Trellis tree-based analysis 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, 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 rarer, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate PDO plasticity—shifting proliferative colonic stem cells (proCSCs) to slow-cycling revival colonic stem cells (revCSCs) to protect cancer cells from chemotherapy. [Display omitted] •>2,500 single-cell signaling responses from drug-treated PDOs and CAFs•Trellis: tree-based treatment effect method for single-cell screening analysis•PDOs have patient- and CAF-specific drug responses that align with PTM signaling•CAFs can polarize PDOs to a chemorefractory revival stem cell (revCSC) fate Single-cell signaling analysis of >2,500 patient-derived organoid (PDO) and cancer-associated fibroblast (CAF) cultures using a method called Trellis reveals patient-specific drug responses. CAFs can protect PDOs by polarizing proliferative colonic stem cells to slow-cycling revival stem cells.