The CFTR PM stability is regulated by phosphorylation. CFTR is predominantly phosphorylated at the R domain and also at nucleotide binding domain 1 (NBD1) and C-terminus residues by protein kinase A (PKA), protein kinase C (PKC), casein kinase II (CK2) and AMP-activated protein kinase (AMPK) for the channel function (Chappe et al., 2003; Kongsuphol et al., 2009; Luz et al., 2011). CK2 is predicted to regulate CFTR PM stability by phosphorylation at Thr-1471 where NHERF1 could interact (Venerando et al., 2013). Lemur tyrosine kinase 2 (LMTK2) phosphorylates CFTR at Ser-737 (Wang and Brautigan, 2006) and its KD or mutation at CFTR Ser-737 suppresses the endocytosis and increases CFTR PM density and stability (Luz et al., 2014). However, LMKT2 KD only modestly improves the PM function of r1F508-CFTR (Luz et al., 2014). Spleen tyrosine kinase (SYK) phosphorylates CFTR at Tyr-512 and decreases CFTR PM levels possibly by triggering endocytosis (Luz et al., 2011; Mendes et al., 2011). Mixed-lineage kinase 3 (MLK3) pathway regulates not only 1F508-CFTR ERQC, but also the PQC by regulating the CFTR proteostasis (Hegde et al., 2015). Inhibition of MLK3 pathway could regulate 1F508-CFTR folding/degradation switch by impairing interaction with PQC machinery such as HSP70/HSP90 Organizing Protein (HOP) (Hegde et al., 2015).

### Chaperones

Molecular chaperones selectively interact with and stabilize unfolded or partially folded protein to acquire a functionally active conformation. Nascent CFTR interacts with a panel of chaperones and co-chaperones including HSC70, HSP70, HSP90, and CNX at the ER (Yang et al., 1993; Pind et al., 1994; Loo et al., 1998; Meacham et al., 1999; Okiyoneda et al., 2004). Even at the post-ER compartments, conformationally defective CFTR such as unfolded r1F508-CFTR is recognized by chaperone/cochaperone complex (Okiyoneda et al., 2010). HSC70/HSP90 complex selectively interacts with unfolded r1F508-CFTR at the post-Golgi and this interaction is crucial for the unfolding dependent ubiquitination (Okiyoneda et al., 2010). KD of HSC70/HSP90 complex (HSP90, HSC70, HOP, AHA1, DNAJB2, DNAJA1, BAG1) increases the r1F508-CFTR PM stability in HeLa cells (Okiyoneda et al., 2010). The HSC70/HSP90 complex is also essential for maintaining kinetic and thermodynamic stability of r1F508-CFTR at the PM by reshaping the CFTR conformation during energetic destabilization (Bagdany et al., 2017). This chaperone activity also maintains r1F508-CFTR channel function at the PM (Bagdany et al., 2017). Thus, modulating the chaperone activity would be a viable target for attenuating the ubiquitination and for stabilizing the CFTR function at the PM.

#### Ubiquitination Enzymes

Ubiquitination determines CFTR elimination not only at the ER, but also from the PM. Ubiquitination is mediated by a sequential action of E1, E2, and E3 enzymes and this modification could be removed by DUB. Specifically, E3 Ub ligase has been proposed to determine the substrate specificity. CHIP is the first identified E3 ligase responsible for the CFTR PQC (Okiyoneda et al., 2010). Consistent with the action at the ER (Meacham et al., 2001), CHIP selectively interacts with unfolded 1F508-CFTR at the post-Golgi through the HSC70/HSP90 chaperones. CHIP KD reduces the ubiquitination of unfolded 1F508-CFTR, resulting in the decelerated endocytosis and lysosomal delivery in HeLa cells (Okiyoneda et al., 2010). CHIP KD also stabilizes r1F508-CFTR at the PM of polarized CFBE cells (Fu et al., 2015).

E3 ligase c-Cbl may play a role in the CFTR peripheral QC, but its contribution could be modest since its KD slightly increases r1F508-CFTR PM stability in CFBE cells (Cihil et al., 2013; Fu et al., 2015). c-Cbl also binds with WT-CFTR and decreases the PM stability without affecting the ubiquitination, suggesting that c-Cbl could regulate constitutive PM turnover of folded CFTR by inducing endocytosis through its C-terminus adaptor function (Ye et al., 2010).

Nedd4-2 is a member of homologous to the E6-AP carboxyl terminus (HECT) E3 which may regulate the CFTR PM expression. Nedd4-2 KD reduces 1F508-CFTR ubiquitination at the ER, and increases the PM expression and function in CF pancreatic adenocarcinoma cell 1 (CFPAC1) and IB3-1 cells (Caohuy et al., 2009). Nedd4-2 binds both WT- and 1F508- CFTR while its role in the WT-CFTR ubiquitination remains controversial (Koeppen et al., 2012). However, Nedd4-2 KD does not stabilize the PM r1F508-CFTR in CFBE cells, implying its marginal contribution to the CFTR PQC (Koeppen et al., 2012; Fu et al., 2015). Nedd4-2 is unlikely a viable CF drug target because its knock out (KO) induces CF-like lung phenotype by excessive function of epithelial Na+ Channel (ENaC) (Kimura et al., 2011; Rotin and Staub, 2012).

A number of DUBs regulate the CFTR turnover. USP10, a DUB localized at early endosomes, interacts with WT-CFTR and reduces the CFTR poly-ubiquination in CFBE cells. The USP10 mediated deubiquitination enhances the endocytic recycling of WT-CFTR (Bomberger et al., 2009). The role of USP10 in the PM stability of conformationally defective CFTR such as r1F508- CFTR remains unclear.

Recently, we have discovered RING finger and FYVE like domain containing E3 Ub protein ligase (RFFL) as a novel component of the CFTR PQC machineries by a comprehensive siRNA screen in CFBE cells (Okiyoneda et al., 2018). RFFL selectively recognizes unfolded r1F508-CFTR through the disordered regions. RFFL promotes K63-linked polyubiquitination of the unfolded CFTR in post-Golgi, resulting in accelerated endocytosis and lysosomal degradation. Importantly, RFFL directly interacts with conformationally defective CFTR such as r1F508-CFTR, but not with folded WT-CFTR at the PM and endosomes. Moreover, the RFFL-mediated ubiquitination is

conformation dependent as it selectively ubiquitinates thermally unfolded NBD1. RFFL KD enhances the functional PM expression of r1F508-CFTR in the presence of VX-809, and this effect is further improved by inhibiting the HSC70-dependent ubiquitination machinery. Thus, RFFL plays an important role in the chaperone-independent CFTR PQC mechanism in HBE cell models.

#### CFTR MODULATORS AFFECTING THE CFTR PM STABILITY

### Pharmacological Chaperones and Chemical Chaperones

Pharmacological chaperones affect the CFTR PM stability by direct stabilization. CFTR corrector VX-809 is the first food and drug administration (FDA) approved CFTR corrector in combination with VX-770/ivacaftor (known as Orkambi). VX-809 selectively improves the processing of misfolded CFTR by stabilizing NBD1-membrane spanning domain (MSD) interface but not other misfolded proteins such as human ether-à-go-go-related gene (hERG) mutants (Van Goor et al., 2011; Okiyoneda et al., 2013; Ren et al., 2013; Farinha et al., 2015). VX-809 repairs not only the CFTR folding defect at the ER but also the CFTR PM instability. VX-809 washout prolongs 1F508-CFTR functional sustainability (Van Goor et al., 2011), suggesting that improvement of the CFTR folding at the ER could increase the thermal stability and proper co- and/or post-translational modifications that renders CFTR more energetic robust conformations even at the PM. VX-809 also promotes 1F508-CFTR and NHERF1 interaction, that may increase the PM stability (Arora et al., 2014). C3 (CFcor-325/VRT-325) and C4 (Corr-4a) also extend r1F508-CFTR PM stability in CFBE cells probably by directing binding (Wang et al., 2007; Varga et al., 2008) although their effect could be not specific to the conformationally defective CFTR (Van Goor et al., 2011). Chemical chaperones such as glycerol also increases the r1F508-CFTR PM stability probably by non-specifically improving the conformational stability (Okiyoneda et al., 2013).

#### CFTR Potentiators

The first FDA approved CFTR potentiator VX-770 improves the gating defect of some CFTR mutants. However, chronic VX-770 treatment destabilizes the PM r1F508-CFTR in CFBE and 1F508 homozygous CF patient HBE (CF-HBE) cells (Cholon et al., 2014; Veit et al., 2014). Importantly, chronic VX-770 treatment diminishes the VX-809 therapeutic efficacy by stimulating the elimination of PM r1F508-CFTR (Cholon et al., 2014; Veit et al., 2014). In addition to VX-770, several CFTR potentiators including P1 (VRT-532) and P2 (PG-01) also decrease the r1F508-CFTR PM stability (Veit et al., 2014). VX-770 and other potentiators could destabilize a variety of CFTR rare mutants referred to as CFTR2 mutants including E92K and L1077P at the PM (Avramescu et al., 2017). Thus, several CFTR potentiators may decrease the thermal stability of metastable mutant CFTR at the PM by inducing conformational change that positively affects for channel gating but negatively affects stability. High-throughput screening has identified several novel CFTR potentiators such as class A analog 4 (A04) and class P analog 12 (P12) that could not destabilize the PM r1F508-CFTR (Phuan et al., 2015).

