Correlation of Electrophysiological and Fluorescence-Based Measurements of Modulator Efficacy in Nasal Epithelial Cultures Derived from People with Cystic Fibrosis

Cells · 9 authors, 7 centres

AI SUMMARY

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POPULATIONNasal epithelial cultures derived from 39 individuals with cystic fibrosis: 31 homozygous for F508del, 3 homozygous for W1282X, and 5 heterozygous for Class III mutations (G551D or G178R).

INTERVENTIONCFTR modulator treatments: lumacaftor (L), tezacaftor (T), elexacaftor plus tezacaftor (ET), ivacaftor (I), and for W1282X cultures: SMG1i + G418 + elexacaftor/tezacaftor (QUAD combination). Chronic drug treatments were applied basolaterally for 48 hours before functional studies.

COMPARISONFluorescence-based membrane potential (FMP/Fl-ACC) assay versus Ussing chamber electrophysiological measurements of CFTR-mediated apical chloride conductance in matched, fully differentiated nasal epithelial cultures.

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TL;DR

This study compared two methods—Ussing chamber electrophysiology and a fluorescence-based plate reader assay (Fl-ACC)—for measuring CFTR modulator drug responses in nasal epithelial cultures from 39 people with cystic fibrosis. The fluorescence assay correlated well with the Ussing chamber for F508del homozygous cultures (Pearson r = 0.65, p < 0.0001) and detected significant responses to modulator combinations across multiple genotypes, including rare mutations. These findings support the Fl-ACC assay as a complementary, higher-throughput approach for CFTR theratyping that may be particularly sensitive for nonsense mutations like W1282X.

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**Background:** CFTR modulator theratyping using patient-derived tissue cultures aims to support precision medicine for people with cystic fibrosis (CF), particularly those with rare mutations. Nasal epithelial cells are readily accessible and can be differentiated into 2D cultures at air-liquid interface on transwell filters. The Ussing chamber is the standard method for measuring CFTR function in these cultures but is time-consuming and requires specialized equipment. A fluorescence-based assay of apical chloride conductance (Fl-ACC) using a membrane potential-sensitive dye offers a potential higher-throughput alternative. This study aimed to systematically compare these two methods across multiple CFTR genotypes.

**Methods:** Nasal epithelial cells were obtained by brushing from 39 CF individuals (31 F508del/F508del, 3 W1282X/W1282X, 5 with Class III mutations G551D or G178R). Cells were expanded and differentiated on transwell inserts: 24-transwell (6.5 mm diameter) for Ussing chamber studies and 96-transwell (4.26 mm diameter) for plate-reader studies. F508del and Class III cultures were studied at ALI day 14; W1282X cultures required longer differentiation and were studied at ALI day 28. Chronic drug treatments were applied basolaterally for 48 hours. The Fl-ACC assay used FLIPR dye (R8034, Molecular Devices) in chloride-free NMDG buffer, with CFTR activation by 10 µM forskolin and 1 µM VX-770, and inhibition by 10 µM CFTRinh172. Fluorescence was measured using either SpectraMax i3X or FLIPR Tetra instruments. Ussing chamber studies were performed in open-circuit mode with symmetrical chloride concentrations. Statistical analyses used GraphPad Prism 9.4.1, with D'Agostino and Pearson normality tests, Student's paired t-test, one-way ANOVA with Tukey's multiple comparison test, and Pearson and Spearman correlations.

**Key Results:** For F508del homozygous cultures (n=31), the Fl-ACC assay detected statistically significant increases in CFTR-mediated apical chloride conductance after 48-hour correction with lumacaftor, tezacaftor, or elexacaftor plus tezacaftor, followed by forskolin and ivacaftor stimulation. The assay successfully reported the superior efficacy of the triple combination (ETI) relative to LI and TI. For Class III mutation cultures (n=5), ivacaftor caused a significant increase in G551D-CFTR-mediated apical chloride conductance. For W1282X homozygous cultures (n=3), the QUAD combination (SMG1i + G418 + elexacaftor/tezacaftor) produced a statistically significant augmentation of ivacaftor-stimulated channel function. The correlation between Fl-ACC and Ussing chamber measurements for F508del cultures was significant by both Pearson (r = 0.65, p < 0.0001) and Spearman (ρ = 0.67, p < 0.0001) tests. For Class III mutations (n=5), correlation was significant only by Pearson test. For W1282X (n=3), no significant correlation was found; notably, the Fl-ACC assay showed relatively robust responses while Ussing chamber responses were modest.

**Clinical Implications:** The Fl-ACC fluorescence-based assay correlates well with the established Ussing chamber method for measuring CFTR modulator responses in F508del homozygous nasal epithelial cultures, supporting its use as a complementary theratyping tool. The assay's higher throughput (96-well format) and use of commonly available plate readers could facilitate inter-institutional studies and expand access to CFTR functional testing. The finding that Fl-ACC may have greater sensitivity for detecting functional rescue of W1282X-CFTR compared to Ussing chamber measurements is particularly noteworthy, as this may relate to the assay's ability to measure apical chloride conductance independently of paracellular pathway effects. This sensitivity could be valuable for therapy discovery programs targeting nonsense mutations. The observed inter-donor variability in F508del cultures, despite identical genotypes and culture protocols, highlights the influence of non-CFTR genetic and environmental factors on drug response and underscores the importance of patient-specific testing.

PICO

PPOPULATION

Nasal epithelial cultures derived from 39 individuals with cystic fibrosis: 31 homozygous for F508del, 3 homozygous for W1282X, and 5 heterozygous for Class III mutations (G551D or G178R).

IINTERVENTION

CFTR modulator treatments: lumacaftor (L), tezacaftor (T), elexacaftor plus tezacaftor (ET), ivacaftor (I), and for W1282X cultures: SMG1i + G418 + elexacaftor/tezacaftor (QUAD combination). Chronic drug treatments were applied basolaterally for 48 hours before functional studies.

OOUTCOME

Correlation between patient-specific drug responses measured by the two methods; detection of significant CFTR channel function improvement after modulator treatment across genotypes.

STUDY TYPE

other

SPECIALTY

pulmonology

SUMMARISED BY

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FIDELITY CHECK

100% · A

ORIGINAL PAPER

PMC10136647

Correlation of Electrophysiological and Fluorescence-Based Measurements of Modulator Efficacy in Nasal Epithelial Cultures Derived from People with Cystic Fibrosis

Cells

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ABSTRACT

It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del ( n = 31) or W1282X ( n = 3), or heterozygous for Class III mutations G551D or G178R ( n = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X.

CITATION EXPORT

Tarini N. A. Gunawardena, Zoltán Bozóky, Claire Bartlett et al.. Correlation of Electrophysiological and Fluorescence-Based Measurements of Modulator Efficacy in Nasal Epithelial Cultures Derived from People with Cystic Fibrosis. Cells. (2023). https://doi.org/10.3390/cells12081174

AUTHORS

9 · 7 CENTRES

Tarini N. A. Gunawardena, Zoltán Bozóky, Claire Bartlett, Hong Ouyang, Paul D. W. Eckford, Theo J. Moraes, Felix Ratjen, Tanja Gonska, Christine E. Bear

1Program of Molecular Medicine, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
2Program of Translational Medicine, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
3Providence Health Care, Vancouver, BC V6Z 1Y6, Canada
4Department of Paediatrics, Division of Respiratory Medicine, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
5Department of Paediatrics, Division of Gastroenterology, Hepatology and Nutrition, University of Toronto, Toronto, ON M5S 1A8, Canada
6Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada
7Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada

EDITOR / REVIEWER

Elio Castagnola