An Allogeneic PBMC–Organoid Co-culture Platform for Evaluating Immunotherapy Efficacy and T-Cell-Mediated Antitumor Immunity

Traditional 2D cell lines and mouse xenograft models have limitations in recapitulating the complexity of the tumor microenvironment (TME) and dynamic interactions between tumor and immune cells, creating an urgent need for models to evaluate immunotherapies. Patient-derived organoids (PDOs), which retain key features of original tumors, offer a platform for dissecting the TME when co-cultured with peripheral blood mononuclear cells (PBMCs). However, challenges such as inter-batch variability of PBMCs and difficulties in autologous PBMC acquisition hinder their clinical translation. This study established four stably passaged colorectal cancer organoid models and developed an allogeneic PBMC-organoid co-culture platform to investigate the immune activation and cytotoxic potential of allogeneic PBMCs. Results showed that vacuolated-structured organoids are associated with the increased proportion of CD8+ T cells in PBMCs within the co-culture system. PBMCs exhibited stronger cytotoxicity against microsatellite-stable (MSS) organoids with low PD-L1 expression, while the anti-PD-1 antibody Keytruda significantly enhanced their antitumor effects on high PD-L1-expressing organoids. The allogeneic co-culture model developed here overcomes the limitations of autologous PBMC acquisition, providing an efficient and versatile proof-of-concept platform for pre-clinical screening of immunotherapeutic drugs. Distinct from prior allogeneic co-culture systems that rely on healthy donor immune cells, our platform uniquely integrates patient-derived allogeneic PBMCs with tumor organoids, providing preliminary proof-of-concept evidence that tumor-intrinsic features may influence immunotherapy responses based on tumor-intrinsic features while preserving the pathological relevance of cancer patient immune profiles.