ESTRO 2025 - Abstract Book

S4026

Radiobiology - Tumour radiobiology

ESTRO 2025

4409

Digital Poster FLASH Effect Mechanisms: Radiation Parameters and miRNA Insights from In Vitro Studies Akvilė Šlėktaitė-Kišonė 1,2 , Paulina Kazlauskaitė 2,3,4 , Marius Burkanas 1 , Džiugilė Valiukevičiūtė 1 , Aleksandras Cicinas 1 , Mindaugas Džiugelis 1,2 , Rasa Sabaliauskaitė 4,5 , Ričardas Rotomskis 2 , Jonas Venius 1,2 1 Department of Medical Physics, National Cancer Institute, Vilnius, Lithuania. 2 Biomedical Physics Laboratory, National Cancer Institute, Vilnius, Lithuania. 3 Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania. 4 Laboratory of Genetic Diagnostics, National Cancer Institute, Vilnius, Lithuania. 5 Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania Purpose/Objective: FLASH radiotherapy (FLASH-RT) is a technique where radiation is delivered at ultra-high dose rates (UHDR). It reduces damage to healthy tissue while maintaining tumor control, making it a promising alternative to conventional radiotherapy (CONV-RT). This study investigates beam parameters’ influence on FLASH-RT effect by analysing reactive oxygen species (ROS) production in water. Additionally, miRNAs expression differences in cancer and normal 3D cell cultures provide insights into the mechanisms behind FLASH-RT’s unique therapeutic effects. Material/Methods: Irradiation was conducted using a modified VARIAN TrueBeam linear accelerator, producing UHDR electrons. For dosimetry gafchromic EBT-3 films were used. The instantaneous dose rate was 0.55 MGy/s (2 Gy/pulse). Doses (10 50 Gy) and dose rates (up to 150 Gy/s) adjusted via impulse amount and frequency. Dihydrorhodamine 123 was used as a fluorescent ROS marker. 3D spheroids of prostate cancer (C4-2) and normal colon (CRL-1541) cell lines were irradiated with 17 Gy using FLASH-RT and CONV-RT. Post-irradiation, miRNA-21-5p, miRNA-29a-3p, and miRNA-222-3p levels were analyzed by RT-qPCR at 4, 12, and 24 hours, normalized to cel-miRNA-39-3p, and expressed relative to untreated controls using the 2^-ΔΔCt method. Results: Fluorescence measurements showed that higher dose rates significantly decrease ROS generation, with the effect magnitude correlating to total dose. The greatest ROS reduction (62%) occurred at highest dose and dose rate (50 Gy, 150 Gy/s), while 10 Gy at 2 Gy/s led to 17% decrease relative to CONV-RT. However, increasing dose rate from 50 Gy/s to 150 Gy/s did not sigificantly decreased ROS production. A pronounced 55% ROS decrease occurred when transitioning from CONV-RT (0.0003 Gy/pulse) to UHDR (2 Gy/pulse, one pulse/s), even without reaching FLASH dose rate (40 Gy/s). The expression of miRNAs was significantly higher at 4 and 12 hours post-irradiation with CONV-RT compared to FLASH-RT in both cell lines. By 24 hours, CRL-1541 cells showed significant downregulation of the studied miRNAs following CONV-RT compared to FLASH-RT, while no significant differences were found in C4-2 cells. Conclusion: The physicochemical processes of FLASH-RT depend on dose rate and total dose. The significant reduction in ROS at average dose rate of 2 Gy/s (single pulse) compared to CONV-RT suggests that instantaneous dose rate may be more critical to FLASH effect than the average dose rate. MiRNAs response at 24 hours suggests that while FLASH-RT impacts cancer cells similarly to CONV-RT, it may involve a selective mechanism that spares healthy cells.

Keywords: FLASH-RT, ROS generation, miRNA