Melanoma phenotype plasticity underlies tumour dissemination and resistance to therapy, yet its regulation is incompletely understood. In vivo switching between a more differentiated, proliferative phenotype and a dedifferentiated, invasive phenotype is directed by the tumour microenvironment. We found that treatment of partially dedifferentiated, invasive A375M2 cells with two structurally unrelated p38 MAPK inhibitors, SB2021920 and BIRB796, induces a phenotype switch in 3D collagen, as documented by increased expression of melanocyte differentiation markers and a loss of invasive phenotype markers. The phenotype is accompanied by morphological change corresponding to amoeboid-mesenchymal transition. We performed RNA sequencing with an Illumina HiSeq platform to fully characterise transcriptome changes underlying the switch. Gene expression results obtained with RNA-seq were validated by comparing them with RT-qPCR. Transcriptomic data generated in the study will extend the present understanding of phenotype plasticity in melanoma and its contribution to invasion and metastasis.

• Keywords
• amoeboid invasion, cancer, melanoma, metastasis, phenotype switch,
• MeSH
• Cell Differentiation drug effects   genetics   MeSH
• Phenotype MeSH
• Gene Ontology MeSH
• Imidazoles pharmacology   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Collagen metabolism   MeSH
• Humans MeSH
• Melanoma genetics   pathology   MeSH
• p38 Mitogen-Activated Protein Kinases antagonists & inhibitors   metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Microenvironment drug effects   genetics   MeSH
• Naphthalenes pharmacology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Cell Proliferation drug effects   genetics   MeSH
• Pyrazoles pharmacology   MeSH
• Pyridines pharmacology   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• RNA-Seq methods   MeSH
• Gene Expression Profiling methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole MeSH Browser
• doramapimod MeSH Browser
• Imidazoles MeSH
• Protein Kinase Inhibitors MeSH
• Collagen MeSH
• p38 Mitogen-Activated Protein Kinases MeSH
• Naphthalenes MeSH
• Pyrazoles MeSH
• Pyridines MeSH

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.

• Keywords
• Interleukin-6, cytokine, extracellular matrix, fibroblasts, heterogeneity, melanoma, senescence-associated secretory phenotype, single-cell sequencing, spheroids, subpopulation,
• Publication type
• Journal Article MeSH

The invasive behaviour of cancer cells underlies metastatic dissemination; however, due to the large plasticity of invasion modes, it is challenging to target. It is now widely accepted that various secreted cytokines modulate the tumour microenvironment and pro-inflammatory signalling can promote tumour progression. Here, we report that cells after mesenchymal-amoeboid transition show the increased expression of genes associated with the type I interferon response. Moreover, the sustained activation of type I interferon signalling in response to IFNβ mediated by the Stat1/Stat2/IRF9 complex enhances the round amoeboid phenotype in melanoma cells, whereas its downregulation by various approaches promotes the mesenchymal invasive phenotype. Overall, we demonstrate that interferon signalling is associated with the amoeboid phenotype of cancer cells and suggest a novel role of IFNβ in promoting cancer invasion plasticity, aside from its known role as a tumour suppressor.

• Keywords
• amoeboid, inflammation, interferon, invasion, melanoma, mesenchymal, plasticity,
• Publication type
• Journal Article MeSH