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American Journal of Physiology - Lung Cellular and Molecular Physiology logoLink to American Journal of Physiology - Lung Cellular and Molecular Physiology
. 2021 May 19;321(1):L119–L129. doi: 10.1152/ajplung.00639.2020

CFTR function and clinical response to modulators parallel nasal epithelial organoid swelling

Justin D Anderson 1,2,*, Zhongyu Liu 1,2,*, L Victoria Odom 2, Latona Kersh 1, Jennifer S Guimbellot 1,2,✉
PMCID: PMC8321858  PMID: 34009038

Abstract

In vitro biomarkers to assess cystic fibrosis transmembrane conductance regulator activity are desirable for precision modulator selection and as a tool for clinical trials. Here, we describe an organoid swelling assay derived from human nasal epithelia using commercially available reagents and equipment and an automated imaging process. Cells were collected in nasal brush biopsies, expanded in vitro, and cultured as spherical organoids or as monolayers. Organoids were used in a functional swelling assay with automated measurements and analysis, whereas monolayers were used for short-circuit current measurements to assess ion channel activity. Clinical data were collected from patients on modulators. Relationships between swelling data and short-circuit current, as well as between swelling data and clinical outcome measures, were assessed. The organoid assay measurements correlated with short-circuit current measurements for ion channel activity. The functional organoid assay distinguished individual responses as well as differences between groups. The organoid assay distinguished incremental drug responses to modulator monotherapy with ivacaftor and combination therapy with ivacaftor, tezacaftor, and elexacaftor. The swelling activity paralleled the clinical response. In conclusion, an in vitro biomarker derived from patients’ cells can be used to predict responses to drugs and is likely to be useful as a preclinical tool to aid in the development of novel treatments and as a clinical trial outcome measure for a variety of applications, including gene therapy or editing.

Keywords: drug response, elexacaftor, ivacaftor, organoids, tezacaftor

INTRODUCTION

Cystic fibrosis (CF) is a genetic disorder with significant morbidity, especially in the lung, that limits life span (1, 2). CF transmembrane conductance regulator (CFTR) modulators, including correctors targeted at repairing misprocessed proteins and potentiators intended to restore CFTR gating mutations at the cell surface, have increasingly transformed the care of patients with CF (3–5). Four modulator therapies are in clinical use, expanding modulator therapy to ∼90% of patients: monotherapy with ivacaftor (6–11) and combination therapy with lumacaftor/ivacaftor (12–14), tezacaftor/ivacaftor (15–18), or elexacaftor/tezacaftor/ivacaftor (19, 20). The development of new therapies for CF (www.cff.org/trials/pipeline) will benefit from well-characterized preclinical tools.

Intestinal organoids have shown reproducible responses to CFTR-targeted therapies, including correlation with measures of clinical response at both individual and group levels (21–25). However, in the United States and elsewhere, this assay is not universally available, whereas many CF centers in the United States have sampled human nasal epithelial cells (HNEs). HNEs are obtained inexpensively and safely and require no specialized equipment other than a cytology brush used similarly to a viral nasal swab. Nasal brush biopsies can be used to culture epithelial monolayers for short-circuit current (Isc) measurements, historically the standard in vitro test for CFTR activity (24, 26). However, monolayers require a relatively large amount of material, and the automation is difficult. Furthermore, measurement of the downstream effects of CFTR (fluid and mucus transport), rather than ion transport alone, is crucial to estimating drug response and provides additional mechanistic insights (27–32).

In this study, we describe further development of an in vitro HNE organoid swelling assay (33) as a functional tool and associations with clinical response to modulators, including the recently approved triple-combination CFTR modulator therapy. This assay combines the advantages of intestinal organoid models with nasal cell culture: tissue that is easily, safely, and inexpensively obtained in a reproducible, automated assay that recapitulates the mutation phenotype of the individual. This assay significantly differs from prior publications (34–36) that describe terminally differentiated epithelial cells that self-organize into free-floating spheres with the apical membrane exposed to the media bath, with limited applications. Another model (37) is similar but with significant differences in methodology and analysis and a lack of evaluation against clinical outcomes. Here, we describe an assay that could be useful as a biomarker for CFTR function not only for precision selection of drug therapy for individual patients but also as a patient-derived model in clinical studies, as a surrogate biomarker for clinical trials, and as a new model for understanding CF pathophysiology. Some of the results of these studies have been previously reported in the form of an abstract (38).

