Nasospheroids permit measurements of CFTR-dependent fluid transport
Jennifer S. Guimbellot, … , Ilona Jaspers, Martina Gentzsch
Jennifer S. Guimbellot, … , Ilona Jaspers, Martina Gentzsch
Published November 16, 2017
Citation Information: JCI Insight. 2017;2(22):e95734. https://doi.org/10.1172/jci.insight.95734.
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Research Article Cell biology Pulmonology

Nasospheroids permit measurements of CFTR-dependent fluid transport

Abstract

Expansion of novel therapeutics to all patients with cystic fibrosis (CF) requires personalized CFTR modulator therapy. We have developed nasospheroids, a primary cell culture–based model derived from individual CF patients and healthy subjects by a minimally invasive nasal biopsy. Confocal microscopy was utilized to measure CFTR activity by analyzing changes in cross-sectional area over time that resulted from CFTR-mediated ion and fluid movement. Both the rate of change over time and AUC were calculated. Non-CF nasospheroids with active CFTR-mediated ion and fluid movement showed a reduction in cross-sectional area, whereas no changes were observed in CF spheroids. Non-CF spheroids treated with CFTR inhibitor lost responsiveness for CFTR activation. However, nasospheroids from F508del CF homozygotes that were treated with lumacaftor and ivacaftor showed a significant reduction in cross-sectional area, indicating pharmacologic rescue of CFTR function. This model employs a simple measurement of size corresponding to changes in CFTR activity and is applicable for detection of small changes in CFTR activity from individual patients in vitro. Advancements of this technique will provide a robust model for individualized prediction of CFTR modulator efficacy.

Authors

Jennifer S. Guimbellot, Justin M. Leach, Imron G. Chaudhry, Nancy L. Quinney, Susan E. Boyles, Michael Chua, Inmaculada Aban, Ilona Jaspers, Martina Gentzsch

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Figure 2

Non-CF nasospheroids show reduction in cross-sectional area over time when CFTR is stimulated by forskolin.

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Non-CF nasospheroids show reduction in cross-sectional area over time wh...
(A) Representative non–cystic fibrosis (CF) nasospheroids are stained with calcein green. Z-stacks are taken sequentially using confocal microscopy and the maximal projection generated. The same nasospheroids are shown at 30 and 75 minutes. Scale bars: 50 μM. (B) The starting size of each nasospheroid was set at 1, and the fractional reduction of each was calculated at all time points. The average fractional reduction was plotted at each time point. Stimulated nasospheroids were treated with 10 μM forskolin, amiloride, and IBMX. Nonstimulated spheroids were treated with an equal concentration of vehicle dimethyl sulfoxide (DMSO). Stimulated spheroids + inhibitor were treated with CFTRinh-172 at time of testing. Analysis of variance was calculated for the groups. Mean values ± SD are shown.*P < 0.00001 and ‡P < 0.001 for 1-way ANOVA and paired t tests between time points. (C) Each nasospheroid absolute size of maximal projection at each time point was plotted over time for all time points in the “Raw” graph. The “Fitted” graph represents the fitted slopes for each nasospheroid after linear regression (see Methods). (D) Summary data of slopes for all non-CF nasospheroids and non-CF nasospheroids treated with CFTRinh-172. Fitted slopes are represented by a box and dot plot. Each dot is a single nasospheroid. The black box is the mean; the central line is the median; the box borders are the 25th and 75th percentiles. *P < 0.001. (E) Summary data of time-averaged AUC unadjusted for starting size for all non-CF nasospheroids and non-CF nasospheroids treated with CFTRinh-172. AUC is represented by a box plot. *P < 0.0001. n = 9 subjects. A minimum of 3 up to a maximum of 18 nasospheroids were analyzed per subject.