### Proteostasis Regulating Drugs

Proteostasis regulating drugs that affect array of proteins regulating CFTR folding and QC also affect the CFTR PM stability. Histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) alters expression of a subset of CF-interacting gene products (e.g., chaperones and DAB2) and sustains PM expression of 1F508-CFTR in CFBE cells (Hutt et al., 2010). Tissue transglutaminase (TGM2) inhibitor cystamine also stabilizes 1F508-CFTR at the PM of airway epithelial cells by restoring BECN1 interactome which is sequestrated by CFTR dysfunction (Luciani et al., 2012; Villella et al., 2013). MLK3 pathway inhibitor oxozeaenol has been reported to be effective in correcting the 1F508-CFTR proteostasis defect in the primary HBE cells (Trzcinska-Daneluti et al., 2012). Oxozeaenol could stabilize 1F508-CFTR at the PM as MLK3 KD reduces mature 1F508-CFTR elimination by PQC (Hegde et al., 2015).

#### Cavosonstat and CAL Inhibitor

HSP70/HSP90 Organizing Protein is adaptor protein which coordinates HSP70 and HSP90 function in protein folding and regulates CFTR maturation and PM stability (Odunuga et al., 2004; Okiyoneda et al., 2010). HOP S-nitrosylation by S-nitrosoglutathione (GSNO) induces HOP degradation and increases 1F508-CFTR PM expression (Marozkina et al., 2010). Levels of S-nitrosothiols such as GSNO are low in CF airway (Grasemann et al., 1999) and S-nitrosothiol decreases the internalization rate of r1F508-CFTR in HBE cells (Grasemann et al., 1999, 2000; Zaman et al., 2014). Cavosonstat (N91115) is an orally bioavailable inhibitor of GSNO reductase and restores GSNO levels (Donaldson et al., 2017). Cavosonstat is the first CFTR stabilizer in phase II trials, but it was not beneficial for improvement of lung function in combination with ivacaftor.

CFTR-associated ligand binds CFTR via a PDZ interaction domain and targets CFTR for lysosomal degradation (Cheng et al., 2004). CAL inhibition increases the PM stability of 1F508- CFTR (Cushing et al., 2010) and cell penetrating CAL inhibiting peptide is established (Qian et al., 2015). CAL inhibitor has been developed as a cell surface CFTR stabilizer in pre-clinical level while its therapeutic efficacy and conformational selectivity remain unclear.

#### Ub Ligase Inhibitors

RING finger protein 5 (RNF5/RMA1) is an ER associated E3 Ub ligase that regulates early stage CFTR proteostasis at the ER (Younger et al., 2006). A RNF5 inhibitor Inh-2 identified by homology modeling and virtual ligand screening causes

significant rescue of 1F508-CFTR in immortalized and primary HBE cells from CF patients (Sondo et al., 2018). Intriguingly, Inh-2 modestly increases mature 1F508-CFTR half-life and this stabilization effect is further improved by VX-809. While the contribution of RNF5 in the CFTR peripheral QC remains unclear, RNF5 inhibitor may be useful to overcome the CFTR instability.

Currently, CHIP and RFFL are the only Ub ligases responsible for the CFTR peripheral QC (Okiyoneda et al., 2010, 2018). Thus, inhibiting their activity could selectively reduce the ubiquitination and elimination of unfolded CFTR from the PM, improving the limited efficacy of CF combination therapy. CHIP binds and regulates a number of substrates via chaperones (Connell et al., 2001). Moreover, inhibiting the CHIP activity induces deleterious effect as the CHIP KO mice result in the abnormal phenotypes including ataxia and pre-mature death1 . In contrast, RFFL could bind and regulate a limited number of substrates because of its nature of direct binding to the CFTR through the disordered regions (Okiyoneda et al., 2018). More importantly, inhibiting the RFFL activity seems to have no venomousness since the RFFL KO mice exhibit no abnormal phenotype (Ahmed et al., 2009). Therefore, counteracting RFFL activity may provide a preferable therapeutic approach as a CFTR stabilizer that is a class of drugs that extends the PM resident time of CFTR class VI mutants. Although future studies

1 http://www.informatics.jax.org/marker/phenotypes/MGI:1891731

# REFERENCES

are needed to validate the impact on 1F508-CFTR in CF-HBE cells, developing agents selectively inhibiting RFFL-mediated CFTR ubiquitination may help improve the efficacy of CF pharmacological therapy.

# CONCLUSION AND PERSPECTIVE

Beside the progresses of CF pharmacological therapy, stabilizing the cell surface CFTR remains challenging and is necessary to improve the limited therapeutic efficacy. Recent studies have revealed some of the CFTR PQC mechanism eliminating the functional but conformationally defective CFTR from the PM. Understanding the CFTR PQC mechanism help the development of the CFTR stabilizer, a novel class of CFTR modulator necessary to establish the robust CF pharmacological therapy.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# FUNDING

This work was supported by JSPS KAKENHI grant numbers JP25893275, 15H05643, and 15H01192.

ubiquitin proteolytic system. PLoS Pathog. 7:e1001325. doi: 10.1371/journal. ppat.1001325

fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones. Biochem. J. 410, 555–564. doi: 10.1042/BJ20071420

**Conflict of Interest Statement:** TO has a patent pending in Japan for methodology to identify inhibitors of RFFL-mediated CFTR ubiquitination (2017-047626).

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Fukuda and Okiyoneda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Network Biology Identifies Novel Regulators of CFTR Trafficking and Membrane Stability

*Cláudia Almeida Loureiro1,2†, João D. Santos1,3†, Ana Margarida Matos1,2†, Peter Jordan1,2†, Paulo Matos1,2†, Carlos M. Farinha1,3† and Francisco R. Pinto1,3\**

*1 BioISI—Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, Lisbon, Portugal, 2 Department of Human Genetics, National Health Institute "Dr. Ricardo Jorge," Lisbon, Portugal, 3 Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Lisbon, Portugal*

In cystic fibrosis, the most common disease-causing mutation is F508del, which causes not only intracellular retention and degradation of CFTR, but also defective channel gating and decreased membrane stability of the small amount that reaches the plasma membrane (PM). Thus, pharmacological correction of mutant CFTR requires targeting of multiple cellular defects in order to achieve clinical benefit. Although small-molecule compounds have been identified and commercialized that can correct its folding or gating, an efficient retention of F508del CFTR at the PM has not yet been explored pharmacologically despite being recognized as a crucial factor for improving functional rescue of chloride transport. In ongoing efforts to determine the CFTR interactome at the PM, we used three complementary approaches: targeting proteins binding to tyrosine-phosphorylated CFTR, protein complexes involved in cAMP-mediated CFTR stabilization at the PM, and proteins selectively interacting at the PM with rescued F508del-CFTR but not wt-CFTR. Using co-immunoprecipitation or peptide–pull down strategies, we identified around 400 candidate proteins through sequencing of complex protein mixtures using the nano-LC Triple TOF MS technique. Key candidate proteins were validated for their robust interaction with CFTR-containing protein complexes and for their ability to modulate the amount of CFTR expressed at the cell surface of bronchial epithelial cells. Here, we describe how we explored the abovementioned experimental datasets to build a protein interaction network with the aim of identifying novel pharmacological targets to rescue CFTR function in cystic fibrosis (CF) patients. We identified and validated novel candidate proteins that were essential components of the network but not detected in previous proteomic analyses.

Keywords: CFTR, interactome, membrane traffic, network biology, plasma membrane

# INTRODUCTION

CFTR is a polytopic integral membrane protein that belongs to the ATP-binding cassette (ABC) transporter superfamily functioning as cAMP- and phosphorylation-regulated chloride and bicarbonate at the apical membrane of epithelial cells. Mutations in the *CFTR* gene cause cystic fibrosis, the most common autosomic recessive disorder among Caucasians (Riordan, 2008). The most prevalent CF-causing mutation is F508del, which is present in about 90% of CF patients and generates a mutant protein recognized by endoplasmic reticulum (ER) quality control (ERQC)

#### *Edited by:*

*Anthony Kicic, Telethon Kids Institute, Australia*

#### *Reviewed by:*

*Marc Chanson, Université de Genève, Switzerland Oscar Moran, Istituto di biofisica (IBF), Italy*

> *\*Correspondence: Francisco Pinto<EMAIL_ADDRESS>
*†These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Pharmacology of Ion Channels and Channelopathies, a section of the journal Frontiers in Pharmacology*

> *Received: 11 March 2019 Accepted: 15 May 2019 Published: 04 June 2019*

#### *Citation:*

*Loureiro CA, Santos JD, Matos AM, Jordan P, Matos P, Farinha CM and Pinto FR (2019) Network Biology Identifies Novel Regulators of CFTR Trafficking and Membrane Stability. Front. Pharmacol. 10:619. doi: 10.3389/fphar.2019.00619*

mechanisms (Farinha and Amaral, 2005; Farinha et al., 2013; Farinha and Canato, 2017) and targeted for proteasomal degradation (Jensen et al., 1995).

