Enhanced radiation sensitivity, decreased DNA damage repair, and differentiation defects in airway stem cells derived from patients with chronic obstructive pulmonary disease

Abstract

Radiation therapy (RT) is a common treatment for lung cancer. Still, it can lead to irreversible loss of pulmonary function and a significant reduction in quality of life for one-third of patients. Preexisting comorbidities, such as chronic obstructive pulmonary disease (COPD), are frequent in patients with lung cancer and further increase the risk of complications. Because lung stem cells are crucial for the regeneration of lung tissue following injury, we hypothesized that airway stem cells from patients with COPD with lung cancer might contribute to increased radiation sensitivity. We used the air-liquid interface model, a three-dimensional (3D) culture system, to compare the radiation response of primary human airway stem cells from healthy and patients with COPD. We found that COPD-derived airway stem cells, compared to healthy airway stem cell cultures, exhibited disproportionate pathological mucociliary differentiation, aberrant cell cycle checkpoints, residual DNA damage, reduced survival of stem cells and self-renewal, and terminally differentiated cells post-irradiation, which could be reversed by blocking the Notch pathway using small-molecule γ-secretase inhibitors. Our findings shed light on the mechanisms underlying the increased radiation sensitivity of COPD and suggest that airway stem cells reflect part of the pathological remodeling seen in lung tissue from patients with lung cancer receiving thoracic RT.

Keywords: COPD; DNA damage response; NOTCH pathway; basal airway stem cells; lung; radiation therapy.

Graphical Abstract

Figure 1.

Growth and differentiation of human COPD BSC. (A) Representative immunofluorescent staining of 3D ALI culture at day 28 from COPD BSC in mucous cells (MUC5AC), ciliated cells (Ac-TUB), and DAPI nuclear stain. (B) Quantification of Ac-TUB+, MUC5AC + cells from non-COPD and COPD ALI cultures as a percentage of total cells. (C) Doubling time of non-COPD and COPD BSC cell cultures with Incucyte cell imaging. N = 3 independent donors non-COPD and COPD per group *P < .05; **P < .01; ***P < .001; ****P < .0001. Scale bar = 50 μm.

Figure 2.

Differential sensitivity to COPD- and non-COPD PBEC irradiation in ALI culture. (A) Schematic representation of the treatment plan and representative examples of immunofluorescent staining of 3D ALI culture at day 21 ALI from COPD for TP63, CK5, MUC5AC, and Ac-TUB. (B) Quantification of MUC5AC and (C) AcTUB, and (D) TP63 in the non-COPD and COPD cultures upon irradiation (0-2-4 Gy) at 21 days in ALI expressed as ratio of total cells. (E) Replating efficiency of non-COPD and COPD from ALI shows a reduced capacity for replating before and after 2, 4 Gy irradiation. N = 3 independent donors non-COPD and COPD per group. *P < .05; **P < .01; ***P < .001.; ****P < .0001. Scale bar = 50 μm. Created with Biorender.

Figure 3.

Irradiated chronic obstructive pulmonary disease (COPD) BSCs have an impaired cell cycle distribution with increased TP53, P21 levels compared to non-COPD BSCs. (A) COPD BSCs have an increased accumulation in the G2-M phase upon 2 Gy irradiation and decreased S phase distribution compared to non-COPD. At 4 Gy, a strong and significant increase in G0/G1 and a reduction in G2/M are observed. (B) Reduction in percentage of TP63 + cells upon 2-4 Gy irradiation (C) Annexin-V-PI FACS staining in COPD and non-COPD BSC’s upon irradiation. Decreased percentage of living cells and increased apoptosis in COPD stem cells upon 4 Gy irradiation. (D) Cleaved PARP, TP53, P21^CIP Western blot in non-COPD and COPD BSC upon 4 Gy irradiation (E) Representative images and quantification of the alkaline comet assay of BSC’s upon 4 Gy irradiation. Right panel. Tail-moment quantitation shows irradiated COPD BSCs have increased tail moment compared. N = 3 independent donors non-COPD and COPD per group *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 4.

Irradiated COPD BSCs have impaired non-homologous end joining and increased DNA damage. (A) 53BP1 nuclear Foci in TP63 + cells in COPD and non-COPD BSCs. (B) Schematic treatment plan and immunofluorescent staining of healthy and COPD BSCs for 53BP1 and EdUR 24 hours after irradiation (0-4 Gy) (C) Quantification of the 53BP1 foci in the EdU-positive cells 6 and 24 hours after irradiation. (D) Total ATM, phospho-ATM Western blot in non-COPD and COPD stem cells 24 hours post-irradiation (0-4 Gy). Lamin A/C is loading control. (E) Viability of non-COPD and COPD BSC upon phospho-ATM inhibition (10 µM KU55933) added 24 hours before irradiation and analysis 24 hours post-irradiation (0-4 Gy). (F) RAD51 nuclear foci in non-COPD vs COPD BSC 1 and 24 hours post-irradiation with 4 Gy. N = 3 independent donors non-COPD and COPD per group *P < .05; **P < .01; ***P < .001; ****P < .0001. Scale bar = 50 μm.

Figure 5.

NOTCH inhibition reverts the pathological secretory phenotype and reduces radiation-induced DNA damage in COPD BSCs. A) Quantification of Ac-TUB+, MUC5A + cells in the ALI system shows that COPD cultures have a perturbed differentiation phenotype compared to non-COPD ALIs with an excess of MUC5A + cells at the expense of ciliated cells, which can be reverted with NOTCH inhibition. (B) Schematic representation of the treatment plan and quantification of the 53BP1 staining in TP63 + cells, 24 hours after RT in the presence or absence of NOTCH inhibition at day 19 to 21, 48 hours before RT (2-4 Gy). (C) Quantification of TP63, 24 hours after RT in the presence or absence of NOTCH inhibition at day 19 to 21, 48 hours prior to RT (2-4 Gy). N = 3 independent donors non-COPD and COPD per group *P < .05, **P < .01; ***P < .001; ****P < .0001.