Heterogeneous spheroids have recently acquired a prominent position in melanoma research because they incorporate microenvironmental cues relevant for melanoma. In this study, we focused on the analysis of microenvironmental factors introduced in melanoma heterogeneous spheroids by different dermal fibroblasts. We aimed to map the fibroblast diversity resulting from previously acquired damage caused by exposure to extrinsic and intrinsic stimuli. To construct heterogeneous melanoma spheroids, we used normal dermal fibroblasts from the sun-protected skin of a juvenile donor. We compared them to the fibroblasts from the sun-exposed photodamaged skin of an adult donor. Further, we analysed the spheroids by single-cell RNA sequencing. To validate transcriptional data, we also compared the immunohistochemical analysis of heterogeneous spheroids to melanoma biopsies. We have distinguished three functional clusters in primary human fibroblasts from melanoma spheroids. These clusters differed in the expression of (a) extracellular matrix-related genes, (b) pro-inflammatory factors, and (c) TGFβ signalling superfamily. We observed a broader deregulation of gene transcription in previously photodamaged cells. We have confirmed that pro-inflammatory cytokine IL-6 significantly enhances melanoma invasion to the extracellular matrix in our model. This supports the opinion that the aspects of ageing are essential for reliable melanoma 3D modelling in vitro.

BIOCEV 1st Faculty of Medicine Charles University Vestec 25250 Czech Republic

Department of Dermatovenereology 1st Faculty of Medicine Charles University and General University Hospital 128 00 Prague Czech Republic

Department of Informatics and Chemistry Faculty of Chemical Technology University of Chemistry and Technology 160 00 Prague Czech Republic

Institute of Anatomy 1st Faculty of Medicine Charles University 128 00 Prague Czech Republic

Institute of Pathology 1st Faculty of Medicine Charles University 128 00 Prague Czech Republic

Laboratory of Genomics and Bioinformatics Institute of Molecular Genetics of the Czech Academy of Sciences 142 20 Prague Czech Republic

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