A highly adaptable protocol for mapping spatial features of cellular aggregates in tissues

Multiplex imaging technologies have developed rapidly over the past decades. The advancement of multiplex imaging has been driven in part by the recognition the spatial organisation of cells can represent important prognostic biomarkers and that simply studying the composition of cells in diseased tissue is often insufficient. There remains a lack of tools that can perform spatial analysis at the level of cellular aggregates (a common histopathological presentation) such as tumors and granulomas, with most analysis packages focussing on smaller regions of interest and potentially missing patterns in the overall lesion structure and cellular distribution. Here we present a protocol to quantify the cellular structure of entire tissue lesions built around two novel metrics. The Total Cell Preference Index reports whether a lesion tends to change in density in its central vs peripheral areas and can indicate the extent of necrosis across the entire lesion. The Immune Cell Preference Index then reports whether each immune cell-type is located more centrally or peripherally across the entire lesion. The output of both indexes is a single number readout for simple interpretation and visualization, and they can be applied to lesions of any size or shape. Additionally, this protocol can be applied to any slide-scanning multiplexed imaging system, either based on protein and nucleic acid staining. Finally, the protocol uses the open-source software QuPath and can be utilized by researchers with a basic understanding of QuPath with the full protocol able to be applied on pre-generated images within one hour.