S2630
Radiobiology - Tumour radiobiology
ESTRO 2026
iron levels. Accordingly, we hypothesize that an altered antioxidant capacity underlies this observation. The present and ongoing in vitro results support an in vivo validation. Follow-up studies using liproxstatin-1 and whole-abdominal irradiation will further elucidate the role of ferroptosis in the FLASH response. References: 1. Froidevaux, P. et al. FLASH irradiation does not induce lipid peroxidation in lipids micelles and liposomes. Radiation Physics and Chemistry205, 110733 (2023).2. Grilj, V. et al. Average dose rate is the primary determinant of lipid peroxidation in liposome membranes exposed to pulsed electron FLASH beam. Radiation Physics and Chemistry222, 111887 (2024).3. Vilaplana-Lopera, N., Abu- Halawa, A., Walker, E., Kim, J. & Moon, E. J. Ferroptosis, a key to unravel the enigma of the FLASH effect? BJR95, 20220825 (2022). Keywords: Ferroptosis, CONV-RT, FLASH-RT Digital Poster 1266 MitoTam reduces hypoxia and enhances radiation- induced DNA damage in spheroids Anne P.M. Beerkens 1 , Paolo S. Taracatac 1 , Johannes P.W. Peters 1 , Gosse J. Adema 1 , Sandra Heskamp 2 , Paul N. Span 1 , Johan Bussink 1 1 Radiation Oncology, Radboud University Medical Center, Nijmegen, Netherlands. 2 Medical Imaging, Radboud University Medical Center, Nijmegen, Netherlands Purpose/Objective: Tumor hypoxia is associated with an aggressive phenotype and reduced responsiveness to radiotherapy. Reducing tumor oxygen consumption has been proposed as a promising durable strategy to alleviate hypoxia and thereby radiosensitize tumors. The aim of this study was to investigate whether OXPHOS inhibition-mediated hypoxia reduction in tumor spheroids indeed enhances radiation-induced DNA damage, indicative for radiosensitization and to examine how this translates to pre-clinical models. Material/Methods: Spheroids were grown from murine melanoma (B16OVA) and murine oral carcinoma (MOC1.3D5) cells. At 72h after spheroid formation, half of the culture medium was refreshed and after an additional 24 h spheroids were treated with MitoTam (5 µM) or vehicle control. 24h after administering MitoTam, spheroids were irradiated with 4 Gy. At 2 h post-RT, spheroids were fixed, sections were cut and stained for γ H2AX. Mice were inoculated with MOC1.3D5 cells on the hindleg. When tumors became palpable, mice were treated with MitoTam (10 mg/kg) via peritumoral injections (p.t.), administered five times at three-day
FLASH-RT (400Gy/s or 300Gy/s, DPP: 2.5Gy or 1Gy) or conventional (CONV) RT (0.13Gy/s) using the FLASHKNiFE (TheryQ) or ElectronFLASH (SIT). The C11BODIPY and FerroOrange dye were respectively used to assess lipid peroxidation and measure intracellular ferrous iron, a key trigger of ferroptosis. PTGS2 expression, a ferroptosis marker, was assessed by qPCR. To further evaluate ferroptosis involvement, cells were treated with the ferroptosis inhibitor, ferrostatin-1, and radiomodulatory properties were investigated via colony formation assay (0Gy, 2Gy, 4Gy, 8Gy and 10Gy) (DLD-1) or live-cell imaging (HIEC6). Results: In DLD-1 cells, both CONV and FLASH-RT similarly increased lipid peroxidation (1.5-fold, Fig.1A), and PTGS2 expression (2-fold, Fig.1B). In HIEC6 cells, lipid peroxidation increased moderately after both CONV and FLASH-RT (1.18-fold, Fig.1A), while PTGS2 expression increased (1.53-fold, Fig. 1B) after CONV-RT but not following FLASH-RT. Ferrous iron levels increased in both cell lines post-irradiation (up to 1.7-fold in DLD-1, and 1.3-fold in HIEC6, Fig.1C), regardless of the RT modality.
In DLD-1 cells, ferroptosis inhibition by ferrostatin-1 induced radioprotection after both CONV and FLASH- RT (Fig.2A). In HIEC6 cells, ferrostatin-1 was radioprotective following CONV-RT, but this effect was abolished after FLASH-RT (Fig.2B), suggesting reduced ferroptosis induction in healthy cells post FLASH- RT.
Conclusion: Our results suggest that ferroptosis contributes equally to the FLASH and CONV-RT responses of CRC cells, but is less prominently induced in healthy intestinal cells after FLASH-RT. This is supported by reduced PTGS2 expression in HIEC6 cells following FLASH-RT, despite unchanged lipid peroxidation or
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