Abstract 5222: Establishment of a patient-derived organoid-PBMC co-culture model to investigate immune responses and immunotherapy sensitivity in advanced gastric cancer

Advanced gastric cancer (AGC) exhibits significant molecular heterogeneity with aggressive behavior and treatment resistance. Immunotherapy (IO), such as PD-1 or PD-L1 inhibitors, has emerged as an important treatment for AGC, but only a small subset of patients responds to these therapies. Therefore, it would be of great benefit to understand mechanisms of anti-tumor immune response in AGC patients. This study aims to establish a co-culture model of patient-derived GC organoids and PBMCs that can recapitulate tumor-immune interactions PBMCs were isolated from healthy donors, pooled equally, and cryopreserved. They were activated with anti-CD3/CD28 Dynabeads at ratios of 1:1 (Bead : Cell) for 48 hours and stained with 1 µM CellTrace Far Red. Organoids with a diameter of ≥50 μm were harvested, filtered through a 50 μm strainer, and seeded (50 per well) into 96-well plates at ratios of 1:1 to 200:1 (PBMCs : Organoids). Pembrolizumab was treated in mono-culture and co-culture assays at a ratio of 50:1 for 48 and 72 hours, and sensitivity was measured with CCK-8. PBMC recruitment was analyzed using the Operetta CLS system and Harmony 4.9 software. Supernatants were collected post-co-culture and analyzed for cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A) using BD Cytometric Bead Array, with values below the detection limit excluded from the analysis. In co-culture conditions, the organoids exhibited variable responses depending on the ratio in the co-culture conditions. YCC-GO10-1 showed 63.1% viability with a 5-fold increase in PBMC recruitment, while YCC-GO63 showed 14.8% viability with a 1.6-fold increase. This indicates that increased PBMC recruitment is not necessarily associated with organoid death. In the co-culture supernatant of YCC-GO10-1, higher levels of inflammatory cytokines were observed compared to YCC-GO63, including IL-6, a cytokine that promotes tumor progression, as well as IFN-γ, TNF and IL-2. Pembrolizumab had no impact on the viability of organoids or PBMCs under monoculture conditions. However, under co-culture conditions, it exhibited variable effects on organoid viability, reducing the viability of YCC-GO63, while YCC-GO10-1 remained resistant to the treatment. Even under identical immune environments, organoids displayed heterogeneous responses, resulting in distinct immune reaction patterns and cytokine secretion profiles. The established organoid-immune cell co-culture model demonstrates the potential to investigate tumor-immune interactions and study the mechanisms underlying sensitivity to IO, antibody-drug-conjugates, and other anticancer agents in vitro. Citation Format: Han Byeol Mun, Woo Sun Kwon, Tae Soo Kim, Sun Young Rha. Establishment of a patient-derived organoid-PBMC co-culture model to investigate immune responses and immunotherapy sensitivity in advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5222.