METHODS

Participants

Patients with CF (n = 18; Supplemental Table S1; all Supplemental material is available at https://doi.org/10.6084/m9.figshare.13497210.v1) and non-CF subjects (n = 5) volunteered for the donation of nasal epithelial biopsy (University of Alabama at Birmingham Institutional Review Board No. 151030001), as previously described (33). Written informed consent was obtained from all human subjects. Patient information was extracted from medical records.

Culture of Organoids and Monolayers

Brush biopsies were processed, expanded using conditional reprogramming culture, and seeded as monolayers or organoids, as previously described (33, 39). Because repeated passaging can reduce CFTR expression, we did not use cells higher than passage 3 (40). Briefly, for organoid formation and culture, cells were seeded as a single-cell suspension at a concentration of 500 cells/µL in 20% Matrigel (minimum protein concentration of 9 mg/mL, Corning, New York, NY) into 15-well angiogenesis slides (ibidi USA, Fitchburg, WI) precoated with 100% Matrigel. After 1 h of consolidation of the 20% Matrigel cell suspension in the culture incubator at 37°C, 50 µL of Ultroser-G-based cell culture medium was exchanged in each well every other day to maintain and differentiate the organoids for 14–21 days (41).

Forskolin-Induced Swelling Assays

Forskolin (FSK)-induced swelling (FIS) assay methods were modified from previously described protocols (21, 33). With the exception of the FSK dose-response curves shown in Fig. 1, organoids were pretreated for 48 h with vehicle (DMSO), tezacaftor (3 µM, Selleck Chemicals, Houston, TX), and/or elexacaftor (3 µM, Cystic Fibrosis Foundation Research Laboratory, Bethesda, MD); incubated in media containing DAPI (Invitrogen, Waltham, MA) with or without 10 µM CFTR inhibitor 172 (INH172; Selleck Chemicals) for 1 h; and treated with a stimulation cocktail containing FSK (10 µM) and 1-methyl-3-isobutylxanthine (IBMX; 100 µM) with or without ivacaftor (1 µM, Selleck Chemicals) diluted in PBS and media (1:1). Due to the concern for detrimental effects on CFTR with chronic administration of ivacaftor (42–46), as well as previously published data that measured (47) or estimated (44) in vivo concentrations, we applied 1 µM ivacaftor compared with commonly used 10 µM ivacaftor in the Isc assay. We define baseline CFTR function as the swelling response from the stimulation by FSK and IBMX (FI) in the absence of any CFTR modulators. Organoids were imaged in an automated inverted microscope (Lionheart FX, Biotek, Winooski, VT) at 37°C with 5% CO2 every 15–20 min over 8 h.

Figure 1.

Figure 1.

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Non-cystic fibrosis (non-CF) forskolin (FSK) dose response. A: area under the curve (AUC) of non-CF subjects (n = 5 subjects) responding to FSK dose. One-way ANOVA indicated that the AUC was significantly increased in the FSK [1 µM, 10 µM, and 10 µM + 1-methyl-3-isobutylxanthine (IBMX)]-treated group compared with vehicle treatment (P < 0.0001). B: comparison of short-circuit current (ΔIsc) due to FSK treatment for non-CF subjects (n = 4 subjects). One-way ANOVA indicated that ΔIsc was significantly increased in the FSK (1 µM, 10 µM, and 10 µM + IBMX)-treated group compared with vehicle treatment (P < 0.0001). C: Pearson’s correlation of ΔIsc for FSK and CF transmembrane conductance regulator inhibitor-172 (INH) vs. FSK-induced swelling (FIS) AUC for the same individuals and concentrations used in B. Each dot represents the mean of those individuals at the different FSK concentrations. D: FSK dose-response curves (three parameters) of the data shown in C.

Isc Measurements

Isc measurements of HNE monolayers were performed as previously described (48) and were treated with tezacaftor (3 µM), elexacaftor (3 µM), and their combination for 48 h before stimulation. After baseline Isc stabilized, amiloride (100 µM) was used to inhibit apical epithelial Na+ channels, a Cl− gradient was created by replacement of apical Ringer’s solution with low-Cl− Ringer’s solution and amiloride, the cAMP agonist FSK (10 µM) was used to stimulate cAMP-sensitive CFTR channels, ivacaftor (10 µM, unless otherwise stated) was used to potentiate CFTR activity, and the CFTR-specific INH172 was added at 10 µM.

