For example, neoplastic melanocytes induce hyperproliferation and impair differentiation of keratinocytes. Nonetheless, already at this point, they affect cellular behavior in their local environment.
Analysis of invasion 3d culture nih imagej software skin#
In this period, which is also termed as radial growth phase, pigmented patches of skin (nevi) increase laterally in size and become palpable, but melanoma cells typically still reside within the epidermis and are not metastasis competent. When diagnosed in its early ‘non-tumorigenic’ stages, resection of the lesion results in very high survival rates. The majority of melanoma mutations are C > T transitions caused by ultraviolet light irradiation, which mostly affect the mitogen-activated protein kinase pathway. Its incidence continues to increase each year and is currently responsible for more than 80% of deaths from skin cancer. Malignant melanoma is the most lethal form of skin cancer. Normally, melanocytes are located in the most basal epidermal layer, attached to a collagen-IV rich basement membrane, which separates epidermal and dermal compartments of the skin. Melanoma is a malignancy that arises from uncontrolled proliferation and metastasis of neoplastic melanocytes.
This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells. In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Regularly, some melanoma cells were also found to invade the fibroblast core. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages.
Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student’s t-test. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. This study used low-attachment based generation of spheroids composed of up to three cell types. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive. Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies.
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