17 High-throughput functional screening of cellular phenotypes in the tumor microenvironment using massively parallel microwell co-culture and in-well mass cytometry

Background The tumor microenvironment (TME) comprises a complex, dynamic mixture of immune and stromal cells whose phenotypic diversity and plasticity shape tumor behavior and immunotherapy response. While advances in single-cell transcriptomic and proteomic technologies have discovered novel phenotypic markers, these markers are often insufficient to define functional states due to the lack of corresponding co-culture experiments for validation.Methods To address this, we developed DEFINE, Direct Evaluation of Functional Immune Networks via Ex vivo co-culture, a high-throughput co-culture and profiling platform that integrates spatially resolved ex vivo microwell assays with high-plex imaging mass cytometry (IMC). This system leverages chamber slides printed with thousands of barcoded microwells using photo-crosslinkable gelatin methacrylate, each capable of longitudinal tumor killing assays by live cell imaging (TROVO, Enrich Biosystems). These microwells are compatible with IMC ablation, enabling >50-parameter single-cell phenotyping within each microwell, directly correlating phenotype with tumor cytotoxicity outcomes. We applied this platform to investigate the functional significance of myeloid phenotypic markers in a mouse model of immunotherapy-resistant pancreatic ductal adenocarcinoma (PDAC). CD11b + myeloid cells were isolated from KPC 6419c5 tumors and co-cultured with OVA-expressing PDAC tumor cells and OVA-specific OT-I CD8+ T cells. Using a tailored antibody panel for myeloid cell profiling and optimized in-well staining procedures, we identified distinct phenotypic features associated with suppression or promotion of T cell-mediated tumor killing.Results Initial discovery from 81 microwells at 1 μm resolution were cross-validated by data acquisition of 338 microwells at 5 μm resolution of the same slide. Notably, tumor-associated macrophages (TAMs) expressing high levels of FcγRII, CD262, and CSF1R were enriched in microwells with lower cytotoxicity, suggesting an immune-suppressive function. In contrast, elevated iNOS expression on myeloid derived suppressor cells correlated with enhanced tumor cell clearance, pointing to an antitumor effector function.Conclusions This integrated platform enables direct functional mapping of thousands of discrete cellular phenotypes, bridging the gap between high-dimensional phenotyping and immune functionally.