Cancer-Associated Fibroblasts Regulate Patient-Derived Organoid Drug Responses

Patient-derived organoids (PDOs) model personalized cancer therapy responses. However, 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 barcoding in situ (TOB is ) 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 developed Trellis – 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. ### Competing Interest Statement S.K. is on the scientific advisory board of KovaDx and AI Therapeutics.