Highly Adaptable Analysis Tools 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 that the spatial organization 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 focusing on smaller regions of interest and potentially missing patterns in the overall lesion structure and cellular distribution. Here, we present protocols to quantitatively describe 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 versus 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 these indexes can be applied to lesions of any size or shape. This simplifies cross-lesion comparison compared to traditional Euclidian distance-based analysis, which outputs multiple values for each lesion (one for output for each band used in the infiltration analysis). Additionally, this approach can be applied to any slide-scanning multiplexed imaging system, based on either protein or nucleic acid staining. Finally, the approach uses the open-source software QuPath and can be utilized by researchers with a basic understanding of QuPath, with the full analysis able to be applied to pre-generated images within 1 hr. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Image preparation and lesion selection Basic Protocol 2: Total Cell Preference Index and Immune Cell Preference Index.