Human Induced Lung Organoids: A Promising Tool for Cystic Fibrosis Drug Screening

Abstract

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. Currently, CFTR modulators are the most effective treatment for CF; however, they may not be suitable for all patients. A representative and convenient in vitro model is needed to screen therapeutic agents under development. This study, on the most common mutation, F508del, investigates the efficacy of human induced pluripotent stem cell-derived lung organoids (hiLOs) from NKX2.1+ lung progenitors and airway basal cells (hiBCs) as a 3D model for CFTR modulator response assessment by a forskolin-induced swelling assay. Weak swelling was observed for hiLOs from NKX2.1+ lung progenitors and hiBCs in response to modulators VX-770/VX-809 and VX-770/VX-661, whereas the VX-770/VX-661/VX-445 combination resulted in the highest swelling response, indicating superior CFTR function restoration. The ROC analysis of the FIS assay results revealed an optimal cutoff of 1.21, with 65.9% sensitivity and 71.8% specificity, and the predictive accuracy of the model was 76.4%. In addition, this study compared the response of hiLOs with the clinical response of patients to therapy and showed similar drug response dynamics. Thus, hiLOs can effectively model the CF pathology and predict patients' specific response to modulators.

Keywords: CFTR modulators; airway basal cells; cystic fibrosis; forskolin-induced swelling assay; human induced pluripotent stem cells; lung organoids.

Figure 1

A schematic pathway for obtaining airway basal cells and lung organoids from hiPSCs. Scale bar, 100 μm. Small molecules for inducing differentiation are shown above the arrows, and cellular markers that characterize the resulting cell type are written under the images.

Figure 2

Forskolin-induced swelling of hiLOs from NKX2.1+ lung progenitors from a healthy donor. (A) A schematic representation of the image analysis of the FIS assay. The analysis comprises the following steps: z-stacking of organoids in different planes in Photoshop (Ps), then compilation of probabilistic masks to determine the area of the organoid in ilastik software, then measurement of the organoid area and the relation of objects over time in CellProfiler software and finally statistical analyses using GraphPad Prism. (B) Fluorescence microscopic images of hiLOs (P17L16 cell line) at three time points (0, 5 and 24 h) under the influence of different concentrations of forskolin. Scale bar, 100 μm. (C) Quantification of the normalized swelling area of the hiLOs (P14L1 and P17L16 cell lines) at 0, 5 and 24 h, calculated from the area at 0 h. The data are presented as the median, Q1-Q3, with n = 9 technical replicates, and the mean number of organoids analyzed in each group was 63 individual organoids. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Forskolin-induced swelling of hiLOs obtained from NKX2.1+ lung progenitors and hiBCs with a homozygous F508del mutation in CFTR in response to CFTR modulators (mixture 1:1 of the potentiator VX-770 with the correctors VX-809, VX-661 and VX-661/VX-445). (A) Fluorescence microscopic images of hiLOs (P5L5 cell line) at two time points (0 and 24 h) under the influence of modulators. Scale bar, 100 μm. (B) Quantification of the normalized (based on time point 0) swelling area of hiLOs derived from NKX2.1+ lung progenitors and hiBCs at time points = 0 and 24 h. Data are presented as the mean ± 95% CI, n = 9 technical repeats, and the mean number of organoids analyzed in each group was 51 individual organoids. * p < 0.05, ** p < 0.01, **** p < 0.0001. (C) Two therapeutic endpoints (ppFEV1 (percent predicted forced expiratory volume in the first second) and sweat chloride concentration before and during CFTR modulator administration) of donor #4 with a homozygous F508del mutation in the CFTR before and during CFTR modulator administration.

Figure 4

Predictive capacity analysis of hiLOs derived from NKX2.1+ lung progenitors and hiBCs from patients with a homozygous F508del mutation in the CFTR gene (P1L5, P2L2 and P7L2) and from healthy donors (P14L1 and P17L16). (A) Normalized area of the hiLOs after 24 h of incubation with forskolin. The mean numbers of organoids analyzed in each group were 226 for the F508del/F508del group and 125 for the WT/WT group. (B) ROC curve of the change in hiLO area in response to forskolin. The red dotted line represents an AUC (area under the curve) of 0.5. (C) The mean difference in the normalized area of hiLOs with CFTR modulators (mixture 1:1 of the potentiator VX-770 with the correctors VX-809, VX-661 and VX-661/VX-445) relative to the control group (without modulators) is represented by the mean difference ± 95% CI, GLMM. The zero line represents the statistical significance of the difference. The dotted value represents the threshold evaluated by ROC analysis.