Multiplex hyperspectral IHC: A 12-plex assay for high-dimensional analysis of the tumor microenvironment.

The immune component of the tumor microenvironment (TME) is highly prognostic of patient outcome, further understanding of TME and the spatial organization of the immune cells within the TME is needed for better patient prognosis and treatment planning. Current brightfield immunohistochemistry techniques quantify immune cell counts and density but generally cannot assess the spatial relationship between tumor and immune cells due to the limited number of markers on a single slide. Here, we present a brightfield-based multiplexed immunohistochemistry workflow that combines multiple labels per staining round with iterative cycles of staining, destaining, and hyperspectral imaging to enable high-dimensional immune profiling within a single tissue section. This approach is directly compared with a sequential immunofluorescence technique applied to adjacent tissue sections to compare and evaluate its performance. By integrating serial imaging, sequential labeling, and image registration, we are able to spatially map the TME using standard brightfield microscopy.