Cell segmentation of hematoxylin-stained tissue. Example of segmented cells in the sub-capsular region, showing both small round nuclei in lymphocyte-rich areas (B cell follicle, top left) and elongated nuclei in macrophage-rich areas (sub-capsular sinus, middle).
ARTICLE ABSTRACT
Recent clinical observations have emphasized the critical role that the spatial organization of immune cells in lymphoid structures plays in the success of cancer immunotherapy and patient survival. However, implementing sequential chromogenic immunohistochemistry (scIHC) to analyze multiple biomarkers on a single tissue section has been limited due to a lack of a standardized, rigorous guide to the development of customized biomarker panels and a need for user-friendly analysis pipelines that can extract meaningful data. In this context, we provide a comprehensive guide for the development of novel biomarker panels for scIHC, using practical examples and illustrations to highlight the most common complications that can arise during the setup of a new biomarker panel, and provide detailed instructions on how to prevent and detect cross-reactivity between secondary reagents and carryover between detection antibodies. We also developed a novel analysis pipeline based on non-rigid tissue deformation correction, Cellpose-inspired automated cell segmentation, and computational network masking of low-quality data. We applied this biomarker panel and pipeline to study regional lymph nodes from head and neck cancer patients, identifying novel contact interactions between plasmablasts and plasmacytoid dendritic cells in vivo. Given that Toll-like receptors, which are highly expressed in plasmacytoid dendritic cells, play a key role in vaccine efficacy, the significance of this cell-cell interaction decisively warrants further studies. In summary, this work provides a streamlined approach to the development of customized biomarker panels for scIHC that will ultimately improve our understanding of immune responses in cancer.
