Organoids are powerful biomimetic tissue models. Despite their widespread adoption, methods to analyse cell-type specific post-translational modification (PTM) signalling networks in organoids are absent. Here we report multivariate single-cell analysis of cell-type specific signalling networks in organoids and organoid co-cultures. Simultaneous measurement of 28 PTMs in >1 million single small intestinal organoid cells by mass cytometry reveals cell-type and cell-state specific signalling networks in stem, Paneth, enteroendocrine, tuft, goblet cells, and enterocytes. Integrating single-cell PTM analysis with Thiol-reactive Organoid Barcoding in situ (TOB is ) enables high-throughput comparison of signalling networks between organoid cultures. Multivariate cell-type specific PTM analysis of colorectal cancer tumour microenvironment organoids reveals that shApc , Kras G12D , and Trp53 R172H cell-autonomously mimic signalling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type specific signalling analysis of healthy and cancerous organoids.

The process of metastasis is highly complex 1. In the case of breast cancer, there are frequently long timespans between cells leaving the primary tumour and growth of overt metastases 2, 3. Possible reasons for disease indolence and subsequent transitioning back to aggressive growth include interplay with myeloid and fibroblastic cells in the tumour microenvironment and ongoing immune surveillance 4-6. However, the signals causing actively growing cells to enter into an indolent state, and enabling them to survive for extended periods of time, are not well understood. In this work, we reveal how the behaviour of indolent breast cancer cells in the lung is determined by their interactions with alveolar epithelial cells, in particular AT1 cells. This crosstalk promotes the formation of fibronectin (FN) fibrils by indolent cells that drive integrindependent pro-survival signals. Combined in vivo RNA sequencing and drop-out screening identified Secreted frizzled-related protein 2 (Sfrp2) as a key mediator of this interaction. Sfrp2 is induced in breast cancer cells by signals emanating from lung

This multiplexed mass cytometry protocol uses Thiol-reactive Organoid Barcoding in situ (TOBis) and a CyTOF siGNalling AnaLysis pipeline (CyGNAL) to enable 126-plex single-cell analysis of cell-type, cell-state, and posttranslational modification signalling network in organoids.Tweet A multiplexed mass cytometry protocol using Thiol-reactive Organoid Barcoding in situ (TOBis) and a CyTOF siGNalling AnaLysis pipeline (CyGNAL) for 126-plex single-cell analysis of cell-type-specific PTM signalling in organoids (from @QinXiao1990 @FerranC96 and @christophertape).