Simple SummaryGlioblastoma is the most common malignant brain tumor in adults, entailing a very short survival. New therapeutic strategies are desperately needed. Immunotherapeutic approaches seem promising, yet their breakthrough is hindered by interactions of the tumor with its immunological tumor environment. In order to understand these complex interactions, innovative glioblastoma models are needed. We aimed to investigate whether patient-derived tumor models are able to maintain the tumor’s microenvironment signature and composition. Secondly, we added immune cells to our model in order to reflect a more realistic tumor microenvironment, which could be used for preclinical testing of novel immunotherapeutic approaches. Thus, we hope to contribute to the challenging task of advancing glioblastoma therapy.AbstractWhile glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future.