Molecular and Cellular Effects of FLASH Versus Conventional Radiotherapy on Human Pulmonary Cells: An In Vitro Comparative Study

Lung cancer is one of the most diagnosed cancers and the second leading cause of cancer-related death worldwide. Radiotherapy (RT) plays a major role in its treatment, remaining a key therapy option. Still, conventional types of radiotherapy (CONV-RT) can imply damage to the healthy tissues surrounding the tumor, potentially leading to severe complications such as pulmonary inflammation and fibrosis, an incurable condition that can lead to death. In recent years, FLASH-RT has emerged as a promising alternative; by delivering radiation at dose rates above 40 Gy/s, FLASH-RT can induce the so-called FLASH effect that reduces toxicity in healthy tissues while maintaining the same antitumor efficacy as CONV-RT. However, the biological mechanisms underlying this effect remain unclear. This Ph.D. thesis investigates the biological responses to FLASH-RT compared with CONV-RT using in vitro lung cell models representing tumor and healthy tissues. Human lung adenocarcinoma cells (A549) and healthy bronchial epithelial cells (16HBE) were irradiated using a linear electron accelerator capable of delivering both conventional and FLASH dose rates. Cellular responses were evaluated using clonogenic survival assays, oxidative stress measurements, cell cycle analysis, cell death pathway analysis, migration assays, and the expression of inflammatory and fibrosis-related markers. In A549 cells, both irradiation modalities caused cell cycle arrest in the G0/G1 phase, while, compared with CONV, FLASH irradiation led to lower ROS production, no oxidation of macromolecules such as proteins and lipids, increased activation of the apoptotic pathway rather than pro-inflammatory forms of cell death, and reduced metastatic activity. Concerning 16HBE cells, FLASH-RT resulted in lower levels of ROS, along with no oxidation In macromolecules, accompanied by an activation of the SOD enzyme, lower cell death and the absence of a cell cycle arrest, specifically with the lowest dose; moreover, FLASH resulted in lower levels of inflammatory and early fibrosis markers, suggesting a protective effect on normal tissue. This data was also supported by the wound healing assay, where FLASH-irradiated cells behaved very similarly to the control, while CONV-exposed cells exhibited an accelerated wound closure, suggesting the onset of pro-fibrotic activity. The influence of irradiation parameters within the FLASH regime was also explored, including dose rate, dose per pulse, temporal dose delivery, and spatial fractionation. While dose-per-pulse variations did not significantly affect outcomes, a dose rate of 350 Gy/s resulted in lower survival of tumor cells and higher survival of healthy cells, indicating a potential optimization window. Additionally, almost every delay, except for 0.5 and 60 seconds, delay between dose trains increased survival in healthy cells, and spatial fractionation using the MiniBeam technique further enhanced normal tissue sparing. Overall, these findings provide additional in vitro evidence of the differential effects of FLASH-RT on tumor and healthy lung cells and highlight the importance of treatment parameters in modulating the FLASH effect.