Double-pillar co-culture (DPC) platform for evaluating cancer invasion and immunotherapy efficacy

Background: Co-culture is an essential in vitro technique for evaluating cancer invasion and immunotherapy efficacy, as tumor-stroma and tumor-immune interactions critically influence cancer progression and therapeutic response. Conventional assays, such as Transwell-based migration or invasion systems, are limited because they cannot simultaneously quantify cell invasion and viability, reducing physiological relevance. To address this, we developed a double-pillar co-culture (DPC) platform enabling high-throughput screening while culturing two distinct cell types in a spatially defined 3D architecture. Cancer cells embedded in Matrigel are seeded onto a central pillar to form a 3D tumor-like spot, while stromal cells, including fibroblasts or immune cells, are embedded in Matrigel on an outer, slightly lower pillar surrounding the core. This configuration allows direct visualization of invasion from the central mass and concurrent monitoring of cell viability. Methods : A549 lung cancer cells and stromal cells (fibroblasts or lymphocytes) were co-cultured using the DPC platform. For drug screening, compounds including immune checkpoint-targeting drugs were applied to assess their effects on invasion and viability. To evaluate immune cytotoxicity, A549 cells were stabilized in the central pillar for three days, followed by addition of Jurkat cells to the outer pillar at a 1:5 ratio and co-cultured for an extended period. Invasion and viability were quantified using live-cell imaging and fluorescence-based assays and compared to monoculture or vehicle-treated controls. Results: Co-culture of A549 cells with fibroblasts increased invasion over time. High-throughput screening using multiple compounds showed inhibitory effects on cancer cell invasion. Among them, the EGFR inhibitor Dacomitinib reduced invasion by <40% while maintaining >95% viability, demonstrating anti-invasive activity independent of cytotoxicity. Co-culture with Jurkat cells decreased cancer cell viability by ~55% compared to monoculture, confirming significant immune-mediated cytotoxicity. Conclusions: The DPC platform provides a physiologically relevant, high-throughput system for simultaneous assessment of cancer invasion and immune cytotoxicity. It enables identification of compounds with selective anti-invasive effects and evaluation of immunotherapy efficacy, offering a versatile tool for preclinical cancer research.