Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type–specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type–specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for >40 antibodies from millions of cells across 126 organoid cultures in a single MC run.

类器官是包含多种细胞类型和细胞状态的仿生组织模型。翻译后修饰 (PTM) 信号网络控制细胞表型，并且在癌症等疾病中经常失调。尽管信号网络因细胞类型而异，但研究异细胞类器官中细胞类型特异性 PTM 的技术有限。在这里，我们提出了一个多路复用质谱细胞术 (MC) 协议，用于单细胞分析 PTM 信号和类器官中的细胞状态以及与成纤维细胞和白细胞共培养的类器官。我们描述了原位硫醇反应性类器官条形码（TOB是) 能够对类器官培养物进行 35 重和 126 重单细胞比较，并提供飞行时间 (CyTOF) 信号分析管道 (CyGNAL) 的细胞计数，用于计算细胞类型特异性 PTM 信号网络。TOB是MC 协议从类器官固定到数据采集需要大约 3 天的时间，并且可以在单次 MC 运行中为来自 126 个类器官培养物的数百万个细胞的 >40 抗体生成单细胞数据。