Radiotherapy (RT) is used in approximately 50% of cancer patients [1]. While highly effective, its potential is limited by toxicity to healthy cells, restricting doses that can be safely administered [2]. To address this issue, research has focused on studying the unique radiobiological responses at ultrahigh dose rates, known as FLASH-RT [3]. Recent studies show that FLASH-RT reduces damage to healthy tissues while maintaining tumor control, making it a promising alternative to conventional RT (CONV-RT) [4]. However, the molecular mechanisms underlying distinct responses to FLASH and CONV irradiation remain insufficiently understood. Therefore, the aim of this study was to compare the effects of FLASH-RT and CONV-RT on reactive oxygen species (ROS) production in water and intracellular environment-mimicking solutions, as well as their impact on miRNAs expression and cell viability in prostate cancer and normal colon 3D spheroids.
All samples were irradiated using a clinical linear accelerator modified to achieve the required dose rates for FLASH-RT. Water and Bovine Serum Albumin (BSA)-enriched solutions received 10, 15, and 20 Gy doses, and ROS production was measured via fluorescence spectroscopy. Prostate cancer (C4-2) and normal colon (CRL-1541) cell spheroids were irradiated with 17 Gy doses. MiRNA-21-5p, miRNA-29a-3p, and miRNA-222-3p levels were measured at 12, 24, 48, and 72 hours post-RT using reverse transcription quantitative PCR with expression determined relative to untreated controls using the \(2^{-\Delta\Delta\text{Ct}}\) method. Cell viability was assessed on days 1, 4, 7, and 11 post-RT using the XTT assay, with results expressed as a percentage relative to untreated controls.
FLASH-RT produced notably lower ROS levels compared to CONV-RT in both water (~3 times) and BSA-enriched (~1.8 times) solutions. At 24 hours post-RT, there were no differences in miRNAs expression between CONV-RT and FLASH-RT in either cell line. By 72 hours, CRL-1541 cells showed significant downregulation of the studied miRNAs following CONV-RT compared to FLASH-RT, while no significant differences were found between two treatments in C4-2 cells. Additionally, CONV-RT significantly reduced cell viability compared to FLASH-RT on day one post-RT in CRL-1541 cells, whereas no significant differences in FLASH-RT and CONV-RT treatment effects were observed in C4-2 cells.
The results suggest FLASH-RT reduces ROS production, minimizing ROS effects on cell damage. Distinct miRNA expression and cell viability patterns suggest differing mechanisms between FLASH-RT and CONV-RT in healthy and cancerous cells. Notably, miRNAs expression at 72 hours post-RT and cell viability on day one post-RT reveal that while FLASH-RT affects cancer cells similarly to CONV-RT, it shows unique protective effects on normal cells. These findings suggest that FLASH-RT may selectively spare healthy cells, highlighting the need for further investigation. This study was supported by the National Cancer Institute Research Fund and the Research Council of Lithuania (LMTLT), agreement No. S-MIP-24-134.