FIS Analysis

Organoid analysis was performed using Gen5 ImagePrime software (Biotek). Organoids were masked using bright-field or DAPI channels. Automated total surface area of the organoids was measured for each well. Quality control exclusion criteria for masking included organoid masks smaller than 90 µm or larger than 500 µm, masking of bubbles, masking of cellular debris, and whole organoid not in frame for the entirety of the 8 h. The change in sum total surface area for all organoids in each well from 0 to 8 h was graphed over time, and the average fractional change (AFC) was calculated as the total surface area at the end of the indicated imaging time divided by the total surface area at beginning of imaging. The net area under the curve (AUC) was calculated for the full 8 h using GraphPad Prism 8 (baseline = 1 and accounting for peaks that went below baseline, reducing AUC, and representing fluid transport out of the lumen). Because there are substantial technical challenges with the analysis of similar assays (49), we incorporated additional quality control measures. To standardize quality control screening of individual wells, results for each condition that were more than 1 SD from the mean of that condition (n = 5 replicate wells) were excluded to account for inevitable technical errors (e.g., debris, introduction of bubbles, and evaporation of media from wells). A subset of wells was manually checked in every experiment to ensure any exclusions were consistently due to such technical errors. On average, approximately one of five wells was excluded due to these technical errors. The sum area of an average of 40 organoids is measured per well, and five replicate wells were treated for every condition. To be included in the final analysis, a minimum of three replicate wells meeting all quality control criteria was required. Unless otherwise specified in the text, each data point for each condition from a single donor represents the mean of three to five replicate wells. Baseline lumen ratio, a measurement of the lumen compared with total surface area in the absence of any treatments, was calculated as previously described (33). The percentage of non-CF [wild-type (WT)] control in Figs. 4, 5, and 6 was calculated using the mean FIS AUC and ΔIsc values for the non-CF (n = 4) individuals in Fig. 2B.

Figure 2.

Figure 2.

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Forskolin-induced swelling (FIS) assessment of baseline cystic fibrosis (CF) transmembrane conductance regulator activity. A: representative images of a non-CF subject and a CF subject (F508del/F508del) after 8 h of FIS. Red arrows point to a few of the many fluid-filled lumens in non-CF organoids (left) after 8 h of imaging compared with no fluid-filled lumens in the CF organoids on the right. B: comparison of non-CF (n = 4) and CF (n = 14) baseline 8-h FIS responses using an unpaired t test. ****P < 0.0001. C: comparison of non-CF (n = 4) and CF (n = 9) baseline changes in short-circuit current (ΔIsc) using an unpaired t test. ****P < 0.0001. For this study, not all samples available for the area under the curve (AUC) were used for subsequent Isc testing (B and C). Each symbol represents a different subject, with colored symbols representing subjects with CF and black circles representing subjects without CF (middle key).

Statistical Analysis

Results are presented as means ± SD or SE where noted. Analysis was performed using Pearson’s correlation, one-way ANOVA (with Tukey’s multiple-comparisons test when appropriate), or Student’s t test, as indicated using GraphPad Prism 8. Due to this study’s small sample size, normality was assumed where the quantiles of the residuals were similar to quantiles for a normal distribution (50). If the assumption of normality was not reasonable on visual assessment, then the values were log-transformed and reassessed. All datasets were normally or log normally distributed; therefore, all comparisons were made using parametric analysis. P values of <0.05 were considered significant.

RESULTS

HNE Organoid Swelling Assay

We have previously optimized and characterized the differentiated HNE organoid model (33). To improve the dynamic range and discriminating capacity, we extended the length of the assay to 8 h (33) in contrast to the prior similar organoid study (37), which enabled us to clearly distinguish differences between conditions that were not always apparent at earlier time points (Supplemental Fig. S1). The maximal stimulatory FSK dose in non-CF HNE organoids was determined using increasing doses (0.01–10 µM) as well as IBMX (100 µM) to maintain the concentrations of cAMP during the lengthened assay (Fig. 1). Total surface area was measured by automated image analysis and plotted over time to determine AFC (Supplemental Fig. S2A). The AUC was used as the primary measure for detecting differences between conditions. The magnitude of swelling was significantly increased in a linear fashion as FSK concentration increased (Fig. 1A). There was a nonsignificant increase in swelling when IBMX was added to the maximal stimulating FSK concentration (10 µM; Fig. 1A). Given the length of the assay and the larger response when used, we opted to include it in further experiments.

