Abstract
The spatial organization of cells within tissues aids in understanding physiological and pathological processes, as well as elucidating the mechanisms of action underlying treatments. We present a protocol for analyzing image-based spatial proteomics data. To illustrate, we focus on whole-slide images of human multiplexed tumor tissues acquired using the PhenoCycler-Fusion 2.0 platform from Akoya Biosciences. We describe steps for cell segmentation, cell phenotyping, intercellular distance calculation, and data visualization. For complete details on the use and execution of this protocol, please refer to Franken et al.1.