Abstract 5222: Establishment of a patient-derived organoid-PBMC co-culture model to investigate immune responses and immunotherapy sensitivity in advanced gastric cancer
Authors: Han Byeol Mun, Woo Sun Kwon, Tae Soo Kim, Sun Young Rha
Journal: Cancer Research (2025)

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.