Isc is a standard measure of CFTR activity and has been shown to correlate with clinical responses (51), but it requires a large amount of cell material, is laborious, and is difficult to fully automate. Because the FIS assay is automated and requires far fewer cells per condition, we assessed whether the FIS assay may substitute for Isc. Isc was measured across HNE monolayers concurrently performed in cells from the same biopsy from four non-CF subjects to assess the correlation of swelling with Isc in response to increasing FSK concentrations, as illustrated by the representative Isc tracings (Supplemental Fig. S2B) and the summary change in current (ΔIsc; Fig. 1B). There was a significant correlation between mean ΔIsc and AUC in response to increasing concentrations of FSK (r = 0.921, P = 0.009) as well as specific inhibition of CFTR by INH172 (r = −0.935, P = 0.006), as shown in Fig. 1C (n = 3 subjects). AUC and ΔIsc data were used to generate FSK dose-response curves for each and were nearly identical, showing EC50 values of 0.24 and 0.26 µM, respectively (Fig. 1D).

Assessment of Baseline CFTR Function in the Organoid Swelling Assay

We evaluated baseline CFTR function in the FIS assay from patients with CF in addition to those without CF. We noted significant differences in FIS between non-CF and CF organoids of varying CF genotype after 8 h of FI stimulation (Fig. 2). Figure 2A shows example images of a non-CF subject and a subject with CF (F508del/F508del) after 8 h of FI stimulation. The AUC after 8 h of swelling was significantly lower in subjects with CF compared with non-CF subjects (P < 0.0001; Fig. 2B). ΔIsc of monolayers from the same subjects paralleled the response of swelling assays (P < 0.0001; Fig. 2C). Due to a paucity of swelling values in the intermediate range, correlation analysis was not performed.

Individual CFTR Modulator Response

HNEs have been used to assess responses to different CFTR modulators on an individual basis (26, 37, 51). To assess this capacity in our assay, we conducted a series of experiments in individual patients with different mutation combinations. We used three key modulator treatments that are currently in clinical use: ivacaftor alone, tezacaftor/ivacaftor, and elexacaftor/tezacaftor/ivacaftor. A sample of individual patient results is shown in Fig. 3 and provided in Supplemental Fig. S1 for three patients with different CFTR genotypes (F508del/F508del, F508del/G551D, and F508del/P67L). In this assessment, we showed representative experiments including summary 8 h FIS AUC (Fig. 3, A–C, left), summary ΔIsc responses (Fig. 3, A–C, right), AFC (Supplemental Fig. S1, A–C, left), and representative Isc tracings (Supplemental Fig. S3). These results demonstrate the ability of this assay to distinguish an individual response to various modulator combinations. Both FIS and Isc generally agreed as to the pattern of modulator response, which was consistent with clinically observed findings. However, in the case of ivacaftor versus tezacaftor/ivacaftor shown in Fig. 3B, the swelling results were more reflective of the subject’s clinical response (subject A; Supplemental Table S2).

Figure 3.

Figure 3.

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Theratyping by forskolin-induced swelling (FIS) and short-circuit current (Isc) responses to cystic fibrosis transmembrane conductance regulator (CFTR) modulators in patients with CF. F508del/F508del (A), F508del/G551D (B), and F508del/P67L (C) individual areas under the curve (AUCs; left) over 8 h for each condition and changes in Isc (ΔIsc) under each condition (right). All conditions were compared using one-way ANOVA with Tukey’s multiple-comparisons test, although not all comparisons are shown for simplicity. **P < 0.01; ***P < 0.001; ****P < 0.0001. AUC, area under the curve; elex, elexacaftor; INH172, CFTR inhibitor-172; iva, ivacaftor; ns, not significant; tez, tezacaftor.

