A reporter mouse model forin vivotracing andin vitromolecular studies of melanocytic lineage cells and their diseases

Abstract

Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSAmT/mG and Tyr::CreERT2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage.

Targeting FER Kinase Inhibits Melanoma Growth and Metastasis

“…The propensity of melanoma cells to metastasize has been attributed, in part, to their ability to interact with and modify their surrounding extracellular matrix, and to their imprinted high migratory capacity, arising from the embryonic neural crest cells that give rise to melanocytic cells [25]. Cultured melanocytes exhibit marked differences in migratory capacity, depending on the substrate on which they are seeded [26]. Consequently, we first determined the effect of various extracellular substrates on motility of parental 131/4-5B1 cells using time-lapse videomicroscopy.…”

“…Melanoma cell interactions with ECM proteins present in the skin, such as collagen IV and laminin 332, are important for cell migration and invasion. We have observed that both normal primary cultured melanocytes and parental 131/4-5B1 melanoma cells exhibit greater motility on laminin 332 compared to collagen I ([26] and Figure S2). Similarly, A375 and other melanoma lines show enhanced haptotactic migration in response to laminin 332, relative to collagen and other ECM substrates, a response likely mediated through α3β1 integrins [37].…”

“…Paraffin-embedded sections (6 µm) were dewaxed, rehydrated and stained with Mayer’s Hematoxylin (MHS16) and Eosin Y (HT110116), purchased from Millipore Sigma (Oakville, ON, Canada). Melanin in tissue sections was visualized using Fontana-Masson (ab150669, Abcam), as described [26]. Coverslips were mounted onto slides using Permount (SP15100, Fisher Scientific, Hampton, NH, USA).…”

Integrin-linked kinase regulates melanosome trafficking and melanin transfer in melanocytes

“…To investigate the role of ILK in melanosome trafficking, we isolated primary Tyr::CreER T2 ;ROSA mT/mG ;Ilk f/f melanocytes and cultured them to >95% purity, as we previously described (Crawford et al, 2017). In the absence of CreER T2 activation, these cells express a membrane-bound form of Tomato fluorescent protein (mTomato) (Crawford et al, 2017).…”

Cutaneous Melanoma—A Long Road from Experimental Models to Clinical Outcome: A Review

“…Experimental animal models also proved to be reliable sources of data concerning (i) the screening for novel antimelanoma agents: temozolomide (B16F10 metastatic melanoma model) [ 66 , 67 ], thymoquinone (B16F10 intracerebral melanoma model using C57BL/6J mice as host) [ 68 ], oncolytic herpes simplex virus HF10, and dacarbazine combined therapy (DBA/2 mice subcutaneously inoculated with clone M3 mouse melanoma cells) [ 69 ], gliotoxin (a xenograft mouse model using athymic mice) [ 70 ], recombinant methioninase [ 71 ], cancer vaccines [ 72 ], natural compounds [ 73 , 74 ]; (ii) molecular discovery—the comprehension of melanoma metastatic pathway involving microvascular environment [ 75 ]; and (iii) in vivo tracing of melanocytic lineage cells [ 76 ].…”