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

作者：Nicole Groß, Soren Charmsaz, Diana Carolina Vargas Carvajal, Xuan Yuan, Yiyang Gao, Alexei Hernandez, Courtney Cannon, Elizabeth M. Jaffee, Won Jin Ho · 发表于：Regular and Young Investigator Award Abstracts · 年份：2025 · DOI：10.1136/jitc-2025-sitc2025.0017 · 研究领域：Single-cell and spatial transcriptomics、Cancer Cells and Metastasis、Cell Image Analysis Techniques

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 pa...