Recent clinical observations highlight the importance of the spatial organization of immune cells into lymphoid structures for the success of cancer immunotherapy and patient survival. Sequential chromogenic immunohistochemistry (scIHC) supports the analysis of multiple biomarkers on a single tissue section thus providing unique information about relative location of cell types and assessment of disease states. Unfortunately, widespread implementation of scIHC is limited by lack of a standardized, rigorous guide to the development of customized biomarker panels and by the need for user-friendly analysis pipelines able to streamline the extraction of meaningful data. Here, we examine major steps from classical IHC protocols and highlight the impact they have on the scIHC procedure. We report practical examples and illustrations of the most common complications that can arise during the setup of a new biomarker panel and how to avoid them. We described in detail how to prevent and detect cross-reactivity between secondary reagents and carry over between detection antibodies. We 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. The resulting biomarker panel and pipeline was used to study regional lymph nodes from head and neck cancer patients. We identified contact interactions between plasmablasts and plasmacytoid dendritic cells in vivo. Given that TLR 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 conclusion, this work streamlines the development of novel biomarker panels for scIHC, which will ultimately improve our understanding of immune responses in cancer.

<p>Alignment results. Comparison of before and after alignment for CD3 (red, top) and CD20 (red, bottom) superimposed with hematoxylin (green).</p>

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.

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<p>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).</p>