Radiation decreases bronchial epithelial progenitor function as assessed by organoid formation

Respiratory Research, vol. 26, pp. 20

Abstract

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS AND DISCUSSION: 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. CONCLUSION: 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.

Authors 5

1. Merian E. Kuipers corresponding Aachen Department of Pulmonology, Leiden University Medical Centre (LUMC)

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Pulmonology, Leiden University Medical Centre (LUMC), Albinusdreef 2, C2-R-062, 2333 ZA, Leiden, The Netherlands. m.e.kuipers1@lumc.nl

   Department of Pulmonology, Leiden University Medical Centre (LUMC), Albinusdreef 2, C2-R-062, 2333 ZA, Leiden, The Netherlands

2. Dennis K. Ninaber Aachen Department of Pulmonology, Leiden University Medical Centre (LUMC)

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Pulmonology, Leiden University Medical Centre (LUMC), Albinusdreef 2, C2-R-062, 2333 ZA, Leiden, The Netherlands

3. Krista C.J. Wink Aachen Department of Radiotherapy, Leiden University Medical Centre

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Radiotherapy, Leiden University Medical Centre (LUMC), Leiden, The Netherlands

4. Annelies Margaretha Slats Aachen Department of Pulmonology, Leiden University Medical Centre (LUMC)

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Pulmonology, Leiden University Medical Centre (LUMC), Albinusdreef 2, C2-R-062, 2333 ZA, Leiden, The Netherlands

5. Pieter Sicco Hiemstra Aachen Department of Pulmonology, Leiden University Medical Centre (LUMC)

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Pulmonology, Leiden University Medical Centre (LUMC), Albinusdreef 2, C2-R-062, 2333 ZA, Leiden, The Netherlands