Kuipers Merian E, van Liefferinge Floriane, van der Wal Ernst, Rovituso Marta, Slats Annelies M, Hiemstra Pieter S, Van Doorn-Wink Krista C J
Leiden University Medical Center (LUMC), Department of Pulmonology, C02-Q, Albinusdreef 2 2333 ZA Leiden, the Netherlands.
Holland Proton Therapy Center (HollandPTC), Huismansingel 4 2629 JH Delft, the Netherlands.
Clin Transl Radiat Oncol. 2025 Jan 29;52:100927. doi: 10.1016/j.ctro.2025.100927. eCollection 2025 May.
The effects of conventional (CONV) and FLASH proton therapy on primary bronchial epithelial cell (PBEC) organoids from individuals with chronic obstructive pulmonary disease (COPD) were investigated. The primary objective was to compare the effect of FLASH and CONV on COPD PBEC organoids with a focus on DNA damage, organoid formation, and gene expression.
PBECs were obtained from six COPD donors, cultured as three-dimensional (3D) organoids and exposed to 2 and 8 Gy CONV and FLASH proton radiation at the Holland Proton Therapy Center. DNA damage was assessed by γH2AX staining. Organoid formation capacity was assessed by counting the organoids formed after reseeding irradiated cells at 24 h and 7 days. Bulk RNA sequencing (RNAseq) and qPCR analyses were performed to identify pathways and differences in the radiation response.
γH2AX foci analysis showed a significant dose-dependent increase in DNA damage at 1 h for both CONV and FLASH treatments, without differences between the two modalities. Organoid formation assays revealed a dose-dependent decrease in organoid formation capacity at 24 h for both treatments. At 7 days, 2 Gy FLASH-treated samples showed significantly reduced organoid formation compared to 2 Gy CONV (p = 0.008). RNAseq identified CONV and FLASH-induced changes in expression of DNA-damage response and apoptosis pathway genes. A dose-dependent upregulation of MDM2, GDF15, DDB2, BAX, P21, AEN and a decrease in MKi67 expression was confirmed by qPCR analysis.
No significant differences were found in DNA damage or gene expression profiles between CONV and FLASH. The organoid formation assay showed a prolonged detrimental effect in the FLASH-treated organoids, suggesting a more complex interaction of FLASH with lung epithelial cells. The results of this study contribute to the advancement of robust human lung models for investigating the mechanisms of action of FLASH, potentially facilitating the treatment of NSCLC patients with proton FLASH therapy.
研究传统（CONV）质子治疗和FLASH质子治疗对慢性阻塞性肺疾病（COPD）患者的原发性支气管上皮细胞（PBEC）类器官的影响。主要目的是比较FLASH和CONV对COPD PBEC类器官的影响，重点关注DNA损伤、类器官形成和基因表达。
从6名COPD捐赠者获取PBEC，培养为三维（3D）类器官，并在荷兰质子治疗中心接受2 Gy和8 Gy的CONV和FLASH质子辐射。通过γH2AX染色评估DNA损伤。通过计数照射后24小时和7天重新接种的细胞形成的类器官来评估类器官形成能力。进行批量RNA测序（RNAseq）和qPCR分析以确定辐射反应中的途径和差异。
γH2AX焦点分析显示，CONV和FLASH治疗在1小时时DNA损伤均有显著的剂量依赖性增加，两种方式之间无差异。类器官形成试验显示，两种治疗在24小时时类器官形成能力均有剂量依赖性下降。在7天时，与2 Gy CONV相比，2 Gy FLASH治疗的样本类器官形成显著减少（p = 0.008）。RNAseq确定了CONV和FLASH诱导DNA损伤反应和凋亡途径基因表达的变化。qPCR分析证实了MDM2、GDF15、DDB2、BAX、P21、AEN的剂量依赖性上调和MKi67表达的下降。
CONV和FLASH在DNA损伤或基因表达谱方面未发现显著差异。类器官形成试验显示FLASH治疗的类器官有延长的有害作用，表明FLASH与肺上皮细胞的相互作用更复杂。本研究结果有助于推进用于研究FLASH作用机制的强大人类肺模型，可能促进质子FLASH治疗非小细胞肺癌患者。