Although most F508del-CFTR protein is only detected as a non-native (misfolded) core-glycosylated ER-specific intermediate, a small amount reaches the plasma membrane (PM) and is partially functional as a channel (Dalemans et al., 1991; Swiatecka-Urban et al., 2005). Thus, one important focus of basic research in CF involves attempts to rescue F508del-CFTR to the PM, by identifying either the key molecular factors involved in its ER retention or small molecules that promote its folding and release from the ERQC. Recent advances in the field allowed the identification of small molecules with potential to treat the basic defect in CF—either correctors, promoting the relocation of F508del-CFTR from the ER to the PM or potentiators, promoting channel activity (for variants already located at the PM). Among these, the potentiator VX-770 alone or its combinations with the correctors VX-809 or VX-661 made their way to the clinical practice, securing approval from the EMA and the FDA. However, results of these modulators in patients bearing F508del suggest that a physiologically relevant restoration of function will require additional small molecules with distinct corrective properties (Farinha and Matos, 2016). This may be due to the fact that F508del-CFTR, despite its partial pharmacological rescue of intracellular retention and impaired channel regulation, still presents decreased stability at the PM (Sharma et al., 2004; Swiatecka-Urban et al., 2005). Whereas the retention and the gating defects have been extensively studied, and both small-molecule correctors and potentiators are available, CFTR membrane stability is a less understood aspect, albeit crucial to restore F508del-CFTR to levels that correspond to clinical benefit.

CFTR traffic and activity at the PM is the result of numerous interactions that function as molecular switches (Amaral, 2005; Farinha et al., 2013) modulating channel activity and controlling the number of channel present at the membrane, through regulation of the secretory and membrane recycling pathways. This complex network of proteins is the last step in the control of the overall biogenesis/function of the protein and includes other transporters, channels, and trafficking machinery components (Rab GTPases, SNAREs, PDZ-domain-containing proteins) as well as different types of molecular switches (such as kinases, phosphatases, and small GTPases) (Guggino and Stanton, 2006; Farinha et al., 2013; Farinha and Canato, 2017).

Several mechanisms have been described to regulate CFTR anchoring and trafficking at the PM, which include CFTR phosphorylation by different kinases, interaction with cAMP sensors, or response to small GTPase signaling.

Phosphorylation in the regulatory region of CFTR is known for long to be required for its activity and it involves protein kinases A (PKA) and C (PKC) (Schultz et al., 1999). Together with PDZdomain containing proteins, phosphorylation is responsible for the formation of multiprotein signaling complexes that provide spatial and temporal specificity to CFTR function (Guggino and Stanton, 2006). Besides this well-documented role on function, phosphorylation by spleen tyrosine kinase (Syk) in NBD1 (Luz et al., 2011; Mendes et al., 2011) or by lemur tyrosine kinase 2 LMTK2 in the R region (Luz et al., 2014) was shown to control the levels of CFTR at the PM.

cAMP levels in the cell lead to PKA activation and hence promote CFTR function, as described above. cAMP also activates members of guanine nucleotide-exchange factors, called EPACs (exchange proteins directly activated by cAMP), whose activation does not lead to PKA activation (Bos, 2006). Activation of EPAC1, the most abundant EPAC family member expressed in the lung, promotes its interaction with CFTR, leading to its stabilization at the PM through interaction with the PDZ-containing anchor protein NHERF-1 (Lobo et al., 2016).

Overexpression of NHERF1 promotes apical expression of the F508del-CFTR mutant channel (Guerra et al., 2005). This effect relies on the activation of endogenous RAC1 signaling and may also contribute to the increase of F508del-CFTR PM levels (Moniz et al., 2013; Loureiro et al., 2015).

Here, we used network biology to identify relevant protein nodes that may connect the interactomes obtained by analyzing different mechanisms regulating CFTR stability, namely, through phosphorylation by SYK or activation of EPAC1 or RAC1. We focused the network analysis on proteins never described to interact with or regulate CFTR and show that this approach is a valid strategy for finding novel regulators of CFTR function and traffic. Network biology thus proves to be a tool capable of contributing insights into the etiology of other human diseases linked to chloride transport or membrane protein traffic.

#### MATERIALS AND METHODS

#### Protein Interaction Networks

Human physical protein–protein interaction data were extracted from HuRI (Human Reference Protein Interactome Mapping Project) (interactome.baderlab.org) (Rual et al., 2005; Venkatesan et al., 2009; Yu et al., 2011; Rolland et al., 2014; Yang et al., 2016) and APID (Agile Protein Interaction DataAnalyzer) (apid.dep.usal.es) (Alonso-Lopez et al., 2016) databases (accessed in June 2018). APID gathers literature-reported protein interactions, while HuRI results from unbiased large-scale screens for binary interactions. We constructed undirected and unweighted networks using Igraph R-package (Csardi and Nepusz, 2006). Loop and multiple edges were eliminated and only the main component of the network was selected. The network was further filtered to contain only proteins that are not exclusively located in nucleus, mitochondria, or peroxisomes. Gene ontology cellular component annotations obtained from UNIPROT were used to define protein subcellular location. This filter was necessary since the relevant interactions affecting CFTR stability at the PM should not occur within these three cellular compartments. The resulting operational network contained 17,218 proteins and 252,472 interactions.

#### Network Analysis

Candidate proteins were selected through a neighbor enrichment test and a bridge score. The neighbor enrichment test evaluates if the set of direct neighbors of a candidate protein is enriched in members of an input protein set using a hypergeometric test. The bridging score quantifies for every node in a network its relevance in connecting two other nodes (provided as inputs) through short paths of length 2, 3, or 4. It counts the fraction of paths of length 2, 3, or 4 linking the two input nodes that contain a candidate node. These fractions for each length are used in a weighted sum where paths of length 2 have weight 1, paths of length 3 have weight 0.5, and paths of length 4 have weight 0.25. Both methods are implemented as R functions (*specific\_ neighbors* and *bridge\_score*, respectively) publicly available in the NetSetStat repository (github.com/GamaPintoLab/NetSetStat). Proteins selected through both methods were analyzed for biological process enrichment using the DAVID tool (Huang Da et al., 2009a; Huang Da et al., 2009b) with the *Homo sapiens* genome as background and testing terms from the FAT subset of the Biological Process branch of Gene Ontology. Enriched GO terms were considered significant with a False Discovery Rate lower than 0.05 and were excluded if their background frequency was greater than 10% or lower than 5%.

#### Preparation of siRNA-Coated 96-Well Plates

Two siRNAs against each of the following targets (CUL3, GABARAP, GABARAPL1, GABARAPL2, ILK, IQGAP1, LRRK2, NF2, NOS2, SMURF1, and UBASH3B) were acquired as a cherry-pick siGenome library (Dharmacon, Cambridge, UK); 96-well plates were coated with customized siRNAs for solid-phase reverse transfection. For that, a transfection mixture of 300 µl was prepared containing 161.5 µl of the Lipofectamine 2000 (Invitrogen #11668019) and 138.5 µl of 0.4 M sucrose in OptiMEM solution. Then, 1.25 µl of siRNA 20 µM was added per well into 96-conic well plates (siRNA plate); 1.75 µl of transfection mix was added per well to siRNA plate, followed by mixing and centrifugation for 1 min at 50×*g*. After 30 min incubation at room temperature, 1.75 µl of 0.2% (w/v) gelatin solution was added to the siRNA plate and centrifuged at 50×*g*. Then, the total of 4.25 µl of the mix in siRNA plate (siRNA plus transfection mix plus gelatin) was diluted 1:50 in a 96-conic well plate using doubledistilled water. Finally, 50 µl from the diluted mix was lyophilized and stored in an anhydrous atmosphere before cell seeding.

#### Reverse Transfection With siRNA

CFBE cells expressing an inducible doxycycline promotor for mCherry-Flag-CFTR (kind gift of Prof. Margarida Amaral, Lisbon) were grown to confluence and split. Then, after 24 h, cells were seeded in a siRNA-coated 96-well plate (100 µl of cell suspension/well, 7 × 10 7 cells/well) using a Multidrop™ Combi Reagent Dispenser (Thermo Scientific #5840300). After 24 h of reverse transfection, CFTR expression was induced for 24 h for wt-CFTR and 48 h for F508del-CFTR, using 1 µg/ml doxycycline (Sigma #9891) in antibiotic-free medium.

#### CFTR Trafficking Assay

The CFTR trafficking assay was performed as previously documented (Almaca et al., 2011; Botelho et al., 2015). This assay uses a double-tagged version of CFTR with mCherry at the N-terminus and FLAG at the fourth extracellular loop. Usage of the mCherry tag allows for quantification of the total amount of CFTR protein expressed by each individual cell. The Flag tag allows one to quantify CFTR that is exclusively localized at the cell surface by usage of an antibody applied extracellularly without cell permeabilization. Hence, 72 h after seeding and siRNA knockdown, cells were washed once with cold PBS+/+ (1× PBS supplemented with 0.9 mM CaCl2, 0.5 mM MgCl2, pH 7.2) using Tecan Hydrospeed™ and immunostained for the extracellular Flag-tag (in non-permeabilized cells) for 1 h at 4°C with 1:500 anti-Flag® M2 monoclonal antibody (Sigma-Aldrich #F1804). Then, cells were washed three times in ice with cold PBS+/+ and incubated 20 min with 3% (w/v) paraformaldehyde (PFA) at 4°C. Cells were then washed three times in ice with cold PBS+/+ and incubated with anti-mouse Cy5 conjugated secondary antibody (Invitrogen, #A10524). Cells were washed three times in ice with cold PBS+/+ and incubated for 1 h with 1:5000 Hoechst 33342 solution (Sigma #B2261) for nucleus staining. Finally, cells were washed in ice with cold PBS+/+ and immersed in PBS+/+. All solutions were prepared in PBS+/+ and primary and secondary antibodies were diluted in PBS+/+ supplemented with 1% (v/v) BSA.

