Analysis of melanoma co-culture systems effect on cell fate and signalling rewiring

Late-stage melanoma remains a deadly form of skin cancer as many patients become resistant or do not respond to treatment. The tumour microenvironment (TME) is one mechanism by which treatment efficacy can differ between patients. There remains much to be elucidated on how secreted factors influence melanoma cell fate and can potentially be used for therapeutic benefit. Hence we investigate the cellular cross talk of melanoma TME cells. Here co-cultures were created to mimic the TME based on the A375 melanoma cell line, the HaCaT keratinocyte cell line, primary normal human dermal fibroblast (NHDF) and cancer associated fibroblasts (CAFs). From Various co-culture combinations of these cells the secretome or conditioned media (CM) was harvested and analysed using mass spectrometry. The CM from various co-cultures were characterised. Following a differential expression analysis the differentially expressed proteins were subjected to a gene-set enrichment analysis. Metabolic pathways were identified as a major pathway associated with the secreted proteins of the CM’s containing NHDF. The intracellular effect of the CM on A375 was examined. Indeed, there was differentially expressed intracellular intermediates of key metabolic pathways. Thus the metabolic phenotype of various CM from different co-cultures was investigated. CM was added to A375 cell monolayer and using the seahorse ATP real time flux assay, the ATP production in response to CM, indeed all the CM increased the ATP production some significantly. The A375 cells were also treated with selected differentially expressed protein, from the secretome. LAMB3 induced a statistically significant increase in the ATP production in the A375 cells. Taken together we identified various unique and differentially expressed proteins in the co-culture secretomes. An extracellular CM association with metabolism and intracellular metabolic rewiring was identified. There was a change in metabolic phenotype depending on the CM the A375 cells were treated with. We also describe a potentially novel function of LAMB3 in the ATP production of A375 cells. Thus, this thesis describes extracellular secreted factors that influence intracellular rewiring and cell phenotype, that could be further investigated for therapeutic benefit.