MODL-09. SCAFFOLD-BASED (MATRIGELTM) 3D CULTURE TECHNIQUE OF GLIOBLASTOMA RECOVERS A PATIENT-LIKE IMMUNOSUPPRESSIVE PHENOTYPE

Abstract

Abstract 3D (organoid) models in cancer research are considered the novel gold standard for in vitro experiments. Nevertheless, a variety of different protocols exits and the models are often used uncritically in lack of further validation. In this study, we aimed to characterize a scaffold-based (MatrigelTM) 3D glioma cell culture approach. We compared the 3D technique to conventional 2D culturing in its efficacy of generating models and in respect to its exact modeling properties. Models were generated from 62 patients with CNS tumors, most of them with glioblastomas, IDH-wildtype (GBM, n = 43). Pairs of 11 GBM models (2D and 3D) together with corresponding patient material were further characterized using bulk and single-cell RNA sequencing as well as 850k methylation analyses. Functional aspects of 3D tumor organoids were analyzed using a coculture system (Incucyte® SX5 live cell imager) with tumor infiltrating lymphocytes. Compared to conventional 2D culture the MatrigelTM-based 3D protocol was not superior in terms of the successful generation of in vitro models. However, NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in the 3D models, indicating the reoccurrence of an immunosuppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. In the coculturing approach, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release as well as live cell staining and proliferation analysis pointed towards an elevated resistance of 3D models. In conclusion, we found that the Scaffold-based (MatrigelTM) 3D culture technique depicts clinically highly important aspects of tumor-immune cell interactions, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.