#### Image Acquisition

Cell imaging was performed at room temperature with an inverted wide-field fluorescence microscope Leica DMI6000 equipped with a 12-bit 1,344 × 1,024 pixel resolution DFC360FX camera (Leica) and a 10× objective. Exposure times at maximum light brightness for Hoechst, mCherry, and Cy5 were 50, 1,300, and 8,000 ms, respectively. The Hoechst channel was used for contrast-based autofocus. The experiment was conducted in triplicate and five images per well were collected.

#### Image Analysis

Automatic image analysis was performed using open-source software tools [cell image analysis software CellProfiler1 (Carpenter et al., 2006) and R2 ] and a pipeline to measure CFTR traffic efficiency developed previously (Botelho et al., 2015). Using this pipeline, the background subtraction was first applied to each image to correct the image illumination and background fluorescence. Then, a quality control was also applied for each image to exclude cells that do not significantly express CFTR, present abnormal morphology, or contain a significant number of saturated pixels. Finally, CFTR traffic was measured in each cell using the fluorescence quantification according to the following formula:

$$\begin{aligned} \text{CFTR Traflic Efficiency} &= \frac{\text{PM CFTR}}{\text{Total CFTR}} \\ &= \frac{\text{Cy5 Intensity Flavourscene}}{\text{mCherry Integrated Fluorescence}} \text{ (Formula 1)} \end{aligned}$$

CFTR traffic efficiency was calculated using the median CFTR traffic efficiency for all cells in the image. Using this pipeline, we

1 CellProfiler, http://cellprofiler.org/ (last accessed October 19, 2017).

2 R program, https://www.r-project.org/ (last accessed March 09, 2018).

also remove out-of-focus images, images with high background fluorescence, and images with a lower number of cells (less than 20 cells). Then, the average of CFTR traffic efficiency for all images in wells transfected with siRNAs, which passed the quality control (traffic efficiency test), was compared with the one measured under control conditions—siRNA EGFP (Traffic Efficiencycontrol) according to the following formula:

DeviationScore Traffic Efficiency Traffic Eff test = − iciency SEM control 2 × control ( ) Formula 2

The standard error of the mean for the Traffic Efficiency is given by the SEMcontrol recorded for siRNA EGFP. Deviation scores (DSs) that are positive or negative correspond to traffic enhancers or inhibitors, respectively. The DS equation normalizes the Traffic Efficiency measures to the mean and variation of the negative controls included in the same 96-well plate. This normalization facilitates the comparison between independent replicates, as all values are relative to the mean and standard error of the mean (SEM) of the negative controls in the respective plate. A DS of 1 is the double of the negative control SEM, which means that the latter has a normalized value of 0.5.

#### Analysis of siRNA Knock-Down Efficiency

CFBE cells constitutively expressing F508del-CFTR were seeded in 96-well dishes and transfected with the panel of siRNAs described above (Dharmacon, Cambridge, UK), using Lipofectamine 2000 (Invitrogen). All cells were incubated for 48 h at 37°C with 5% CO2 and then RNA was extracted with the NucleoSpin RNA kit (Macherey Nagel, Düren, Germany). For cDNA synthesis, 0.5 μg of total RNA was then reverse transcribed using random primers (Invitrogen/Life Technologies) and Ready-to-Go You-Prime beads (GE Healthcare, Buckinghamshire, UK). Realtime PCR quantification (qPCR) was performed as previously described (Matos et al., 2008). Primers used in qPCR for each of the targeted transcripts are described in **Table 1**. Each cDNA sample was diluted fivefold to guarantee accurate pipetting and 5 μl was added to each real-time reaction together with 250 nM primers and SYBR Green Master Mix (Applied Biosystems/ Life Technologies). Data were analyzed with the ABI Prism 7000 SDS 1.1 RQ Software (ΔΔCT method, Applied Biosystems/Life Technologies).

#### CFTR Functional Assay by Halide-Sensitive YFP (HS-YFP)

CFBE cells constitutively expressing wt- or F508del-CFTR together with HS-YFP-F46L/H148Q/I152L (Galietta et al., 2001) were seeded in eight-well chamber slides. F508del-CFTR cells were seeded in duplicate slides. Cells were then transfected with either siLUC (Eurofins MWG Operon), siGABARAP\_1, siSMURF1\_2, or siENOS\_2 (Dharmacon) using Lipofectamine 2000 (Invitrogen). All cells were incubated for 48 h and VX-809 (3 μM; Gentaur) was added to one of the F508del-CFTR cell sets. Cells were carefully washed twice with isomolar PBS (WPBS: 137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4), and wt-CFTR cells were incubated for 15 min in WPBS containing 1 μM indomethacin to reduce endogenous cAMP levels. Cells were then transferred to a Leica TCS-SPE confocal microscope for time-lapse analysis. Each well was assayed individually for iodide influx by recording fluorescence continuously (500 ms per point), first for 10 s (baseline) and then for 60 s after the rapid (≤1 s) addition of isomolar PBS, containing either 5 μM Forskolin (Fsk) (wt-CFTR cells) or 5 μM Fsk plus 20 μM Gen (F508del-CFTR cells), in which 137 mM Cl− was replaced by I− (IPBS; final NaI concentration of 100 mM/plate well). Cells were kept at 37°C up until being assayed at room temperature. After background subtraction, HS-YFP fluorescence recordings (F) were normalized to the initial average value measured before addition of I− (F0). Quantification of fluorescence decay was performed on at least 24 individual cells per well, using ImageJ (NIH) as previously described (Matos et al., 2018). The average fluorescence decay was fitted to an exponential decay function to derive the maximal slope [d(*F*/*F*0)/d*t*, at *t* = 0] that is proportional to the initial influx of I− into the cells. (Loureiro et al., 2015).

#### Protein Thermal Destabilization Assay (Thermal Shift Assay, TS)

After Dox-induced CFTR expression (1 μg/ml; Sigma-Aldrich), mCherry-Flag-F508del-CFTR CFBE cells were transfected with either siLUC (Eurofins MWG Operon) or siGABARAP\_1 (Dharmacon), using Lipofectamine 2000. Cells were then incubated for 24 h with VX-809 (3 μM; Gentaur) at 37°C and then moved for another 24 h to 30°C, to increase the thermal stability of rescued F508del-CFTR

at the PM and allow its accumulation at the cell surface. For the thermal shift assay, cells were again transferred to 37°C for 4 h to induce F508del-CFTR thermal destabilization and PM removal, as described previously (Okiyoneda et al., 2010). Cells were then placed on ice, washed three times with icecold WPBS, and left 5 min in cold WPBS to fully arrest CFTR trafficking. Afterwards, they were incubated on ice with anti-Flag M2 Ab (F3165, Sigma-Aldrich) in WPBS for 1 h at 4°C, without permeabilization. Cells were washed three times with ice-cold WPBS and incubated with anti-mouse Alexa Fluor 488 secondary Ab (Thermo Fisher Scientific) for 1 h at 4°C. Cells were then fixed with 4% formaldehyde for 15 min, thoroughly washed in PBS containing DAPI (4´,6-diamidino-2-phenylindole, Sigma-Aldrich), mounted on microscope slides with Vectashield (Vector Laboratories), and analyzed by confocal microscopy.

### Confocal Microscopy Imaging

Slides of mCherry-Flag-F508del-CFTR CFBE cells immunolabeled with anti-Flag followed by Alexa Fluor 488 were analyzed by confocal microscopy. Images were acquired on a Leica TCS-SPE confocal microscope, where mCherry fluorescence is proportional to the total amount of CFTR, Alexa Fluor 488 fluorescence is proportional to the amount of CFTR present at the cell surface, and nuclei were stained with DAPI. A 0.2 μm Z-stack of 1-Airy confocal images was acquired to select the best *XY* plane to analyze the CFTR protein at the cell surface. ImageJ software (NIH) was then used to delimitate each single cell as individual ROIs (regions of interest, to allow measurement of mCherry and Alexa488 fluorescence signal intensities). Quantification of fluorescence was performed on at least 24 individual cells per field, as previously described (Matos et al., 2018), and expressed as mean ± SEM fold variation over control cells.

# Statistical Analysis

*Multiple treatment data analysis—*Statistical significance of the observed variations between different treatments was assessed using two-tailed Kruskal–Wallis tests, followed by paired comparisons using the Mann–Whitney *U* test, considering a 0.05 significance level for both tests. All observations were confirmed in at least three independent experiments, each with two replicates for each individual condition tested.

