Peter van Mourik

143 Personalized treatment of CF using rectal organoids swelling was measured in duplicate at multiple independent culture time points as indicated in supplemental Fig. S1, with 4-8 different concentrations of forskolin as previously described 15,16,21 . The CFTR modulators (3 µM VX-770/ivacaftor (Selleck Chemicals LLC) or a combination of 10 µM genistein (Sigma) plus 50 µM curcumin (Sigma)) were directly added to the organoids with forskolin, except for VX-809/ lumacaftor (3 µM, Selleck Chemicals LLC) that was pre-incubated for 24h. Organoids were fluorescently labeled and total area per well and time point was monitored by a Zeiss LSM800 confocal microscope. A Zen Image analysis software module (Zeiss) was used to quantify the organoid response (area under the curve measurements of relative size increase of organoids after 60 minutes forskolin stimulation, t = 0 min baseline of 100%). Patient selection A total of 24 patients (15 males and 9 females, median age 16.0 years) were included in this study. From these 24 patients, 15 patients had at least one S1251N mutation and were treated with CFTR modulators as part of a clinical trial aiming to compare different CFTR potentiator treatments (NTR4585 and NTR4873). Thirteen of these 15 patients participated in both clinical trials and therefore received two different CFTR modifying treatments. The remaining 9 patients carried at least one rare CFTR mutation and were selected for off-label CFTR modulator treatments based on the organoid response and clinical necessity. A rare mutation was defined as a mutation with a prevalence of less than 1.0 % in the Dutch CF population of which no data on clinical drug responsiveness was available in literature at the time of biopsy 22 . More information on the clinical characteristics of the selected patients is shown in Table 1. All patients (and/or their legal representatives) gave informed consent for rectal biopsies, generating and testing of their individual organoids as well as for (data collection on the effect of) clinical treatment. Clinical endpoints In vivo therapeutic effect in the patients with an S1251N mutation was measured by absolute change after 8 weeks of CFTR modulator treatment in comparison with pretreatment baseline value. Data from people with rare mutations receiving either ivacaftor or lumacaftor/ivacaftor was collected between 4-8 weeks after initiation of treatment. Forced expiratory volume in one second is a widely used readout to assess pulmonary function, and was expressed as percent predicted for body height, age and gender (ppFEV 1 ). SCC measurements were assessed as this is currently the best established in vivo biomarker of CFTR function. 7