ABSTRACT: Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.