*Statistical analysis of CFTR trafficking assay—*Each siRNA was used to measure traffic efficiency of wild-type CFTR, F508del-CFTR, and F508del-CFTR rescued with VX-809. For each siRNA and CFTR type, three independent replicate DSs were obtained. The DS mean was considered a measure of the siRNA capacity to influence the traffic efficiency of a particular CFTR type (<DSi >, i corresponding to CFTR type), and the sum of the three DS mean absolute values (Σ*|*<DSi >|) was considered a global measure of the siRNA effect. To evaluate statistical significance of these effects, a random permutation test was performed, where the data matrix containing all the individual DS (all replicates for all CFTR types and all siRNAs, including siRNAs without knockdown effect, functioning as additional controls) was randomly shuffled 10,000 times. Full matrix permutation is possible because all DS values are normalized by EGFP control traffic efficiency mean and SEM values. For each random matrix, each siRNA random Σ*|*<DSi >| was computed, generating distributions according to the null hypothesis that siRNAs have no effect. Null hypotheses were rejected when the fraction of random values greater than or equal to the observed Σ*|*<DSi >| was lower than 0.05.

# RESULTS

#### Network Connectivity between CFTR Interactors Potentially Modulating Stability at the PM

Studies from our team have identified five sets of CFTR interactors through three parallel approaches (under consideration elsewhere): a) proteins selectively interacting at the PM with either wt-CFTR (set 1: wt) or rescued F508del-CFTR (set 2: rescued), b) protein complexes involved in cAMP-mediated CFTR stabilization (set 3: EPAC), and c) adaptor proteins binding to tyrosine-phosphorylated (set 4: pep-py) or un-phosphorylated (set 5: pep-wt) CFTR peptide (unpublished data). All these complementary approaches were designed to identify potential modulators of CFTR PM stability through different pathways. We hypothesized that proteins simultaneously interacting with these different pathways may also have a significant influence on CFTR stability at the PM.

First, we mapped these sets of interactors on a human protein physical interaction network and explored how strongly they were connected through this network (**Table 2**). To evaluate the statistical significance of network connectivity, we had to consider that these interactor sets were selected by their presence in immunoprecipitated CFTR complexes. Therefore, we generated 500 random sets of interactors (for each of the five observed sets), with the same number of proteins at each observed network distance from CFTR. For each connectivity property (number of overlapping proteins between sets, number of direct interactions between set members, and number of common direct neighbors between sets), we recorded the average value across the 500 randomizations and a *p* value, which corresponds to the fraction of random set pairs showing a value higher than that observed with the experimental sets. The five sets have a low number of proteins in common, although 8 out of 10 pairs have a significantly higher overlap than expected by chance. The strong connectivity between these CFTR interactor sets is much more evident by looking at the numbers of direct physical interactions and common neighbors that are much higher than randomly expected and reach statistical significance for all set pairs. This strong connectivity supports our attempt to find new proteins simultaneously interacting with multiple CFTR PM stability-related pathways.

### Specific Common Neighbors of CFTR Interactor Sets

We have identified 4411 proteins that have direct physical interactions with proteins in at least two CFTR interactor sets (2,420, 1,161, and 443 with direct neighbors in at least 3, 4, and 5 sets, respectively). However, to increase the likelihood that perturbations in one of these proteins will have an effect in these

TABLE 2 | Overlap, direct interactions, and common neighbors between CFTR interactor sets.

specific neighbor pathways and, consequently, on CFTR PM stability, we should require that these proteins are enriched in direct interactions with these CFTR interactor sets. With that aim, we performed a hypergeometric test to determine if these proteins are enriched in interactions with each of the five sets. As we performed this test to all proteins with neighbors in each of the five interactor sets, choosing a trivial *p* value cutoff may be associated with many false positives. To avoid this multiple testing problem, we selected for each set of CFTR interactors the 5% proteins with the lowest hypergeometric *p* values as specific neighbors of that set, and additionally required the selected proteins to have at least three interactions with two or more CFTR interactor sets. This strategy resulted in the selection of 478 proteins. We ranked these proteins by summing the ranks of their neighbor enrichment *p* values for all sets, discarding sets where the protein was not in the top 5% of significantly enriched proteins (protein list and associated statistics are available in **Supplementary Table 1** and the selection process is depicted in **Figure 1**).

#### Important Mediators of CFTR Interaction With EPAC, NHERF1, EZRIN, and SYK

The CFTR interactor sets were experimentally detected under conditions that modulate CFTR PM stability through the influence of factors such as EPAC, NHERF1, EZRIN, and SYK, previously known to be involved in the stabilization process (Mendes et al., 2011; Loureiro et al., 2015; Lobo et al., 2016). To uncover novel proteins that most likely influence CFTR PM stability, we aimed to quantify the contribution of a given protein to interfere (either positively or negatively) with the formation of complexes including CFTR and one of the referred stability factors.

For this purpose, we developed a bridge score that measures the fraction of short length paths (with two to four interactions) connecting two proteins of interest (CFTR and the stability factor) that are mediated by a candidate protein. Mediation of shorter paths is given a higher weight/score. The perturbation of proteins with high bridge scores will disrupt a higher fraction of short network paths linking the two proteins of interest. If the interactions in these paths stabilize or facilitate the assembly of the complex containing the two proteins of interest, the bridging protein may have a positive effect on the functional interaction between the two proteins. Reversely, if the interactions in the short paths compete with the formation of the complex, the bridging protein may negatively affect the functional interaction between the two proteins.

To compute the bridge score, we assembled a connected subnetwork containing CFTR, the four stability factors, and the common neighbors identified in the previous section (process described in **Figure 1**). The resulting subnetwork contained 482 proteins and 16,481 interactions. We considered a protein to be an important mediator between CFTR and one stability factor if its bridge score was in the top 25% of all scores with the corresponding stability factor. This procedure allowed the identification of 194 strong mediator proteins (**Supplementary Table 1**). Gene ontology analysis (**Supplementary Table 2**) showed that the two biological processes most significantly enriched within these 194 proteins were "protein localization to organelle" (GO:0033365, 54 proteins) and "regulation of protein modification process" (GO:0031399, 71 proteins), which supports their hypothetical role in the modulation of CFTR PM stability.

#### Hit Selection

To assess the impact of protein networks in CFTR trafficking, we selected hits for validation from the list of 478 proteins significantly enriched in neighbors from at least two CFTR interactor sets.

From the list of specific common neighbors of CFTR interactors, we started by excluding any proteins detected in the three initial experimentally derived interactor sets. Then, we focused on the top 25% of the list (proteins with higher neighbor enrichment rank sum as defined above). Then, we used data from Human Protein Atlas and excluded proteins with low expression in the "bronchus" (keeping thus only those with medium, high, or n/a protein expression). As we were interested in assessing CFTR PM stability, we then excluded proteins with annotation as ribosomal proteins/ elongation or transcription factors/HNRNP/RNA processing/ nuclear proteins. The next step was to exclude proteins already detected as CFTR interactors in previous studies (Wang et al., 2006; Pankow et al., 2015). This refined our list from 478 to 44 proteins. Finally, we used literature mining and refined this list to 25%, selecting 11 possible hits for validating the proteins listed in **Table 3**.

### Assessment of CFTR Trafficking Under Knockdown of Selected Hits

We ordered two different siRNAs for each of the putative hit proteins from Dharmacon's siGenome library (see Materials and methods). We then used qPCR to assess the knockdown efficiency of the targeted transcript by each siRNA in CFBE cells (**Figure S1**). We verified that most siRNA pairs exhibited considerable differences in their ability to knock down the abundance of the mRNAs coding for the targeted proteins. Notwithstanding, we observed that the siRNAs GABARAP\_1, GABARAPL2\_1, IQGAP1\_1, LRRK2\_2, NF2\_2, NOS2\_2, and SMURF1\_2 were able to significantly (*p* < 0.05) downregulate the hit mRNAs that they targeted (**Figure S1**), and these were chosen for further analysis on CFTR trafficking (wt-, F508del-, and F508del-CFTR rescued with VX-809). We used CFBE cell lines stably expressing a double-tagged (mCherry and FLAG) version of CFTR under the control of a doxycycline-inducible promoter so that CFTR expression could be induced 24 h after transfection with siRNA and immunofluorescence be performed at 24 h post-induction. CFTR at the PM is monitored by the Flag (Cy5) signal and total CFTR by mCherry fluorescence. CFTR trafficking is given by the ratio of PM CFTR to total CFTR (Botelho et al., 2015).

For this assay, cells were also transfected with two previously validated siRNAs targeting CFTR and COPBI (Botelho et al. 2015), as controls of the transfection efficiency and trafficking inhibition, respectively.

Results showed that the different proteins affected differently the three CFTR conditions tested (**Figure 2**). **Table 4** summarizes the top seven siRNAs that, when transfected into the three cell lines, promoted the largest increases in CFTR trafficking efficiency.

Altogether, we observed that siRNAs GABARAP\_1, NOS2\_2, and SMURF1\_2 elicited consistent increments in the trafficking and overall PM levels of VX-809-rescued F508del-, wt-, and untreated F508del-CFTR, respectively (**Figure 2**), prompting us to analyze their impact on overall CFTR function in CFBE cells.

