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. 2021 Dec 31;25(1):103710. doi: 10.1016/j.isci.2021.103710

Figure 2.

Figure 2

Characterization of I37R-CFTR functional response to corrector or potentiator monotherapy in intestinal organoids

Forskolin-induced swelling (FIS) assay in organoids from participants with F508del/F508del (n = 5), G551D/F508del (n = 2), and I37R/F508del (n = 1) CFTR genotypes. Organoids were incubated overnight with 0.03% DMSO (untreated) or 3 μM VX-809 or 3 μM VX-661 or 3 μM VX-445. After 24 h, organoids were stimulated with fsk concentrations ranging from 0.02 to 5 μM, either alone or in combination with potentiator monotherapy (3 μM VX-770 or 3 μM G1837 or 50 μM Gen).

(A) FIS of I37R/F508del organoids stimulated with VX-770, GLPG1837 (G1837), or genistein (Gen) monotherapy, expressed as the means ± standard deviation (SD) of the area under the curve (AUC) calculated from t = 0 (baseline) to t = 60 min.

(B) Representative brightfield images of I37R/F508del organoids at baseline (t = 0) and after 1 h of stimulation (t = 60) at 0.128 μM fsk. Scale bars = 100 μm.

(C) FIS of organoids at 0.128uM fsk following stimulation with VX-770, GLPG1837 (G1837), or genistein (Gen) monotherapy. Data corrected for baseline FIS and represented as violin plots with mean to show distribution.

(D) Representative Ussing chamber recordings of short circuit current in I37R/F508del organoid-derived monolayers. Dot plots of total currents stimulated by DMSO or G1837 plus fsk. Experiments were performed in the presence of 10 μM indomethacin. Arrows indicate the addition of compounds: 100 μM apical amiloride, apical addition of either vehicle control 0.01% DMSO or 10 μM G1837, 5 μM basal fsk, 30 μM apical CFTR inhibitor CFTRinh-172, and 100 μM apical ATP. Each dot represents an individual replicate. Data represented as mean ± standard error of the mean (SEM).

(E) FIS of I37R/F508del organoids pre-incubated with corrector (VX-809 or VX-661 or VX-445) for 24 h, expressed as the means ± standard deviation (SD) of the area under the curve (AUC) calculated from t = 0 (baseline) to t = 60 min.

(F) Representative brightfield images of I37R/F508del organoids at baseline (t = 0) and after 1 h of stimulation (t = 60) at 0.128 μM fsk. Scale bars = 100 μm.

(G) FIS of organoids at 0.128uM fsk following incubation with corrector (VX-809 or VX-661 or VX-445) for 24 h. Data corrected for baseline FIS and represented as violin plots with mean to show distribution. One-way analysis of variance (ANOVA) was used to determine statistical differences except in (D) where unpaired t test was used. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. aP for G1837, bP for Gen and cP for VX-445 of I37R/F508del, ˆP for G1837 vs VX-770, or Gen and #P for VX-445 vs VX-809 or VX-661.