Objective

There has been a rapid growth in the field of tumor immunobiology in recent years as a result of recent successes in cancer immunotherapies, and it is becoming clear that immune cells play many sometimes conflicting roles in the tumor microenvironment. However, obtaining phenotypic information about the various immune cells that play these roles in and around the tumor has been a challenge. Existing methods can either deliver phenotypic information on homogenous samples (e.g., flow cytometry or PCR) or morphologic information on single immunomarkers (standard IHC). We present here a methodology for delivering quantitative per-cell marker expression and phenotyping, analogous to that obtained from flow cytometry, but from cells imaged in situ in FFPE tissue sections. 
This methodology combines:  the sequential multi-marker labeling of up to 8 antigens using antibodies all of the same species in a single section; automated multispectral imaging (MSI) to remove the typically problematic FFPE tissue autofluorescence and correct cross-talk between fluorescent channels; and an automated analysis that can quantitate the per-cell marker expression, determine the cellular phenotype, count these cells separately in the tumor compartment and in the stroma and provide high-resolution images of their distributions. 
We present here examples of this new methodology: the simultaneous labeling, analysis and validation of CD4, CD8, CD20, PD-L1, Foxp3, cytokeratin and DAPI in breast cancer; and CD8, CD34, PD-L1, FOXP3 and DAPI in head and neck squamous cell carcinoma.  Each example will show the application of the multiplexed staining, per-cell quantitation and cellular phenotyping from multispectral images of FFPE tissue sections, as well as methods to explore the spatial distributions of the phenotyped cells in and around the tumor