#### Knockdown of GABARAP and NOS2 Modulates wt- and VX-809 Rescued F508del-CFTR Function in Opposite Ways

To test the effect of knocking down the expression of the three selected candidates on CFTR function, we used a widely used and previously validated functional assay, based on the ability of iodide to quench the fluorescence of the halide-sensitive YFP-F46L/H148Q/I152L mutant (HS-YFP) (Loureiro et al., 2015; Matos et al., 2018). As shown in **Figure 3**, knockdown of either SMURF1, NOS2, or GABARAP had no apparent

#### TABLE 3 | Hits selected for validation.

effect on HS-YFP fluorescence in untreated F508del-CFTR cells, compared to control cells (siLUC). In contrast, following VX-809 treatment of cells to allow some F508del-CFTR protein to reach the PM, the downregulation of GABARAP produced a moderate but clear effect on HS-YFP quenching (**Figure 3A**), corresponding to a significant (*p* < 0.05) 1.4-fold increase in the initial iodide influx rate, as calculated from the exponential decay fit of quantified fluorescence values (**Figure 3A**). Interestingly, whereas the downregulation of either SMURF1 or NOS2 appeared to have no significant effect on rescued F508del-CFTR, the knockdown of NOS2 produced an apparent 40% decrease in influx rate, when compared to control (siLUC) cells (**Figure 3B**). Notably, similar and statistically significant opposite effects were observed upon the downregulation of GABARAP and NOS2 in wt-CFTR cells (**Figure 3A** and **C**). Moreover, despite showing no observable consequence in the trafficking assay, the increment produced by GABARAP knockdown in these cells was clearly pronounced, triggering a 2.9-fold faster CFTR response, even after endogenous cAMP reduction by pretreatment of the cells with 1 μM indomethacin, prior to stimulation with 5 μM Fsk. Also noteworthy, the knockdown of SMURF1, which failed to produce a clear increase in wt-CFTR PM signal in the trafficking assay, produced a significant (*p* < 0.01) over twofold stimulation of the iodide influx rate in these cells (**Figure 3C**). Overall, interference with either of the three candidate proteins interfered with functional activity of wt-CFTR, but only GABARAP and NOS2 affected the clinically relevant rescued F508delCFTR, producing opposite functional outputs.

## GABARAP Knockdown Increases the PM Stability of VX809-Rescued F508del-CFTR

Because the absolute increment of VX-809-rescued F508del-CFTR at the cell surface exceeded that computed for its trafficking efficiency upon downregulation of GABARAP, we asked whether the observed increase in both CFTR PM abundance and ion transport would rather reflect an improved stability of the rescued channels at the PM. To test this, we used the previously described thermal shift assay (Loureiro et al., 2015). This assay employs the mCherry-Flag-F508del-CFTR CFBE cell line used for the trafficking assay but with a different experimental procedure. First, F508del-CFTR is rescued with VX-809 and then stabilized at the cell surface by incubating the cells at 30°C for 24 h. Next, cells are returned to 37°C for 4 h, which destabilizes the rescued mutant channels causing their endocytic removal from the cell surface, unless the tested co-treatment procedure is able to delay or prevent CFTR internalization. Cells are then stained as before and analyzed by confocal microscopy. Images of the cell surface are used to quantify the fluorescence signal corresponding to the amount of CFTR at the membrane (**Figure 4A**; see Materials and methods). We used treatment with HGF as a positive control, since this growth factor was previously shown to induce the retention of VX-809-rescued F508del CFTR at the cell surface (Moniz et al., 2013; Matos et al., 2018). Indeed, co-treatment with HGF not only increased the overall PM abundance of CFTR at 30°C, as it was able to promote the surface retention of ~80% of the rescued protein, upon switching to 37°C (**Figure 4B** and **C**), corresponding to ~70% of the wt protein at this temperature

used as controls. (A–C) Representative images for the three cell lines and one target for each cell line. PM CFTR image contrast was optimized after quantification to allow visualization of detected signal differences. (D) The CFTR traffic efficiency is obtained with the ratio PM/total CFTR with PM CFTR assessed by Flag staining (Cy5 fluorescence) and the amount of total CFTR with the mCherry signal. Data are shown as the deviation score as detailed in Formula 2, where positive values correspond to traffic efficiencies larger than the negative control (siRNA against EGFP) and negative values correspond to traffic efficiencies lower than the negative control, *n* = 3.

(**Figure 4C**). Importantly, the knockdown of GABARAP also partially prevented the internalization of rescued F508del-CFTR (**Figure 4B** and **C**). Albeit having a more moderate effect than HGF, the absence of GABARAP caused the retention of ~46% of the rescued protein at the cell surface at 37°C (~25% of wt-CFTR), which when compared to the ~30% control cells (siLUC; ~17% of wt-CFTR) reflects a ~1.5-fold increase in surface protein stability, in agreement with the observed extent of functional rescue.

# DISCUSSION

In this work, we identified novel candidate proteins that are involved in the regulation of CFTR membrane traffic and/or its retention at the PM. The results were obtained based on experimental data from independent proteomic studies conducted in the host lab, namely, on PM CFTR levels following phosphorylation by SYK or the activation of EPAC1- or RAC1-mediated signaling. Subsequently,

TABLE 4 | Deviation score (DS)-based analysis of CFTR traffic efficiency for wt- or F508del-CFTR (incubated with either DMSO or VX-809) following the siRNAmediated hit protein knockdown.

*DSi (i corresponding to wt-, F508del-, or F508del-CFTR+ VX-809) informs on the siRNA capacity to influence the traffic efficiency of a particular CFTR type. The sum of the three DS mean absolute values (∑*|*<DSi >|) constitutes a global measure of the siRNA effect. Statistical significance (p) of these effects were calculated using a random permutation test (see Materials and Methods for details). Values highlighted in bold when significant.*

the resulting interactomes were explored by computational network biology to identify novel protein nodes, never described before to interact with or regulate CFTR.

Recently, several authors have proposed network analysis methods to find novel proteins in common (or mediating the interactions) between two related network modules (Silberberg et al., 2017; Garcia-Vaquero et al., 2018). These candidate proteins may contribute to the common phenotypes associated with both modules. Here, we considered the different sets of CFTR interactors as network modules that have in common their potential role in regulating CFTR stability at the PM. To seek for novel proteins in common between these modules, we selected proteins specifically enriched in direct neighbors from at least two of the CFTR interactor sets. An analogous strategy has been successfully employed to expand the composition of disease-associated network modules (Ghiassian et al., 2015). This method requires candidates to have known direct interactions with module members, so it is limited by the completeness and accuracy of the accessed interaction databases. Our analysis is restricted to physical protein interactions, which will also limit our ability to find candidates regulating CFTR stability through indirect mechanisms such as expression regulation.

Besides the enrichment in protein localization and modification processes, the list of candidate proteins identified 12 (AHSA1, BAG3, COPS5, HDAC6, HSP90AB1, HSP90B1, HSPA5, LGLALS3BP, PABPC1, RACK1, USP19, and YBX1) out of 25 recently reviewed putative CFTR-interacting targets for restoring CFTR biogenesis and function identified through proteomic approaches (Lim et al., 2017). Both observations support the validity of our approach.

Among the novel protein nodes identified, the proteins encoded by the *GABARAP*, *NOS2*, and *SMURF1* genes were functionally validated in the physiologically relevant bronchioepithelial cell line CFBE. For this, the expression of the endogenous proteins was depleted by RNA interference and the effect on PM localization and function of CFTR was analyzed.

NOS2 encodes the inducible isoform of nitric oxide synthase (NOS), which is expressed maximally following an inflammatory stimulus and produces relatively large micromolar quantities of NO. While macrophages and neutrophils express higher levels of iNOS in the lung, it can also be expressed by bronchial epithelial cells. NOS2 expression in these cells is upregulated by lipopolysaccharide (LPS) and by proinflammatory cytokines, such as interleukin-1, tumor necrosis factor alpha, and gamma interferon (Ermert et al., 2002). Expression of iNOS appears to be nearly absent in bronchial cells from CF patients (Meng et al., 1998; Thomas et al., 2000; Dotsch et al., 2002) and may be caused by abnormalities in the signaling system that normally causes its induction, such as cytokine receptors, second messengers, or transcription factors (Meng et al., 1998; Satitsri et al., 2016). Given the apparent negative

functional effect on both wt and VX-809-rescued F508del-CFTR observed here upon iNOS downregulation, our results suggest that, besides an improved resistance to microbial infection, any therapeutic approach that increased iNOS production in the airways might further benefit CF patients by stimulating residual CFTR function. This has, however, to be further investigated since, in a previous study, micromolar concentrations of NO were shown to inhibit forskolin-stimulated cAMP production by adenylyl cyclase and cAMP-dependent Cl− transport and fluid secretion mediated by CFTR (Spirli et al., 2003).

SMURF1 functions as an E3 ubiquitin ligase protein and has been shown previously to regulate endocytosis of other membrane proteins such as the bone morphogenetic protein type II receptor (BMPR), related to pulmonary arterial hypertension. Blockage of SMURF1 ubiquitin ligase activity increased cell surface expression of BMPR (Murakami and Etlinger, 2019). Here, we observed that depletion of SMURF1 produced a twofold stimulation of the iodide influx rate in CFTR wt cells but did not promote any significant rescue of F508del-CFTR to the PM. Moreover, while endogenous cAMP depletion by pretreatment with indomethacin delayed the response to Fsk in control wt-CFTR CFBE cells, this effect was much less pronounced upon SMURF1 knockdown. This is suggestive that the knockdown of these proteins can have an effect not only in CFTR's PM abundance but also on its gating efficiency. Such an effect might nevertheless have therapeutic potential for class III to V CFTR mutations.

