S2505
Radiobiology - Biology of novel irradiation techniques
ESTRO 2026
Keywords: FLASH radiotherapy, cuproptosis, HNSCC
concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS). Mitochondrial status was evaluated through mitochondrial membrane potential assays and transmission electron microscopy (TEM). The tumor immune microenvironment (TiME) was analyzed using multiplex immunofluorescence (mIF) staining and multicolor flow cytometry (mFCM). Additionally, specific molecule expression levels in cell supernatants or serum were quantified by enzyme- linked immunosorbent assay (ELISA). Results: Both cellular and murine models demonstrated that resistance to cuproptosis significantly diminishes the radiosensitivity of HNSCC. Further experiments revealed that FLASH-RT markedly induces cuproptosis in radioresistant cell lines (SAS_R/CAL27_R) and mouse models (MOC1_R/MOC2_R). TEM analysis showed that FLASH-RT caused pronounced mitochondrial swelling and structural disruption in tumor cells. mIF staining and qPCR analyses revealed a significant increase in mitochondrial DNA (mtDNA) release following FLASH- RT. Transcriptomic profiling suggested hyperactivation of the cGAS-STING signaling pathway. Correspondingly, elevated levels of TNF- α , IFN- β , and IL-1 β were detected in the cell culture supernatant. mFCM analysis of murine tumor tissues revealed that the copper enhancer elesclomol-Cu synergized with FLASH-RT to significantly increase the infiltration of CD8 ⁺ T cells and M1/M2 ratio within the TiME. Mouse model studies confirmed the critical role of the cGAS- STING pathway in mediating FLASH-RT-driven remodeling of the TiME. Finally, the efficacy and safety of combining FLASH radiotherapy, ES-Cu, and PD-1 blockade therapy were validated in vivo. Conclusion: Our study demonstrates that FLASH-RT, combined with ES-Cu, significantly improves the efficacy of radiotherapy in radioresistant HNSCC by inducing cuproptosis, activating the cGAS-STING signaling pathway, and enhancing the infiltration of CD8 ⁺ T cells and M1 macrophages within the tumor immune microenvironment. These findings provide a promising novel therapeutic strategy for clinical application. References: 1. Li H-S, Tang R, Shi H-S, et al. Ultra-high dose rate radiotherapy overcomes radioresistance in head and neck squamous cell carcinoma. Signal Transduction and Targeted Therapy. 2025;10(1):82.2. Lei G, Sun M, Cheng J, et al. Radiotherapy promotes cuproptosis and synergizes with cuproptosis inducers to overcome tumor radioresistance. Cancer Cell. 2025.3. Shi X, Yang Y, Zhang W, et al. FLASH X-ray spares intestinal crypts from pyroptosis initiated by cGAS-STING activation upon radioimmunotherapy. Proceedings of the National Academy of Sciences of the United States of America. 2022;119(43):e2208506119.
Proffered Paper 1027 Fractionation preserves the fibrotic but not the acute skin-sparing FLASH effect Line Kristensen 1,2 , Anna Holtz Hansen 2,3 , Sky Steenholdt 1,3 , Jacob Graversen Johansen 1,3 , Lone Hoffmann 3,4 , Lars Præstegaard 4 , Per Rugaard Poulsen 1,3 , Brita Singers Sørensen 1,2 1 Danish Centre for Particle Therapy, Aarhus University, Aarhus, Denmark. 2 Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark. 3 Department of Clinical Medicine, Aarhus University, Aarhus, Denmark. 4 Department of Oncology, Aarhus University Hospital, Aarhus, Denmark Purpose/Objective: The tissue-sparing FLASH effect of ultra-high dose rate (UHDR) has been studied preclinically for over a decade. Few of these studies have examined the potential for tissue protection under fractionated regimes. With fractionation being the standard for clinical radiotherapy, understanding how fractionation interacts with the FLASH effect is critical for clinical implementation. In a previous study, the FLASH tissue sparing of acute skin damage was shown to diminish with increasing fractionation from 42% at one fraction (Fig. 1A), to 18% at four fractions (Fig. 1B) and 5% at eight fractions (Fig. 1C) (Kristensen et al., 2025). This study investigated the same mice for fibrotic damage, to quantify the fibrotic FLASH sparing effect under hypofractionation using a murine model in a study design comparable to the acute skin damage study. Material/Methods: Radiation-induced fibrosis was assessed in the right hindleg of unanaesthetised female CDF1 mice. Irradiations compared a conventional dose rate of 0.16 Gy/s (CONV) to UHDR (FLASH) of 251 Gy/s with a 16 MeV electron beam from a FLASH-enabled accelerator (experimental Varian TrueBeam).The target legs were irradiated in six arms: 1) single fraction (1fr) using CONV or 2) 1fr UHDR, 3) four fractions (4fr) using CONV or 4) 4fr UHDR, 5) eight fractions (8fr) using CONV or 6) 8fr UHDR. Fractionated treatment was delivered as one daily dose for four consecutive days for four fractions, and two daily doses with six hours separation for four consecutive days for eight fractions. Each dose group included 4-8 mice. Fibrosis was quantified biweekly from 9 to 53 weeks post-treatment with a leg extension assay. Results: The fibrotic protection ratio of FLASH was maintained with increasing fractions, with 1.15 (1.06-1.24) after single-dose irradiations (Fig. 1D), 1.26 (1.14-1.35) after
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