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

Abstract

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.

Pan-cancer analysis reveals NAA50 as a cancer prognosis and immune infiltration-related biomarker

“…In the past, more and more studies have testified that the prognosis of cancer patients was closely interrelated to the tumor microenvironment (TME) (Liu et al, 2017;Chen et al, 2020). Immune responses in the tumor microenvironment are also believed to play a huge role in the progression of cancer and response to treatment (Song et al, 2020;Ugolini et al, 2022). Frontiers in Genetics frontiersin.org…”

Exploring the Complex and Multifaceted Interplay between Melanoma Cells and the Tumor Microenvironment

“…Multiplexed imaging techniques, such as brightfield (BF) multiplexed IHC, can visualize up to eight markers simultaneously by labeling different cell populations on the same section [ 121 ]. Immune cell markers including CD3 (T cell activation), CD8 (cytotoxic T cells), CD20 (B cells), CD68 (macrophages), CD163 (M2 macrophages), and CD16 (cytotoxic macrophages and NK cells) can then be combined with PD-L1, SOX10, and Ki67 [ 110 , 122 ]. Thus, simultaneous determination of the location and interaction between immune cell subpopulations and melanoma cells can be accomplished.…”

An update on methods for detection of prognostic and predictive biomarkers in melanoma

“…Typical approaches to immunohistochemistry (IHC) evaluation of tissue from melanoma patients have several limitations including inter-observer variability and the labeling of just a single biomarker for each tissue section. Emerging techniques, namely, multiplex IHC or immunofluorescence (mIHC/IF), have attempted to address these limitations by detecting multiple biomarkers in a single tissue section through high-throughput staining and quantitative analysis (Tan et al, 2020;Ugolini et al, 2022;Yaseen et al, 2022). This technology deters from using a cocktail of antibodies reared in separate hosts and instead relies on cycles of single antibody stains added in sequential order, which are subsequently removed in order for the next antibody to be added without cross-reaction (Nguyen et al, 2021).…”