Similar effects were also observed upon knockdown of the GABA receptor-associated protein GABARAP, which belongs to the Atg8 family of ubiquitin-like proteins that are involved in the regulation of autophagy. GABARAP proteins associate with the membrane of the autophagosome through conjugation of phosphatidylethanolamine (Ichimura et al., 2000) and are primary drivers of autophagy working upstream of LC3 proteins and operate mainly at the late stages of autophagy driving the fusion of autophagosomes to lysosomes (Nguyen et al., 2016). Our results show that knockdown of GABARAP increased the temperaturedependent stability and function at the PM of F508del-CFTR after correction with VX-809. Furthermore, depletion of GABARAP also increased wt-CFTR function and, similar to SMURF1 knockdown, its response to Fsk stimulation after pretreatment with indomethacin. Since we observed no detectable impact of GABARAP downregulation on wt-CFTR traffic, it is possible that GABARAP may function by stabilizing the molecular machinery required to anchor and activate CFTR at the PM, thus increasing its gating efficiency. While the high levels of wt-CFTR at the surface may have led to an underestimation of fluorescence signals at the cell surface, the PM stabilizing effects observed with VX-809 rescued F508del-CFTR seem to support a functional potentiation hypothesis. In this case, interference with GABARAP activity may also prove beneficial for class III to class V CFTR mutations.

The identification of novel regulators of CFTR levels at the PM has important biomedical implications. The pharmacological rescue of mutant CFTR folding or gating improves symptoms in CF patients (Zhang et al., 2016); however, a truly relevant clinical benefit appears to require the targeting of additional cellular processes: the trafficking of CFTR as well as its retention at the PM (Farinha and Matos, 2016). The proteins identified here suggest that the cellular processes of membrane traffic, protein stabilization at the PM, and NO signaling are suitable targets for novel therapeutic approaches in CF.

Besides providing new insights into CFTR biology and suggesting possible therapeutic targets in CF, the network biology approach delineated in this work provides a framework applicable to other human diseases related to the traffic and function of PM transport proteins.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the manuscript and/or the supplementary files.

# AUTHOR CONTRIBUTIONS

CL, JS, and AM conducted the experiments. PJ, CF, and PM designed the experimental studies. FP provided the network

#### REFERENCES

analysis. PJ, CF, PM, and FP analyzed the data and wrote the manuscript.

### FUNDING

This work was supported by FCT, Portugal, through center grant UID/MULTI/04046/2019 to BioISI and the BioSys PhD program PD65-2012 (fellowships SFRH/BD/52488/2014, SFRH/ BD/106084/2015, and SFRH/BD/52490/2014 to CL, JS, and AM, respectively).

### ACKNOWLEDGMENTS

We would like to acknowledge technical support by the high-throughput imaging facility of BioISI and thank Prof. Margarida Amaral for providing the CFBE cells expressing mCherry-Flag-CFTR.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2019.00619/ full#supplementary-material

FIGURE S1 | qPCR assessment of siRNA efficiency in CFBE cells for the targeted transcript, \**p* < 0.05.

TABLE S1 | Specific common neighbours of CFTR interactor sets.

TABLE S2 | Enriched biological processes in the list of strong mediator proteins.

connectivity patterns of disease proteins in the human interactome. *PLoS Comput. Biol.* 11, e1004120. doi: 10.1371/journal.pcbi.1004120

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Loureiro, Santos, Matos, Jordan, Matos, Farinha and Pinto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Human Primary Epithelial Cell Models: Promising Tools in the Era of Cystic Fibrosis Personalized Medicine

Nikhil T. Awatade1,2†, Sharon L. Wong1,2†, Chris K. Hewson1,2,3, Laura K. Fawcett 1,2,4 , Anthony Kicic5,6,7,8,9, Adam Jaffe1,2,4 and Shafagh A. Waters 1,2 \*

1 Faculty of Medicine, School of Women's and Children's Health, University of New South Wales, Sydney, NSW, Australia, 2 Molecular and Integrative Cystic Fibrosis Research Centre (miCF\_RC), School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia, 3 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia, 4 Department of Respiratory Medicine, Sydney Children's Hospital, Sydney, NSW, Australia, 5 Centre for Child Health Research, Telethon Kids Institute, The University of Western Australia, Nedlands, WA, Australia, 6 Occupation and Environment, School of Public Health, Curtin University, Bentley, WA, Australia, 7 Faculty of Health and Medical Sciences, The University of Western Australia, Nedlands, WA, Australia, 8 Department of Respiratory and Sleep Medicine, Perth Children's Hospital, Nedlands, WA, Australia, 9 Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Nedlands, WA, Australia

#### Edited by:

Diana Conte Camerino, Università degli Studi di Bari, Italy

#### Reviewed by:

Emanuela Caci, Istituto Giannina Gaslini (IRCCS), Italy Anabela S. Ramalho, KU Leuven, Belgium

> \*Correspondence: Shafagh A. Waters<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Pharmacology of Ion Channels and Channelopathies, a section of the journal Frontiers in Pharmacology

Received: 10 September 2018 Accepted: 19 November 2018 Published: 07 December 2018

#### Citation:

Awatade NT, Wong SL, Hewson CK, Fawcett LK, Kicic A, Jaffe A and Waters SA (2018) Human Primary Epithelial Cell Models: Promising Tools in the Era of Cystic Fibrosis Personalized Medicine. Front. Pharmacol. 9:1429. doi: 10.3389/fphar.2018.01429 Cystic fibrosis (CF) is an inherited disorder where individual disease etiology and response to therapeutic intervention is impacted by CF transmembrane regulator (CFTR) mutations and other genetic modifiers. CFTR regulates multiple mechanisms in a diverse range of epithelial tissues. In this Review, we consolidate the latest updates in the development of primary epithelial cellular model systems relevant for CF. We discuss conventional two-dimensional (2-D) airway epithelial cell cultures, the backbone of in vitro cellular models to date, as well as improved expansion protocols to overcome finite supply of the cellular source. We highlight a range of strategies for establishment of three dimensional (3-D) airway and intestinal organoid models and evaluate the limitations and potential improvements in each system, focusing on their application in CF. The in vitro CFTR functional assays in patient-derived organoids allow for preclinical pharmacotherapy screening to identify responsive patients. It is likely that organoids will be an invaluable preclinical tool to unravel disease mechanisms, design novel treatments, and enable clinicians to provide personalized management for patients with CF.

Keywords: cystic fibrosis, organoid, personalized medicine, CFTR, drug development, sweat chloride, CFTR modulator

# INTRODUCTION

Cystic Fibrosis (CF; OMIM 219700) is the most common life-limiting, autosomal recessive monogenic disease in Caucasian populations (Riordan, 2008). It is caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), an anion channel essential for regulating trans-epithelial secretion of chloride (Cl−) and bicarbonate (HCO− 3 ). The loss of CFTR function leads to abnormalities at the mucosal surfaces in multiple exocrine organs including the lungs, pancreas, liver, and intestine. Notably however, progressive lung disease and respiratory failure are the major cause of morbidity and mortality for most patients (Ratjen et al., 2015). While the regulatory role of CFTR in ion and fluid transport is widely known, the exact mechanism of its defect in the development of CF remains debatable. Two competing hypotheses attempt to explain CF pathogenesis; (1) the "high salt" hypothesis postulates that diminished trans-epithelial Cl− conductance associated with CFTR dysfunction results in increased retention of salt at the mucosal surface, which negates the activity of antimicrobial peptides present in the epithelial apical secretions (Zabner et al., 1998; Wine, 1999); (2) the more favorable "low volume" hypothesis suggests the lack of epithelial sodium channel (ENaC) inhibition in the absence of CFTR function causes sodium (Na+) hyper-absorption, with accompanying intracellular flux of Cl− and water (Matsui et al., 1998; Wine, 1999). Mucus becomes dehydrated, viscous and sticky which then leads to decreased airway surface liquid (ASL) height, as well as mucus stasis and obstruction (Riordan, 2008). Irrespective of the mechanism of action, impaired mucociliary clearance in the lung and the innate inability to eradicate inhaled pathological agents are hallmarks of CF. Recurrent cycles of lung infection and exacerbations result in chronic inflammatory responses which then lead to structural lung damage, loss of lung function, and ultimately respiratory insufficiency (Ratjen et al., 2015).

Major advances in symptom management have been instrumental in delaying disease progression in CF. Administration of enzyme replacement therapy, nutritional support, mucolytic agents airway clearance techniques, and antibiotics for bacterial lung infection, along with early detection of disease (newborn screening, pathogen surveillance) has dramatically improved life expectancy in CF patients over the last 4 decades. The median age of death is now above 40 years (Keogh et al., 2018). Importantly, a class of small molecule compounds termed CFTR modulators were recently discovered. These compounds directly correct defective CFTR protein in CF patients and have now transformed the therapeutic landscape of CF in a precision-based fashion. The first part of this review outlines the current developments in CFTR modulators addressing the different dysfunctional CFTR mutation class and highlights the need for individualized strategies to restore their function.