Overall CF Modulator Swelling Response Correlates with Isc Measurements

Baseline organoid FIS and ΔIsc were highly correlated in non-CF, as shown in Fig. 1. Although fluid transport is a complex process incorporating both CFTR activation and other pathways, and the modulator response may not be as simple as restoring CFTR alone, we hypothesized that the response to modulators in organoid swelling should have a similar response to change in current, even if the swelling assay does not always fully reflect the modulator effects, as shown in Fig. 3. A prior study of such correlations included non-CF subjects in similar assessments (37). We assessed whether the FIS assay would correlate with the change in current in the absence of non-CF responses, which would increase sample size at the high end of the range and potentially confound the association. We found significant correlations between baseline and modulator-treated AUC and ΔIsc as percentages of non-CF (WT) control, when combination treatments were evaluated separately and also for all treatments together (Fig. 4, A–D). Treatment with ivacaftor alone did not show a significant correlation with Isc, possibly as a result of the small, homogeneous sample. We also analyzed correlation by sequentially removing the baseline activity of all participants, leaving only the response to modulators in the correlation analysis (r = 0.5515, P = 0.0177; Fig. 4D).

Figure 4.

Figure 4.

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Correlation of short-circuit current (Isc) and forskolin-induced swelling (FIS) area under the curve (AUC) in response to modulator treatment as a percentage of the non-cystic fibrosis response [wild type (WT)]. A: baseline and ivacaftor (iva) responses (n = 10; subjects A, B, G, K, and M). B: baseline and tezacaftor/ivacaftor (tez/iva) responses (n = 14; subjects A, B, C, G, H, J, and L). C: baseline and elexacaftor/tezacaftor/ivacaftor (elex/tez/iva) responses (n = 12; subjects A, B, C, G, H, and J). D: baseline and responses for all modulator therapies (n = 27; subjects A, B, C, G, H, J, K, L, and M). All error bars are ±SE. Letters and corresponding colors match the subject codes provided in Supplemental Table S1.

Comparison of Organoid Swelling with Clinical Measurements in Patients

Ultimately, the goal of this in vitro assay was to attempt to predict the in vivo clinical response to a therapeutic intervention. In Fig. 5, we assessed baseline sweat Cl− (SwCl−) of individual patients with their own baseline HNE organoid FIS AUC (Fig. 5A) and their baseline lumen ratio (Fig. 5B). Baseline SwCl− was not available for non-CF healthy volunteers. Therefore, we used the mean SwCl− value from available clinical trial data with non-CF participants coupled with the mean FIS response or baseline lumen ratio for our non-CF participants. A significant correlation was seen between baseline SwCl− and the FIS response (r = −0.7478, P = 0.0081; Fig. 5A). In the absence of the mean non-CF value, the correlation was lost, similar to that reported by other investigators (23, 37, 52), in part likely due to an absence of more intermediate values. We have previously reported that lumen formation, as measured by baseline lumen ratio, was correlated with CFTR activity, as measured by Isc (33). Baseline lumen ratio was also significantly correlated with baseline SwCl− (r = −0.7625, P = 0.0015; Fig. 5B) and was persistent when the non-CF value was excluded (r = 0.6060, P = 0.0280). Paired pre- and postmodulator treatment SwCl− values were not available for enough patients to analyze, since all clinical data were obtained retrospectively and posttreatment SwCl− is not routinely obtained in our center.

Figure 5.

Figure 5.

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Correlations of the forskolin-induced swelling (FIS) response with clinical data. A: patient baseline sweat Cl− (SwCl−) values (n = 11) compared with baseline FIS area under the curve (AUC) as a percentage of the non-cystic fibrosis (non-CF) response [wild type (WT)]. B: baseline SwCl− values (n = 10) compared with baseline lumen ratio. C: patients’ absolute change in percent predicted forced expiratory volume in 1 s (ΔppFEV1) with the organoid response as a percentage of WT (n = 10). Dashed rectangle represents the in vitro threshold of the response (10% of WT) to yield a clinically relevant response (5%). D: organoid swelling due to modulators as a percentage of WT using the subjects shown in C to compare responders and nonresponders using an unpaired t test. **P < 0.01. The baseline SwCl− values for the mean non-CF control average value in A and B are from a clinical trial healthy control group (PROSPECT; NCT02477319). Letters and corresponding colors match the subject codes provided in Supplemental Table S1. Data from C and D can be found in Supplemental Table S2. †These subjects switched CF transmembrane conductance regulator modulators during their treatment course, so in vitro and in vivo data were included for both.

