Influence of Neutrophils and Extracellular Matrix on the Reactivation of Dormant Melanoma Cells

Abstract

Despite new therapeutic strategies, melanoma patients suffer from recurrence decades after successful treatment. The underlying mechanism is thought to be tumor dormancy, a state of reversible growth arrest in which tumor cells persist in niches. Upon an adequate stimulus, such as sustained inflammation through smoking, metastatic outgrowth may occur. As proven in breast cancer, this may be mediated by neutrophil extracellular traps (NETs), which in turn alter the extracellular matrix (ECM) of the tumor niche. In this study, we aimed to validate the previously established in vitro model of melanoma dormancy. We further hypothesized that stimulated neutrophils release NETs, which reawaken dormant tumor cells in an ECM-dependent manner. Dormancy was induced in SbCl2 and A375 melanoma cells by a 72 h treatment with cisplatin, which lasted for at least 15 days. Regrowth was assessed for up to 6 weeks by light microscopy, cell or colony count and by using the fluorescence ubiquitination-based cell cycle indicator (FUCCI) system in melanoma cells. Stable G0/G1 arrest was only achieved in SbCl2 but not in A375 cells. Expression analysis by qPCR and western blot characterized dormant melanoma cells on the molecular level. While p53 and p21 stabilized the cell cycle arrest for 20 days, ERK activation (MAPK) may contribute to cell survival. Epithelial-mesenchymal transition (EMT) markers further indicated a shift towards a differentiated epithelial phenotype shortly after dormancy induction, contrary to our expectations. Findings of the senescence-associated β-galactosidase assay allow coexistence of a dormant and senescent (irreversible arrest) state. The proliferative capacity of melanoma cells was assessed during cocultivation of melanoma cells with neutrophils or NETs and/or the ECM components Matrigel or a fibroblast matrix. Fibroblast derived ECM (FF-ECM) and Matrigel restricted growth in proliferating cells, while solubilized FF-ECM promoted proliferation. Cocultivation (6-18 h) of PMN or NETs with dormant SbCl2 cells did not induce reawakening when seeded in 2 % Matrigel. NETs induced massive cell death, which may explain why melanoma cells act suppressive on NET formation. All in all, PMN/NETs did not reawaken SbCl2 cells of our in vitro dormancy model under the selected test conditions. Future studies may focus on the effect of sustained neutrophil inflammation in melanoma.