A high-throughput 3D tumor-immune co-culture platform for evaluating immune checkpoint inhibitors

The development of immune checkpoint inhibitors (ICIs) requires preclinical models that accurately reflect interactions between human tumor and immune cells. Conventional 2D systems lack architectural and immunological relevance, and animal models often fail to capture human checkpoint regulation. There is therefore a strong need for physiologically relevant and scalable in vitro platforms to improve the predictive power of early-stage ICI evaluation.To address this need, we developed a high-throughput 3D tumor-immune co-culture platform to model checkpoint activity and quantify immune-mediated tumor responses. Tumor microtissues were generated in the Akura™384 format and treated to induce inhibitory ligand expression, while human immune cells were stimulated through defined co-stimulatory pathways to induce checkpoint receptor expression. After establishing the co-cultures, we applied reference ICIs or control antibodies and assessed responses using multiple assays. Apoptotic tumor cell death was quantified using Caspase-3/7 Glo®, and tumor integrity was monitored through fluorescence-based viability assays. Immune activation was evaluated using multiplex cytokine and cytolytic mediator profiling. High-content imaging was used to visualize immune-tumor interactions, spheroid structural changes, and immune infiltration depth, supported by automated segmentation workflows. Flow cytometry analysis provided phenotypic information on immune activation and checkpoint receptor modulation. To extend the platform beyond lymphoid checkpoints, we established a myeloid module in which pH-sensitive dye-labeled tumor spheroids were co-cultured with fluorescent macrophages to quantify phagocytic uptake.Checkpoint blockade enhanced immune effector function across these readouts. We observed increased caspase-3/7 activation and reduced viability of tumor spheroids. Cytokine profiling revealed elevated inflammatory and cytolytic mediator release, and imaging demonstrated deep immune penetration into the 3D architecture. Flow cytometry confirmed activation-associated immune phenotypes, while the myeloid module showed increased macrophage-tumor engagement and phagocytosis.In summary, this 3D co-culture platform provides a physiologically relevant, robust, and scalable system for characterizing human tumor-immune interactions and evaluating ICI activity. Its integration of microtissue engineering with multiparametric functional and phenotypic assays offered a translational approach for more predictive preclinical assessment and accelerated development of next-generation ICIs and biosimilars.