Background Inter- and intralesional tumor heterogeneity is commonly seen in metastatic melanoma, likely playing a major role in resistance to therapy, immunotherapy included. This research project aims to identify by 6-color multiplex immunofluorescence melanoma cell subpopulations, to reveal those that are insufficiently targeted by current immunotherapies.

Methods In silico analysis of single cell RNAseq data available from the literature for melanoma were performed and matched with a list of known cancer antigens. Genes discriminating between different subpopulations of melanoma cells were selected and included for multiplex immunofluorescence experiments. FFPE sections from pre- and post-immunotherapy (DC vaccination or ipilimumab) treated melanoma patients were stained by multiplex immunofluorescence for AXL, MITF, PRAME, melanoma lineage (comprising Melan-A, gp100 and tyrosinase), CD45 and CD8 expression.

Results Single cell-based analysis of RNAseq data revealed two sets of genes discriminating between different subpopulations of melanoma cells and covering most melanoma cells. Set 1 was shown to be AXL high/MITF low and expressed PRAME, whereas set 2 was shown to be AXL low/MITF high and expressed melanoma lineage markers. The 6-color multiplex immunofluorescence panel could discriminate different melanoma subpopulations, thereby giving information on melanoma heterogeneity. Image analyses of melanoma phenotypes and immune infiltrates is still ongoing. These analyses also include the topographical location of different melanoma cell subpopulations with respect to immune cells, and their changes after immunotherapy.

Conclusions Melanoma heterogeneity pre- and post-immunotherapy can be analyzed by 6-color multiplex immunofluorescence.