Cellular phenotyping of tumor microenvironments with a high-plex fluorescent labeling and spectral unmixing approach 9333

Description Tumor microenvironments are complex and host diverse cell types and interactions. In-depth characterization of these environments requires detecting multiple biomarkers within the same sample, which is limited by traditional fluorescent immunohistochemistry (IHC) due to spectral constraints. Further, many high-plex IHC methods are time-consuming or require multiple imaging rounds. Here we developed a one-step multiplex labeling approach using a cocktail of primary antibody conjugates combined with spectral unmixing, enabling the simultaneous detection of 8 biomarkers and a nuclear counterstain. This streamlined approach offers high-plex biomarker detection within a timeframe similar to traditional single-antibody IHC workflows. Using the EVOS S1000 Spatial Imaging System and quantitative image analysis software, we identified multiple distinct myeloid (CD68+), lymphoid (CD20+, CD4+, FOXP3+, CD8+), and proliferating (Ki67+) cell subpopulations in normal and tumor colon tissue samples and compared their abundance and spatial distribution in relation to tissue architecture markers for smooth muscle actin and pan-cytokeratin. Additionally, we analyzed the cellular composition of a tertiary lymphoid structure in the colon tumor, believed to play a role in the immune response to colorectal cancer. These data highlight the advantages of high-plex biomarker detection for phenotyping complex tumor microenvironments using our streamlined labeling workflow and spectral unmixing approach.