A 3D pooled PBMC-organoid co-culture platform for profiling immune susceptibility and PD-1 blockade response in gastric cancer

BACKGROUND: Peritoneal metastatic gastric cancer (PMGC) is associated with a dismal prognosis and limited benefit from immune checkpoint inhibitors (ICIs). Although PD-1/PD-L1 blockade has improved outcomes in selected patients, therapeutic responses remain highly heterogeneous, even among PD-L1-high tumors. The lack of preclinical models that functionally recapitulate tumor-immune interactions in a three-dimensional (3D) context has hindered the investigation of immune susceptibility and resistance mechanisms in PMGC. METHODS: We established a standardized allogeneic 3D co-culture platform integrating patient-derived PMGC organoids with pooled peripheral blood mononuclear cells (PBMCs) from healthy donors. An optimized Matrigel-embedded configuration was used to enable sustained immune cell infiltration and tumor-immune contact. Immune-mediated cytotoxicity, organoid susceptibility phenotypes, and responses to PD-1 blockade with pembrolizumab were functionally assessed. Comparative proteomic profiling was performed, and differential expression between groups was evaluated using appropriate statistical tests with false discovery rate correction. Group comparisons were conducted using Student's t-test. RESULTS: The optimized 3D embedded system enabled dynamic infiltration of activated T cells into organoid structures. PMGC organoids exhibited heterogeneous susceptibility to immune-mediated cytotoxicity, classifying them into cytolytic and noncytolytic phenotypes independent of HLA mismatching. Comparative proteomic profiling revealed that cytolytic lines were enriched in metabolic pathways, whereas noncytolytic lines showed enrichment of immune-related signaling. Notably, response to pembrolizumab varied even among PD-L1-high organoids. Nonresponsive PD-L1-high lines were characterized by elevated baseline expression of alternative immune checkpoint ligands, specifically CD112 (TIGIT ligand) and galectin-9 (TIM-3 ligand). CONCLUSIONS: We established a robust pooled PBMC-organoid co-culture platform that enables functional assessment of tumor-immune dynamics in PMGCs. This system serves as a functional ex vivo tool for evaluating immunotherapy responses and for examining the expression patterns of alternative immune checkpoint ligands associated with differential PD-1 blockade response. These findings support the utility of this ex vivo PMGC co-culture system for evaluating heterogeneous responses to PD-1 blockade and associated baseline differences in alternative immune checkpoint ligand expression among PD-L1-high organoids.