For lung function assessment, our cohort (n = 8 subjects) produced at least one value for percent predicted forced expiratory volume in 1 s (ppFEV1) in the year preceding and following modulator initiation. Only values in the absence of a pulmonary exacerbation were included in the analysis. If more than one value meeting that criterion was available, the average of these values was used as the baseline. We found a significant correlation between each individual’s in vitro organoid response (AUC as a percentage of WT) and their in vivo response (absolute ΔppFEV1 as a percentage of WT) to modulator treatment (r = 0.7778, P = 0.0081; Fig. 5C). Because of the small sample size, we also evaluated patient response as a categorical variable. Patients were categorized as responders or nonresponders (nonresponder: <5% absolute ΔppFEV1), with 5% being the threshold accounting for the technical variability of FEV1 and a perceived improvement by the patient (53). When classified in this manner, a significant difference (P = 0.0016) was seen for the FIS AUC drug response between responders and nonresponders (Fig. 5D). As shown in Fig. 5C, all subjects (within the dashed rectangle) categorized as nonresponders also had a <10% of WT organoid swelling response (dashed line, Fig. 5, C and D). This 10% of WT CFTR function was considered clinically significant, due to its correlation with mild disease (i.e., higher rates of pancreatic sufficiency, milder respiratory manifestations, and lower SwCl− values) (54, 55).

Correlation of Organoid Swelling with Clinical Measurements from Clinical Trials

To assess clinical correlations at a group level as seen with clinical trial cohorts for CFTR modulator studies, we extracted mean ΔSwCl− (Fig. 6A) and mean absolute ΔppFEV1 (Fig. 6B) from clinical trial results (Fig. 6C) for which our subjects would have been eligible to participate, similar to previous assessments for intestinal organoids (56). FIS drug response data for specific genotype-treatment pairs were averaged and compared with respective clinical trial data (Supplemental Table S2). Correlations for both mean ΔSwCl− (r = −0.8056, P = 0.0287; Fig. 6A) and absolute ΔppFEV1 (r = 0.9574, P = 0.0007; Fig. 6B) are shown.

Figure 6.

Figure 6.

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Mean organoid responses correlated with clinical trial data. Pearson’s correlation of the organoid swelling response to the clinical trial mean change in sweat Cl− (ΔSwCl−; A) and to the clinical trial matched absolute change in percent predicted forced expiratory volume in 1 s (ΔppFEV1; B). The dashed vertical line represents 10% modulator improvement from baseline as a percentage of the non-cystic fibrosis (non-CF) response [wild type (WT)]. C: clinical trial details corresponding to the numbers above each data point in A and B. The forskolin-induced swelling (FIS) area under the curve (AUC) percent in the WT column corresponds to the mean (SD) drug response of the subjects listed in the last column. These subject codes correspond with the subject codes provided in Supplemental Table S1. CFTR, CF transmembrane conductance regulator; elex/tez/iva, elexacaftor/tezacaftor/ivacaftor; iva, ivacaftor; tez/iva, tezacaftor/ivacaftor.

DISCUSSION

Our results show that baseline CFTR function of the HNE organoid model and response to modulators for different mutation combinations correlate significantly with Isc. This illustrates that the organoid model parallels measures of monolayer culture for CFTR ion channel activity, while providing advantages over the more resource-consuming assay. At the same time, the correlation with clinical data suggests that our assay is a candidate ex vivo biomarker that may predict clinical responses to therapeutic interventions for either individuals or groups.

In many ways, our HNE organoid model is similar to the intestinal organoid system that correlates well with clinical phenotype and drug response (23, 56–63). However, rectal biopsy is not easily obtainable in all centers for assessment of CFTR activity. The HNE organoid model in the present study recapitulates many of the desirable characteristics of the intestinal organoid assay but instead uses a tissue type that is easily accessible and acts as a surrogate for the lower airway. Because of the extensive efforts of the Cystic Fibrosis Foundation (CFF) in multiple observational trials that procured nasal cells, the nasal brush biopsy technique is well known across most centers and can be performed in infants, children, and adults without sedation in an outpatient examination room with simple, inexpensive brushes. We have also paid particular attention to using materials that are widely available, including well-published methodology for culturing and commercially available culture dishes, reagents, equipment, and analysis software. Our intent is to allow expansion across centers, using an automated imaging system that helps reduce subjective bias from measurements.

