Bright-Field Multiplex Immunohistochemistry Assay for Tumor Microenvironment Evaluation in Melanoma Tissues

: The tumor microenvironment (TME) plays a crucial role in melanoma development, progression and response to treatment. As many of the most relevant TME cell phenotypes are defined by the simultaneous detection of more than two markers, the bright-field (BF) multiplex immunohistochemistry (IHC) technique has been introduced for the quantitative assessment and evaluation of the relative spatial distances between immune cells and melanoma cells. In the current study, we aimed to validate BF multiplex IHC techniques in the Ventana Discovery Ultra Immunostainer to be applied to the evaluation of the TME in variably pigmented melanoma tissues. The BF multiplex IHC staining was performed using different combinations of six immune-cell markers-CD3, CD4, CD8, CD20, CD68 and CD163-and the melanoma cell marker SOX10. Our results show that the BF double IHC Yellow/Purple protocol guarantees the maximum contrast in all the cell populations tested and the combination SOX10 (Green), CD8 (Yellow) and CD163 (Purple) of the BF triple IHC protocol ensures the best contrast and discrimination between the three stained cell populations. Furthermore, the labeled cells were clearly distinct and easily identifiable using the image analysis software. Our standardized BF IHC multiplex protocols can be used to better assess the immune contexts of melanoma patients with potential applications to drive therapeutic decisions within clinical trials.