Kuipers Merian E, Ninaber Dennis K, van Doorn-Wink Krista C J, Slats Annelies M, Hiemstra Pieter S
Department of Pulmonology, Leiden University Medical Centre (LUMC), Albinusdreef 2, C2-R-062, 2333 ZA, Leiden, The Netherlands.
Department of Radiotherapy, Leiden University Medical Centre (LUMC), Leiden, The Netherlands.
Respir Res. 2025 Jan 18;26(1):20. doi: 10.1186/s12931-025-03105-z.
Radiation-induced lung injury (RILI) is a serious side-effect of radiotherapy for lung cancer, in which effects on the normal lung epithelium may play a key role. Since these effects are incompletely understood, the aim of the present study was to evaluate the effect of ionizing radiation (IR) on cultured well-differentiated primary bronchial epithelial cells (PBEC) with a focus on cytotoxicity, barrier formation, inflammation and epithelial progenitor function.
PBEC were cultured at the Air-Liquid Interface (ALI-PBEC) to allow mucociliary differentiation. Effect of IR (1, 2, 4, 8 Gy [Gy]) on ALI-PBEC cultures was investigated by lactate dehydrogenase (LDH) release, Trans Epithelial Electrical Resistance (TEER; as a measure of barrier function), qPCR (P21/CDKNA1, MKI67, AEN, E2F1, ATF3) and immunofluorescence staining (γH2Ax-foci). The impact on epithelial progenitor function was assessed by studying organoid formation capacity of irradiated ALI-PBEC at 24 h and 7 days after IR.
IR increased the number of γH2Ax-foci (marker of double stranded DNA breaks) in ALI-PBEC, but did not affect markers of toxicity (LDH-release or TEER). IR did also not affect mRNA markers for inflammation or epithelial-mesenchymal transition (EMT), but did increase mRNA levels of the cell cycle inhibitor P21/CDKN1A and resulted in downregulation of the proliferation markers MKI67 and E2F1. Finally, IR of ALI-PBEC had a marked effect on organoid formation capacity, which was markedly impaired following IR in a dose-dependent manner.
In conclusion, epithelial progenitor cell function as assessed by organoid formation capacity is strongly reduced by IR and persists for at least 7 days. Despite an effect on organoid formation capacity, DNA breaks, P21/CDKN1A expression and reduced expression of MKI67 and E2F1, this effect was not accompanied by IR-induced cytotoxicity, or an increase in markers of inflammation or EMT. This study indicates that studying the effects of IR on organoid formation is a valid and sensitive tool to study adverse effects of IR on normal lung epithelial cells and could be used as a tool to study RILI.
放射性肺损伤（RILI）是肺癌放疗的一种严重副作用，其中对正常肺上皮细胞的影响可能起关键作用。由于对这些影响尚未完全了解，本研究的目的是评估电离辐射（IR）对培养的高分化原发性支气管上皮细胞（PBEC）的影响，重点关注细胞毒性、屏障形成、炎症和上皮祖细胞功能。
将PBEC在气液界面（ALI-PBEC）培养以实现黏液纤毛分化。通过乳酸脱氢酶（LDH）释放、跨上皮电阻（TEER，作为屏障功能的指标）、qPCR（P21/CDKNA1、MKI67、AEN、E2F1、ATF3）和免疫荧光染色（γH2Ax灶）研究IR（1、2、4、8 Gy[戈瑞]）对ALI-PBEC培养物的影响。通过研究IR后24小时和7天照射的ALI-PBEC的类器官形成能力来评估对上皮祖细胞功能的影响。
IR增加了ALI-PBEC中γH2Ax灶（双链DNA断裂的标志物）的数量，但不影响毒性标志物（LDH释放或TEER）。IR也不影响炎症或上皮-间质转化（EMT）的mRNA标志物，但确实增加了细胞周期抑制剂P21/CDKN1A的mRNA水平，并导致增殖标志物MKI67和E2F1的下调。最后，ALI-PBEC的IR对类器官形成能力有显著影响，照射后类器官形成能力以剂量依赖性方式显著受损。
总之，通过类器官形成能力评估的上皮祖细胞功能因IR而显著降低，并持续至少7天。尽管对类器官形成能力、DNA断裂、P21/CDKN1A表达以及MKI67和E2F1表达降低有影响，但这种影响并未伴随IR诱导的细胞毒性或炎症或EMT标志物的增加。本研究表明，研究IR对类器官形成的影响是研究IR对正常肺上皮细胞不良影响的有效且敏感的工具，可作为研究RILI的工具。
