Islet and Blood Vessel Segmentation Islet and blood vessel segmentation was performed in a similar manner as cell segmentation with the following modifications: i) for ilastik pixel classification, images were divided into islet, blood vessels, and “other” (that is non-islet, non-vessel) compartments based on substacks containing informative markers (i.e., SYP, CD99, CD31, CD45, AMY2A, KRT19, and Iridium); ii) in CellProfiler, islets and blood vessels were directly defined as primary objects. Distance to islet rim was measured by combining islet and cell masks, using a custom CellProfiler plugin (https://github.com/BodenmillerGroup/ImcPluginsCP)

Cell Segmentation CellProfiler (Kamentsky et al., 2011) was subsequently used to define cell masks and quantify marker expression. To define cell borders, nuclei were first identified as primary objects based on ilastik probability maps and expanded through the cytoplasm compartment until either a neighboring cell or the background compartment was reached. Cell masks were generated for identification of single cells and used to extract single-cell information (marker abundance, spatial and neighborhood data) from the original images. The results were exported as csv tables for further analysis.