Abstract

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyse cell-type-specific signalling networks in organoids are absent. Post-translational modification (PTM) signalling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signalling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 126-plex single-cell comparison of organoid cultures and demonstrate how TOBis MC scales to tumour microenvironment organoid co-culture drug screening applications