UV Exposure of Multipotent Dermal Stem Cells as Potential Target Cells for the Development of UV-induced Malignant Melanoma

Abstract

Malignant melanoma is the most aggressive and lethal type of skin cancer with rising incidence worldwide. The unequivocal risk factor is exposure to solar and artificial UV radiation. Despite the central role of UV-induced cyclobutane pyrimidine dimers (CPDs), particularly in the formation of UV signature mutations, the precise cellular and molecular mechanisms driving melanomagenesis are far from being understood in detail. Moreover, the cell of origin remains a subject of debate, with discussions centering on whether transformation initiates in mature epidermal melanocytes or stem cell precursors. An alternative theory proposes a damaged extrafollicular dermal melanocyte stem cell as the earliest origin. Within human skin, multipotent neural crest-derived dermal stem cells (DSCs) may serve as a reservoir for epidermal melanocytes. Although DSCs are constantly exposed to solar UV radiation, potentially contributing to the onset of melanoma, there is still insufficient investigation into how DSCs react to UV-induced damage. Therefore, this study aimed to establish workflows for the in vitro investigation of DSCs as potential cell of origin and of their response to UV irradiation. The work focused on examining functional stem cell features and understanding protection mechanisms to determine if UV-induced cell damage poses a higher risk in DSCs.

It was demonstrated that DSCs isolated from human foreskin can serve as a model to study their potential relevance in the genesis of malignant melanoma. Cultivated in specific stem cell medium, primary DSC cultures exhibited neural crest stem cell characteristics, including the expression of relevant markers, the ability to form three-dimensional spheres, and the potential for melanocytic differentiation. However, fibroblast contamination was a serious limitation for certain applications, necessitating the enrichment of DSCs. Immunomagnetic separation with negative selection based on CD90 and alternative non-cell-specific approaches, such as Geneticin treatment or selective detachment, proved unsuitable for DSC purification. Highly enriched DSCs with a good recovery rate were obtained using column-based MACS® positive selection targeting NGFRp75. These purified DSCs maintained viability and differentiation potential, although further validation is needed. This in vitro purification marks a crucial advancement in DSC research. A comparative study of the DNA damage response after UVA and UVB irradiation revealed that DSCs reacted similarly to fibroblasts and melanocytes, displaying functional DNA repair and apoptosis induction. Protective mechanisms in DSCs were not superior to those of differentiated skin cells. Interestingly, DSCs exhibited an exceptional cell cycle regulation with a lack of growth arrest following UV irradiation, potentially indicating deficiencies in cell cycle checkpoints. This could lead to replication through unrepaired damage, mutations, and an elevated risk of transformation. Although initial evidence hints at a possibly heightened susceptibility of DSCs to UV-mediated carcinogenesis, further research is needed to establish a causal link between this specific radiation response and melanoma development. UV irradiation predominantly had no impact on stem cell features and epigenetic regulation. Exposure to a single dose of UVA or UVB did not alter the expression of selected melanoma-specific microRNAs or the ability of DSCs to differentiate into melanocytes, neither impairing nor triggering differentiation. The radiation scheme might have been insufficient to induce UV-related effects, requiring multiple irradiations to adequately mimic physiological conditions. Irrespective of irradiation, a set of eight differentially expressed microRNAs allowed a clear distinction between DSCs, melanocytes, and melanoma cells, indicating significant differences in their overall baseline microRNA expression. By contrast, both single and multiple UVB exposures affected the sphere-forming potential of DSCs, resulting in reduced sphere counts. However, uncertainties arose regarding the influence of fibroblasts and cell aggregation in the utilized sphere formation assay, hindering a comprehensive evaluation of the results.

In summary, this study has successfully met its objectives by establishing workflows and methods that enabled an initial in vitro investigation of functional stem cell features and UV protection mechanisms in DSCs. While these findings yield a preliminary assessment of DSCs as potential cell of origin for malignant melanoma, they also raise questions for future investigations that could provide further insights into mechanisms underlying UV-induced melanomagenesis. Understanding how DSCs respond to UV exposure could enhance knowledge of early events in melanoma initiation, potentially improving prevention, early diagnosis, and the development of new treatment strategies.