may qualitatively differ from human melanomas. Mice do not naturally develop melanoma. Indeed, the rate of melanomagenesis remains very low, even after chemical or physical induction. However, molecular genetics have allowed the generation of mouse melanoma models that reflect human melanomas at the molecular, cellular, histological and clinical levels (Damsky and Bosenberg, 2010; Larue and Beermann, 2007). Clinical examinations will include monitoring the appearance and localization of the tumours, their growth rate and the number of independent tumours in each mouse. All procedures have to be carried out according to ethical regulations. Melanomas and their metastasis can also be followed in live mice with non-invasive imaging systems, including PET and NMR scans. Two-photon imaging of pigmented cutaneous melanoma brings higher resolution of primary tumours compared with PET and NMR analysis. However, being not penetrant, deep metastasis cannot be visualized using this optical approach. Bioluminescence approach was already established in melanoma cell culture and will certainly be very useful using appropriate transgenic mice in the future (Craft et al., 2005). At specified times, animals will be fully autopsied (including annotation and photographs) and primary tumours and metastases will be removed (proper fixation and conservation of tissues is required, with liquid nitrogen, paraformaldehyde fixation followed by paraffin embedding, paraformaldehyde fixation followed by inclusion in OCT) (Data S1 and S2). Depending on the question of interest, the complexity and the degree of knowledge of a particular murine melanoma model, samples can be further dissected by macro-dissection, micro-dissection or laser-micro-dissection. The establishment of a pure population of melanoma cells in culture (Data S3) may also be important to generate a full and coherent system with the mice to better understand the specific function of such genes, which will allow a better understanding of melanomagenesis. Classical biochemistry can be performed on these melanoma cell lines, such as the determination of the melanin level (Data S4), the tyrosinase activity (Data S5) and the level of RNA (Data S6). Moreover, cell biology analysis can be performed, such as migration assay (Data S7). The various conserved samples can be used to carry out biochemical, molecular, histological and immunohistological analyses. Biochemical and molecular analyses can be performed on liquid nitrogen frozen tissues. Besides isolation and genotyping of the DNA (Data S8), the quality and quantity of RNA (Data S9) and proteins, including post-translationnal events, can be analysed. Histological analyses can be performed on paraffin-fixed sections (Data S10–S12). Immunohistological analyses can be performed to evaluate melanocyte markers (S100, Mitf and Tyr), signalling markers (S6, Akt, ERK, p65 and GSK3β), proliferative markers (Ki-67 and BrdU), apoptotic markers (Tunel and caspase-3), endothelial markers (PECAM1 – CD34) and cell adhesion markers (E/P/N-cadherin, β-catenin and Slug/Snail/Twist). Markers of surrounding cells can also be of interest, including macrophage markers (F4-80 antibody). The general analyses in melanomagenesis described above help to decipher the main processes involved, and provide insights into the affected molecular pathways. We thank all colleagues of the field, specifically Friedrich Beermann, for their help in the establishment of these protocols. Work in the laboratories is supported by grants from the CNRS, the INSERM, the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, and INCA grant. LL is ‘équipe labellisée’ of the Ligue Nationale contre le Cancer. Data S1. Melanoma/Tissue paraffin embedding. Data S2. Embedding melanoma/tissue in Tissue-Tek. Data S3. Establishing melanoma cell lines in culture from mouse tumors. Data S4. Level of melanin in cells in culture. Data S5. Tyrosinase assay. Data S6. RT qPCR from cultured melanoma cells. Data S7. Single cell migration assay using time-lapse video-microscopy. Data S8. Tissue genotyping – quick DNA extraction followed directly by PCR. Data S9. RT-qPCR from tissue sample. Data S10. Eosin/Haematoxylin coloration on skin or melanoma paraffin sections. Data S11. Immunostaining on cryosections. Data S12. BrdU incorporation followed by tissue-tek embedding and immunostaining. 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