Glioblastoma (GBM) displays a wide range of inter- and intra-tumoral heterogeneity contributing to therapeutic resistance and relapse. Although Tumor Treating Fields (TTFields) are effective for the treatment of GBM, there is a lack of ex vivo models to evaluate effects on patients’ tumor biology or to screen patients for treatment efficacy. Thus, we adapted patient-derived three-dimensional tissue culture models to be compatible with TTFields application to tissue culture. Patient-derived primary cells (PDPC) were seeded onto murine organotypic hippocampal slice cultures (OHSC), and microtumor development with and without TTFields at 200 kHz was observed. In addition, organoids were generated from acute material cultured on OHSC and treated with TTFields. Lastly, the effect of TTFields on expression of the Ki67 proliferation marker was evaluated on cultured GBM slices. Microtumors exhibited increased sensitivity towards TTFields compared to monolayer cell cultures. TTFields affected tumor growth and viability, as the size of microtumors and the percentage of Ki67-positive cells decreased after treatment. Nevertheless, variability in the extent of the response was preserved between different patient samples. Therefore, these pre-clinical GBM models could provide snapshots of the tumor to simulate patient treatment response and to investigate molecular mechanisms of response and resistance.
While 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.
TTFields are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), which are effective and approved for the treatment of glioblastoma (GBM) using 200 kHz frequency. However, there is a lack of ex vivo models to evaluate effects on patients’ tumor biology or to screen patients for treatment efficacy. Therefore, we adapted patient-derived three-dimensional GBM tissue culture models to be compatible with TTFields application and recently published the feasibility of such an approach (Nickl, et al., 2022, doi: 10.3390/cancers14215177). Here, we applied one of those models, i.e. tumor-organoids cultured as microtumors on murine organotypic hippocampal slice cultures (OHSCs), to additional brain tumor entities, namely a sample of an anaplastic ependymoma (AE) patient and an oligodendroglioma patient. Organoids were generated from fresh intra-operatively obtained tumor tissue and cultured for 2 weeks. OHSCs were prepared by slicing the brains of mice 5-8 days postpartum to sections with a thickness of 350 µm using a vibratome, and culturing them for 2 weeks as well. Subsequently, organoids were placed onto the OHSCs. The inovitro™ laboratory research system was used for TTFields administration at 200 kHz and 1.5 V/cm for 72 h. Microtumor growth was evaluated on fluorescence images. Viable organoids formed from the GBM, AE and oligodendroglioma sample and grew to microtumors when placed onto OHSCs. Application of TTFields at 200 kHz led to a significant decrease of microtumor size of the GBM and AE (both p<0.0001), but not the oligodendroglioma sample. This proof-of-principle investigation proved that the application of patient-derived organoids cultured on OHSCs is feasible to investigate the effects of TTFields on different kinds of brain tumors. To our knowledge, this is the first evaluation of TTFields efficacy on patient derived AE and oligodendroglioma tissue cultures. While TTFields at 200 kHz led to a decrease in the microtumor size of the AE sample, the non-responsiveness of the oligodendroglioma sample may be due to different inter-patient sensitivity to TTFields or a suboptimal TTFields frequency. Citation Format: Vera Nickl, Ellina Schulz, Ellaine Salvador, Laureen Trautmann, Leopold Diener, Almuth F. Kessler, Camelia M. Monoranu, Ralf-Ingo Ernestus, Mario Löhr, Carsten Hagemann. Evaluation of tumor treating fields (TTFields) effects at 200 kHz on a glioblastoma, an anaplastic ependymoma and an oligodendroglioma sample in a patient-derived ex vivo organoid model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4573.
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