There has been a rapid grown in the field of tumor immunobiology in recent years as a result of recent successes in cancer immunotherapies, and it is clear that immune cells play many sometimes conflicting roles in the tumor microenvironment. However, obtaining phenotypic information about the various immune cells in and around the tumor has been a challenge. Existing methods can deliver phenotypic information on homogenous samples (e.g., flow cytometry or PCR) or morphologic information in single stain 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 6 antigens using antibodies all of the same species (with a goal of reaching 8 markers); automated multispectral imaging to remove problematic FFPE tissue autofluorescence and correct cross-talk between fluorescent channels; and an automated image 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, provide high-resolution images of their distributions and provide x,y coordinate data from which spatial distance calculations can be made. We will show a 6-plex assay in breast cancer showing the application of the multiplexed staining, per-cell quantitation and cellular phenotyping in FFPE tissue sections, as well as methods to explore the spatial distributions of the phenotyped cells in and around the tumor.
Citation Format: James R. Mansfield, Clifford C. Hoyt, Edward Stack, Michael Feldman, Carlo Bifulco, Bernard Fox. Imaging in cancer immunology: Phenotyping of multiple immune cell subsets in-situ in FFPE tissue sections. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A133.
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