Abstract Introduction Ependymomas (EPN) are malignant glial tumors of the central nervous system and remain challenging to treat despite advances in molecular classification. Standard therapy relies on surgery and radiotherapy, while chemotherapy has shown limited benefit. Notably, approximately half of high-risk pediatric ependymomas relapse after treatment, underscoring the need to better understand radiation response characteristics and to identify rational radiosensitization strategies, including potential differences between radiation qualities. Methods We conducted a comprehensive in vitro characterization of the cellular responses of the pediatric ST-EPN-ZFTA ependymoma cell line EP1NS. Cells were irradiated with photons (X-rays) or protons (spread-out Bragg peak, SOBP) over a defined dose range (0-10Gy). Short-term treatment effects were quantified using viability assays (ATP-based luminescence readout) and flow cytometry for cell death (Annexin V/7AAD). Long-term survival was assessed using limiting dilution assays (LDA) and spheroid assays. In addition, basal and irradiation-associated metabolic parameters were assessed by Seahorse extracellular flux analysis as a complementary functional phenotype. To explore pharmacological modulators of radiation response, a DNA-damage response (DDR)-focused library screen (n = 823) was performed in combination with photon and SOPBP-proton irradiation, and top candidates were prioritized for follow-up in the assays above. Results EP1NS exhibited comparable short- and long-term radiation response profiles following photon and proton irradiation. Conversely, seahorse profiling revealed reproducible differences in metabolic parameters at baseline and after irradiation, suggesting differences in energy metabolism and associated metabolic dependencies. The DDR-focused screen revealed multiple candidate compounds with differential radiosensitizing activity following photon or proton irradiation, which are currently under evaluation. Conclusion Our data demonstrated that pharmacological modulation of DDR pathways can radiosensitize EP1NS ependymoma cells across multiple experimental platforms. These findings support further systematic evaluation of radiation quality-dependent vulnerabilities and provide a framework for prioritizing candidate radiosensitizers for translational follow-up. Acknowledgements This work was supported by funds from the DFG (grant number GRK2762/1), Wilhelm Sander Stiftung (grant number 2023.138.1)
Butenko et al. (Tue,) studied this question.