A high-throughput 3D culture microfluidic platform for multi-parameter phenotypic and omics profiling of patient-derived organoids

Patient-derived organoids (PDOs) are poised to become central tools both in clinical practice, to preemptively identify patient optimal treatments, and in drug discovery, overcoming the limitations of cancer cell lines. However, the use of PDOs in these settings has been hampered by several bottlenecks, including sample requirements, assay time, and handling in the context of high-throughput assays. We developed a M icrofluidic P latform for O rganoids culture (MPO) that miniaturises and simplifies PDOs cultures in a 384-plate format. Both retrospective and prospective clinical studies demonstrate MPO predictive value and the straightforward implementation in the clinical setting. MPO allows subcellular phenotypic screenings, as imaging-based applications like Cell Painting, target engagement analyses, alongside the comprehensive definition of PDOs’ genomic, transcriptomic, proteomic, lipidomic, and metabolomic landscapes. Harnessing the pleiotropic capabilities of MPO, we uncovered the role of EZH2 inhibitors to prevent the long-term emergence of resistance to RAS inhibitors in metastatic colon cancer. In all, we demonstrate the potential of MPO to impact clinical practice, alongside exploration of the mechanisms underlying compound response and resistance.