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Multiplex Immunofluorescence Staining Facilitates the Understanding of Tumor Immune Microenvironment



Recent regulatory approval of numerous immunotherapies has made a greater understanding of the tumor microenvironment essential for contextualizing clinical response, informing drug combinations, and selecting appropriate indications. Due to the complexity and dynamic nature of the tumor immune microenvironment, there is a need for more advanced techniques to capture specific subsets of immune cells and look at cell-cell relationships in patient samples.


The PD-1/PD-L1 axis blockade offers a good example of this complexity. In spite of the success of the checkpoint inhibitors for PD-1 (nivolumab and pembrolizumab) and PD-L1 (atezolizumab and durvalumab) in solid tumor indications and melanoma, resistance and relapse were also observed in spite of PD-L1 expression in the tumors. More recently, combined analysis of CD8+ cytotoxic cells and PD-L1 expression in tumors has been described as a more reliable prognostic marker than PD-L1 alone in gastric cancer. Further understanding of the immunological responses to drugs in the tumor microenvironment and the development of more sophisticated prognostic biomarkers are key to designing treatment regimens tailored for individual patients.


Currently, the most commonly used tissue-based techniques in immuno-oncology research include RNA sequencing (RNAseq), flow cytometry, and multiplex immunofluorescence (mIF). RNAseq is a high-throughput technique that has been used to generate immune signatures and prognostic classifiers for multiple tumor types. However, the low RNA quality of formalin-fixed, paraffin-embedded tissues limits its application in archival or diagnostic samples. Additionally, tissue architecture is destroyed in sample preparation resulting in loss of spatial context. Flow cytometry is multi-channel, sensitive, and able to quickly identify different cell types by size and markers, and it has been used for immunophenotyping of peripheral blood and tumor samples. However, it requires fresh samples and dissociation of cells which, similarly to RNAseq, leads to loss of tissue architecture and relevant spatial relationships between immune cells and tumor cells. Multiplex immunofluorescence, on the other hand, can reveal the expression of multiple markers in individual cells while preserving spatial relationships between immune cells and tumor cells and has therefore become a powerful tool for the characterization of the tumor microenvironment.


Multiplex IF is unique in its ability to provide both expression and location of several immune markers while preserving tissue architecture and spatial context. In developing a multiplex panel, several technical considerations are important, such as a limited number of markers per panel, steric hindrance of the antibody binding to epitopes, auto-fluorescence, leaking of light between different fluorophore channels, and challenges pertaining to scanning images and analyzing the results. A number of published reports have described methods for optimization of multiplex IF for immuno-oncology research. Briefly, whether all primary antibodies are amenable to the same antigen retrieval conditions will be first considered. If the primary antibodies were limited and sequential staining had to be performed, the second consideration is the staining order and antibody-fluorophore pairing, which is based on 3 main points: 1) epitope heat resistance relating to the maximum number of heat-mediated stripping cycles a specific molecular target could withstand, 2) steric hindrance or masking effect resulting in suboptimum detection of markers that share very close subcellular location, and 3) the abundance of the targets. A very abundant target should be preferentially associated with a fluorophore of a relatively weaker intensity. In multiplex fluorescence image acquisition, inadequate signal-to-noise ratios caused by auto-fluorescence contamination and fluorescence bleed-through affect the quality of image visualization and the detection of lowly expressed antigens. It is therefore essential to carefully design and optimize each multiplex panel, not only for the selected markers but also with regard to the detection system as well as appropriate image capture and analysis.




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