Abstract 2199: Ready-to-use oxygen gradient microplates for customized in vitro assays for the phenotypic and functional characterization of primary macrophages in immuno-oncology drug development

Ready-to-use oxygen gradient microplates for customized in vitro assays for the phenotypic and functionalcharacterization of primary macrophages in immuno-oncology drug developmentChristoph Schifflers1, Martijn Vlaming1, Emmanuelle Sidot1, Jezabel Lefèvre1, Elise Pepermans2, EllenBoelen1, Enrico Grassili2 and Sofie Pattyn11IQVIA Laboratories, In Vitro Immunology (formerly known as ImmunXperts), Rue Clément Ader 10, 6041Gosselies Belgium2ImmuneSpec, Galileilaan 18 bus 6, 2845 Niel, Belgium3InSimili srl, Viale Giuseppe Fanin 48, Bologna, 40050, ItalyOxygen level fluctuations in tumor micro-environment (TME) can alter the phenotype and function ofcancer cells as well as tumor-infiltrating immune cells, such as macrophages. For the functional evaluationof new immuno-oncology drug candidates, it can be key to take into consideration the effects of varyingO2 concentrations. In fact, it is well established that varying oxygen concentrations and tumor hypoxia candrive treatment resistance and cancer recurrence. Modeling this layer of functional complexity of thetumor microenvironment, typically requires the use of animal models or tumoroid systems which can becomplex to establish, time-consuming and expensive. InSimili developed a new technology that providesready-to-use microplates that model spatially controlled O2 levels at fixed O2 concentrations or O2 gradients(adaptable for individual wells). Combining this technology with highly optimized in vitro assays using highquality primary immune cells can provide a useful tool with improved predictive value for the cost-effectivefunctional characterization and lead selection of new immuno-oncology drug candidates.In this study, the impact of varying O2 concentrations (ranging from 20% to 1% O2) was assessed usingcustomized microplates on the phenotype of macrophages derived from healthy donor PBMCs.Furthermore, the impact of different O2 concentration on the capacity of M2-like macrophages to suppress T cell activation was explored. Finally, the impact of several oxygen concentrations on the reprogrammingof M2-like macrophages was evaluated.Taken together, this study showed that InSimili’s plates provide a ready-to-use tool that can be used incustomized in vitro assays to model varying O2 concentrations. Integrating this technology with highlycustomized in vitro bioassays can serve as a valuable tool for immuno-oncology drug development whiletaking into account an additional degree of the complexity of the TME in 2D and 3D systems. Citation Format: Christoph Schifflers, Martijn Vlaming, Emmanuelle Sidot, Jezabel Lefevre, Elise Pepermans, Ellen Boelen, Enrico Grassilli, Sofie Pattyn. Ready-to-use oxygen gradient microplates for customized in vitro assays for the phenotypic and functional characterization of primary macrophages in immuno-oncology drug development [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 2199.