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).

Patient-derived organoids (PDOs) model personalized cancer therapy responses. How-ever, existing bulk PDO screening technologies cannot reveal drug response mechanisms or model how cells of the tumor microenvironment alter therapy performance. To address this, we developed a highly-multiplexed thiol-reactive organoid barcodingin situ(TOBis) mass cytometry platform to perform single-cell post translational modification (PTM) signaling analysis of colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies. To compare patient- and microenvironment-specific treatment effects in thousands of single-cell PTM datasets, we developedTrellis— a highly-scalable, hierarchical tree-based treatment effect analysis method. Trellis analysis of>2,500 single-cell PTM PDO-CAF organoid cultures revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is patient-specific. We found drug-induced apoptosis does not correlate with genotype or clinical staging, but does align with cell-intrinsic PTM signaling in PDOs. We observe that CAFs protect chemosensitive PDOs by shifting cancer cells into a slow-cycling cell-state in a patient-specific manner and show that CAF chemoprotection can be reversed by inhibiting YAP. These results reveal that PTM signaling flux is a major determinant of chemosensitivity and demonstrate CAFs regulate patient-specific drug responses by altering cancer cell-state.

changes occurring in T-cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T-cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared to FMC63 CAR T-cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T-cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B-cells present in the manufacture.