Phenotypic profiling of CFTR modulators in patient-derived respiratory epithelia

Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype–phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the “gold-standard” but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures.

Supplementary figure 2: Rescue of F508del misprocessing in CFBE41ocells with low temperature and chemical correction. Whole cell lysates from CFBE41ocells overexpressing Wt CFTR, when probed for the CFTR protein on a western blot show both mature Band C and immature Band B forms of the protein (first lane from left). In contrast, CFBE41ocells overexpressing F508del CFTR appears only as Band B. However, upon rescue with low temperature both Band C and B are now seen. CFTR specific corrector C18 along with low temperature rescue of F508del CFTR, further increases the mature Band C of the protein (last lane).
Supplementary figure 3: HBE-knockout cell line lacks CFTR expression. Western blotting of lysates from human bronchial epithelial (HBE) cells, were loaded in decreasing amount (first 3 lanes on the left). Lysate from HBE-KO cell line (CFTR KO), was ran in the last lane. Electrophoretic pattern consistent with Wt CFTR, seen as a lower band B and a higher band C, is seen in the first three lanes (HBE Wt). Both bands are absent in the lysates derived from the HBE-CFTR(-/-) cell line. Na + /K + ATPase was used as loading control.
Supplementary figure 4: Nasal cultures from three CF patients have no significant differences in expression of epithelial cell differentiation markers. Nasal epithelial cells from three different CF patients were differentiated to measure CFTR function using the ACC assay ( figure  2a). These cultures were tested for markers of epithelial differentiation -ZO-1 and pancytokeratin (PanCK). Analyses of these western blots is shown in Figure 2c, showing no significant differences in these markers across the three CF patients.

Supplementary figure 5: Representative Ussing chamber studies of bronchial epithelial cultures.
(a) Bronchial epithelial cells from CF patient homozygous for F508del CFTR were studied using the Ussing chamber. The mutant F508del protein was rescued with either corrector VX-809 or vehicle DMSO. As seen in the figure, acute stimulation of CFTR with potentiator VX-770 (1 µM) followed by cAMP agonist forskolin (10 µM) led to a greater response in the VX-809 rescued cultures compared to vehicle treated. Also, the response to CFTR specific inhibitor CFTRinh-172 was higher after maximal stimulation in the VX-809 rescued cultures compared to control. (b) Bar graph represents maximal CFTR stimulation achieved with forskolin and potentiator VX-770, when mutant CFTR is rescued with VX-809 or vehicle alone. Error bars represent SEM from 3 biological replicates. Asterisk indicates statistical significance using unpaired two-tailed t-test (*p = 0.029).  Figure  4b, each transwell of a 96 well transwell plate was monitored for change in fluorescence in response to CFTR modulators, using the ACC assay. Responses were monitored overtime simultaneously in multiple regions in each well (n=9). The grey lines depict change in fluorescence relative to baseline for each region of the well analyzed. The black line represents mean value at each time point and red lines indicate standard deviation. The line graph shows the response to CFTR agonist forskolin (10 µM) + VX-770 (1 µM) followed by CFTRinh-172 (10 µM). The responses were measured in nasal cultures derived from F508del homozygous patient, rescued with corrector VX-809 or vehicle.

Supplementary figure 7: Nasal cultures grown on 96 transwell plate express epithelial differentiation markers.
(a) Nasal cultures grown on 96 transwell plate were analyzed for CFTR function using the ACC assay. qRT-PCR studies were performed on them. The expression levels of CFTR, FOXJ1 and SOX17 across all patients was within a 2 fold range, confirming that the cultures were similarly differentiated. (b) Western blotting analyses of ZO-1 and pancytokeratin (PanCK) protein also showed comparable levels of epithelial differentiation. Calnexin (CNX) analysis assessed protein loading.
Supplementary figure 8: MRP4 expression in primary nasal cultures. PCR amplification for detecting expression of MRP4 was performed, and it showed that, MRP4 is expressed in differentiated nasal cultures. TBP (TATA binding protein) was used as loading control.
Supplementary figure 9: CFTR protein expression in primary nasal cultures. CFTR protein expression assessed using western blotting of nasal cultures from non-CF (Wt), G551D/2622+1G>A and F508del homozygous patient (rescued with VX-809 or vehicle DMSO). As seen on the blot, mature band C is abundantly present in cultures derived from Wt and G551D subjects and is relatively low in cultures from the F508del homozygous patient, with partial rescue in presence of corrector VX-809.

Supplementary figure 10: CFTR and F508del-CFTR localization in airway cultures differentiated from ESC and iPSCs respectively.
Immunofluorescence studies of non-CF (Wt) airway cultures derived from CA1 embryonic stem cell (ESC) show that CFTR is expressed on the plasma membrane. Similar studies on airway culture derived from CF iPSC -GM00997 show changes in expression of F508del-CFTR with VX-661 pretreatment, which partially rescues the mutant protein compared to vehicle (DMSO).
Supplementary figure 11: Z prime test of ACC on 96 transwell plate of nasal epithelial cultures. Nasal epithelial cells derived from a non-CF (Wt) individual were differentiated on a 96 well transwell plate. Each alternating column was stimulated with CFTR agonist Forskolin (10 µM) and VX-770 (1 µM), or vehicle (DMSO) alone. The mean and standard deviation was calculated for both positive control (Forskolin + VX-770 stimulated transwells) and for negative control (DMSO). Calculated Z score was 0.16. There was also a statistically significant difference between negative and positive controls, using unpaired t-test (p < 0.0001). Table 1: Reproducibility of apical CFTR conductance (ACC) assay between two different platings of nasal cultures. Maximum change in fluorescence relative to baseline (ΔF/F 0 ) from one plating was plotted against the ΔF/F 0 from another plating of nasal cultures derived from the same four different subjects as shown in Figure 5c. Table 2: Correlation of fluorescence based assay of apical CFTR activation (∆RFU) versus Ussing chamber measurements of CFTR activation (∆ Ieq µA/cm 2 ) in nasal cultures from the same patients (homozygous for F508del CFTR). Paired measurements were performed for nasal cultures pretreated with VX-809 (3 µM) or vehicle (DMSO) for 48 hours and acutely stimulated with either forskolin alone or forskolin plus VX-770.