Peter van Mourik

46 Chapter 3 Incubation of VX-770, genistein and curcumin in rectal organoids derived from F508del / S1251N CF subjects To investigate interactions between different potentiators at larger scale, we analyzed effects of VX-770, genistein, curcumin and their combinations on forskolin-induced swelling (FIS) of organoids derived from CF subjects compound heterozygous for F508del and the gating mutation S1251N. As described previously 29 , organoid swelling was presented as absolute area under the curve (AUC) calculated from 60 min time tracings of the surface area increase of calcein-green-labeled organoids relative to t = 0 (Fig. 2a,b). We assessed titrations of VX-770, genistein and curcumin using suboptimal forskolin concentrations (0.128 and 0.5 μM) (Fig. 2c-e) to remain within the dynamic range of the swelling assay (Fig. 2f,g) and to facilitate an optimal detection of potentiator activity. In line with other studies 28 (Dekkers et al . manuscript submitted), we consistently observe that the FIS assay reaches it’s upper detection limit around 3000-3500 AUC units, most likely because basolateral ion transport becomes rate-limiting above a certain threshold of CFTR function. We observed that all compounds dose-dependently increased FIS of organoids, with highest potency for VX-770 and lowest potency for curcumin (Fig. 2c-e). Next, we studied VX-770 and genistein combination therapy using a forskolin dose- range (0.008 - 5 μM) (Fig 2f,g) and observed that near-saturating (3 or 50 μM; Fig. 2f) and suboptimal (0.1 or 10 μM; Fig. 2g) concentrations of VX-770 or genistein increased basal FIS in a forskolin dose-dependent manner. Interestingly, genistein greatly enhanced VX-770-repaired FIS and synergistic effects were observed at low forskolin concentrations (Fig 2f,g). As observed previously (Dekkers et al . manuscript submitted), the drug-induced FIS of S1251N-expressing organoids reached maximal rates (= AUC of ~3000) at high forskolin levels (Fig 2f,g). The genistein dose- dependent increase in FIS of VX-770-treated organoids also indicated synergy between VX-770 and genistein, even at genistein levels <10 μM (Supplementary Fig. S1a). Subsequently, combinations of VX-770, genistein and curcumin were investigated at near-saturating (Fig. 2h; 3, 50 and 50 μM) and suboptimal (Fig. 2i; 0.1, 10 and 10 μM) potentiator dosages using fixed forskolin concentrations, which were defined from Fig. 2f,g. At near-saturating potentiator concentrations, most FIS responses upon combination treatments (except for genistein + curcumin) greatly exceeded the calculated additive values of the single treatments, indicating synergistic effects for these potentiator combinations (Fig. 2h). Stimulations with suboptimal potentiator concentrations indicated a strong increase of the VX-770 response by genistein, but not by curcumin, albeit that synergy was detected for the