We have shown a significant correlation between HNE monolayer current and the HNE organoid swelling assay. Our data provide evidence that this organoid swelling assay can serve as a substitute for or an adjunct to HNE monolayers. Several studies have shown substantial utility of HNE monolayers for not only theratyping (52, 64, 65) but also studies of the pathophysiology of CFTR mutations, phenotype of CF, and impact of various potential therapeutic approaches, among other uses. We have also shown a correlation between FIS in organoids treated with CFTR modulators and the Isc response of the same cells. The HNE organoid assay itself requires less material (∼5,000 cells/replicate) and less labor than traditional current measurements due to automation of the analysis. It is further amenable to additional automation and higher throughput approaches than shown here, as the imaging slides used come as 96-well plates and the imaging systems have options to use cell-culture robotics to treat and image many plates. Most importantly, the swelling response in our assay is attributable to CFTR activation and correlates well with clinical responses. This is in contrast to a previously published model that did not correlate the in vitro CFTR modulator response with in vivo measures of the clinical response (37). This is significant for the HNE organoid assay to be used as both a model for precision medicine approaches as well as generalizing drug response to broader CF patient cohorts.

This study is limited by the retrospective nature of the clinical observations and a small sample size. A prospective, larger theratyping study should be performed to confirm reproducibility of the assay and correlations with clinical measures. The small sample size enabled optimization of the assay and concurrent monolayer studies. Future studies will focus on developing this model using a larger sample size into a biomarker that can ultimately be implemented as a theranostic, prognostic, and/or diagnostic tool in clinical laboratories.

In conclusion, we describe here an HNE organoid assay that recapitulates many characteristics of the intestinal organoid system but may be feasible for more laboratories to complete. In addition to individual theratyping, it may be useful for the screening of novel compounds that improve CFTR response to non-CF levels, for assessing additive effects of additional modulators such as those in development, and for the screening of drugs that address nonsense and splice mutations in CFTR. It has further utility as a clinical trial biomarker for patients already on a modulator to assess for ex vivo responses to novel interventions, including gene therapy. Correlations of baseline CFTR activity could be useful for predicting manifestation of CFTR dysfunction in individuals who have novel mutations with unknown clinical significance or for those with inconclusive diagnoses. Strong correlations between organoid response to modulators and clinical responses are essential to use this model as a clinical decision tool or as a trial biomarker.

SUPPLEMENTAL DATA

Supplemental Tables S1 and S2 and Supplemental Figs. S1–S3: https://doi.org/10.6084/m9.figshare.13497210.v1.

GRANTS

This work was supported by National Institutes of Health (NIH) Grant K23HL143167 (to J.S.G.), Cystic Fibrosis Foundation (CFF) Grants GUIMBE18A0-Q and GUIMBE1710 (to J.S.G.), the Gregory Fleming James Cystic Fibrosis Center [NIH Grants R35HL135816 and DK072482 and the CFF University of Alabama at Birmingham (UAB) Research and Development Program (Rowe19RO)], and the UAB Center for Clinical and Translational Science (NIH Grant UL1TR001417).

DISCLAIMERS

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

DISCLOSURES

J.S.G. is listed as an inventor on a patent application 20170242033 from the University of North Carolina (UNC) that describes a similar model. When licensed technology from UNC produces royalties, the inventors receive a share of the revenue. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

J.S.G. conceived and designed research; J.D.A., Z.L., and L.K. performed experiments; J.D.A., Z.L., L.V.O., and J.S.G. analyzed data; J.S.G. interpreted results of experiments; J.D.A. and Z.L. prepared figures; J.D.A., Z.L., and J.S.G. drafted manuscript; J.D.A., Z.L., and J.S.G. edited and revised manuscript; J.D.A., Z.L., L.V.O., L.K., and J.S.G. approved final version of manuscript.

ACKNOWLEDGMENTS

We acknowledge Dr. Steven M. Rowe, Director of the Gregory Fleming James Cystic Fibrosis Research Center, for critical review of this manuscript, support, and funding the automated live cell microscope without which this work would not have been possible. We also thank Dr. Sarah Guadiana (Biotek) for technical assistance and training. We thank Dr. William T. Harris for critical review of the manuscript and Jennifer Natt for assistance in preparing the manuscript. Elexacaftor (VX-445) was provided as a generous gift from Dr. Martin Mense (CFF Research Laboratory).

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