Trellis Single-Cell Screening Reveals Stromal Regulation of Patient-Derived Organoid Drug Responses

Patient-derived organoids (PDOs) can model personalized therapy responses, however current screening technologies cannot reveal drug response mechanisms or how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly-multiplexed mass cytometry platform to measure post translational modification (PTM) signaling, DNA-damage, cell-cycle activity, and apoptosis in >2,500 colorectal cancer (CRC) PDOs and cancer associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment specific drug responses in thousands of single-cell datasets, we developed Trellis — a highly-scalable, hierarchical tree-based treatment effect analysis method. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is rare, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate cancer cell plasticity — shifting proliferative stem cells to slow-cycling revival stem cells via YAP to protect cancer cells from chemotherapy. Overall design: Single-cell RNA-seq was performed using the SPLiT-seq protocol on 2 human PDO (PDO21 and PDO27) 3D cell models in triplicate samples of PDO and CAF mono- and co-cultures. The sample preparation was performed in two batches, one for each PDO. For each batch, 9 samples were multiplexed, corresponding to each cell type's mono culture and co-cultures, all perfomed in triplicate (3x3). In total, 18 samples across the 2 SPLiT-seq experiments were analyzed.The dataset contains 4 sublibraries with 39852 cells, these correspond to the 2 sublibraries from each SPLiT-seq barcoding experiment. Further information on the SPLiT-seq barcoding layout can be found at : https://github.com/TAPE-Lab/Ramos-et-al-Trellis