The second part of this review discusses the evolving use of different epithelial cell models and reporter assays for assessing clinical response to CFTR-directed drugs on a personalized basis, and highlights the strengths and limitations of each application. This review also elaborates on the breakthrough discoveries of long-term cultures of patient-derived 3-dimentional (3-D) airway and rectal organoids. We also discuss their emerging use as ex vivo biomarkers and preclinical predictive tools for the disease.

# CURRENT CFTR-BASED THERAPY WITH CFTR MODULATORS

More than 2,000 CFTR mutations have been identified so far and at least 336 of these are reported to lead to symptoms characteristic of CF (Cystic Fibrosis Mutations Database report 31 August 2018 (US CF Foundation, 2011). It is thus perceivable that a multi-pronged approach is required to target the different defective mechanisms that each CFTR mutation confers. For the past 15 years, high-throughput screening (HTS) has accelerated the process of drug discovery. To identify candidate CFTR modulators, hundreds of thousands of chemical compounds with diverse structures were screened and the potential of a compound to rescue or activate CFTR was tested in cellular-based assays (Pedemonte et al., 2005; Van Goor et al., 2006; Sutanto et al., 2018). The promising compounds, called "hits," underwent further medicinal and chemical optimization to improve potency and minimize potential off-target activities of the compounds. This process has led to successful identification of multiple compounds, some of which have moved forward to human clinical trials.

The different approaches targeted toward correcting each CFTR mutation class as well as the compounds currently tested in clinical trials are summarized in **Table 1**. CFTR-modulating compounds are classified into five main groups: read-through agents, correctors, potentiators, stabilizers, and amplifiers. Of these, two classes of modulators (potentiators and correctors) have gained regulatory approval to treat CF patients with specific CFTR mutations.

# Potentiators

Potentiators were developed for CFTR mutant proteins that are expressed at the apical membrane of epithelial cells but are functionally impaired. HTS performed by Vertex pharmaceuticals involving 280,000 small molecule compounds led to the discovery of the potentiator VX-770 (Generic name: Ivacaftor; Trade name: Kalydeco) (Van Goor et al., 2010). Ivacaftor corrects the gating impairment of Class III mutations. Its use has now been extended beyond the most common Class III G551D-CFTR mutation, to additional Class III mutations, as well as CFTR mutations with conductance (Class IV−R117H) or biosynthesis (Class V−3,849 + 10 kb C>T) defects. In clinical studies of participants with G551D-CFTR mutations, the impact of Ivacaftor on CFTR was evidenced by normalization of sweat Cl−, improved lung function (10% mean increase in FEV1), reduced episodes of pulmonary exacerbations and improved body mass index (BMI) (Accurso et al., 2010; Ramsey et al., 2011; Davies et al., 2013).

Compounds starting with VX are developed by Vertex Pharmaceuticals, GLPG by Galapagos NV and AbbVie, FDL, Flatley discovery lab; QBW, Novartis pharmaceuticals; PTI, Proteostasis therapeutics; SPX, Spyryx biosciences; AZD, AstraZeneca; BI, Boehringer ingelheim; Orkambi, VX-809+VX-770; Symdeko, VX-661+VX-770; Ivacaftor, VX-770; #Triple combination therapy with VX-661 and VX-770. PTC, Premature termination codons; PM, Plasma membrane; ∼, classification is based on (Marson et al., 2016).

through the endoplasmic reticulum, with very little or no mutant protein reaching the apical membrane of epithelial cell. Effective rescue of the F508del mutation thus requires chaperones that can repair defective protein folding and rescue trafficking of the mature CFTR to the plasma membrane. VX-809 (Generic name: Lumacaftor; available as Lumacaftor-Ivacaftor combination therapy—Trade name Orkambi) restored F508del-CFTR channel activity to 15% of wild-type CFTR in in vitro preclinical testing performed in primary airway epithelial cells (Van Goor et al., 2012). This result led to Lumacaftor monotherapy clinical trials in CF patients homozygous for the F508del-CFTR mutation, where a significant improvement in sweat Cl− concentrations were observed but lung function remained unchanged (Clancy et al., 2017). Considering that F508del channel gating defect, administration of Lumacaftor-Ivacaftor combination therapy was proposed as a solution that may augment correction of CFTR function to clinically significant levels.

#### Combination Therapy

Lumacaftor-Ivacaftor combination therapy increased CFTR activity at the plasma membrane in vitro (Van Goor et al., 2012). However, results from phase III trials in children and adults homozygous for the F508del-CFTR mutation showed that the combination therapy failed to produce the magnitude of clinical improvements observed with Ivacaftor. While a reduction in pulmonary exacerbations and improved BMI was observed, there was only a modest improvement in lung function (2– 3%) (Boyle et al., 2014; Wainwright et al., 2015). In addition, CF patients receiving Lumacaftor-Ivacaftor combination therapy reported unwanted side effects such as dyspnoea, liver damage, and bronchospasm. Lumacaftor is also associated with significant drug-drug interactions which alter its pharmacokinetic profile and potentially hamper its therapeutic efficacy (Talamo Guevara and McColley, 2017). Tezacaftor is a new CFTR corrector with an improved pharmacokinetic profile, longer half-life and less drug-drug interactions compared to Lumacaftor. It has recently been approved as a combination therapy with Ivacaftor (Trade name: Symdeko/Symveki) for the treatment of patients homozygous for F508del mutation by the Food and Drug Administration (FDA) and European Medicines Agency. Phase III clinical trials data showed that improvement in lung function with Tezacaftor-Ivacaftor combination therapy was generally comparable or better than those observed in patients treated with Lumacaftor-Ivacaftor combination therapy (Rowe et al., 2017; Taylor-Cousar et al., 2017). It is notable however that while overall benefit was demonstrated, individual patient responses have been heterogeneous in the clinical trials of both Lumacaftor-Ivacaftor and Tezacaftor-Ivacaftor.

#### Theratyping

It is now known that patients display a spectrum of responses to CFTR-modulator drugs despite having the same CFTR mutation variant (Wainwright et al., 2015; Donaldson et al., 2018). This suggests that although the current classification system may be an important indicator for prognosis and disease severity in CF, it is inadequate for predicting how individual patients respond to therapy. Indeed, in vitro experimental studies have shown that contrary to findings in the Class II F508del-CFTR mutation, other Class II processing mutations such as N1303K, R560S, and G85E could not be rescued by Lumacaftor treatment (Awatade et al., 2015, 2018; Dekkers et al., 2016b; Lopes-Pacheco et al., 2017). These results suggest that the underlying pathomechanism of each CFTR mutation is distinct and that individualized strategies to restore their function may be warranted.

To address this, CFTR mutations have been classified according to their response to modulator compounds. This approach termed "theratyping," groups together patients who harbor different CFTR mutations but respond to the same CFTRdirected compounds. It is clear that treatment regimens of the future will need to take into consideration the individual's genetic makeup and not just their CFTR mutations. A personalized approach will optimize patient's clinical outcomes by accounting for the specific genetic mutations of the individual patient.

#### TOWARD PERSONALIZED MODULATOR THERAPY

One of the major hurdles to the development of novel treatment regimens in CF is the bench-to-bedside translation of scientific knowledge. Many drugs that perform well in a laboratory setting fail to advance in clinical trials, largely due to inappropriate selection of in vivo and in vitro models for HTS. In the context of CF, animal models and immortalized epithelial cell lines do not fully portray patient-specific disease phenotypes. Animal models of CF have provided insights into disease pathophysiology. However, their generation is time consuming, expensive, and more importantly they poorly represent the full repertoire of disease manifestations in individual patients. For instance, mouse models of CF do not have lung disease and bacterial infections, attributed to the compensatory effect of a secondary Cl− ion channel (Lavelle et al., 2016). On the other hand, immortalized epithelial cell lines derived from primary patient material have contributed tremendously to CF research, but not without limitations. Their generation from primary patient material is very inefficient. It involves extensive adaptation and selection to in vitro 2-D culture conditions, as only rare clones are able to expand and be maintained over many passages. Furthermore, these cell lines may have undergone substantial genetic changes and no longer retain features of the original parental cells. Drug development pipeline for new CFTR-directed compounds have relied heavily on unpolarized Fischer rat thyroid (FRT) epithelial cell lines. It is thus not surprising that this model has a higher propensity of false-positive and false-negative "hits" compared to that performed in primary human bronchial epithelial (HBE) cells.

### PATIENT-DERIVED ORGANOTYPIC CELLULAR MODELS FOR PERSONALIZED MEDICINE

Although CF is a monogenic disease, the diverse mutation variants identified within the CFTR gene as well as the presence of modifier genes (known and unknown), warrants adoption of new technologies to extend research capabilities. There is a clear unmet need for a representative library of patient-specific epithelial cell models for disease modeling, preclinical testing of drug response, and biobanking for future drug discovery. The cell models can be derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or tissue-resident adult stem cells. Whereas, ethical concerns pertaining the source of human ESC, limit their use in research, generation of CFTR gene corrected iPSC lines enable disease modeling, drug discovery and toxicology studies [reviewed in (Pollard and Pollard, 2018)]. In this Review, we focus on airway and intestine epithelium models derived from adult stem cells.

## Human Airway Epithelial (HAE) Cells

The pulmonary epithelium is divided into three regions; upper (nasal and oral cavities), lower (trachea and primary bronchi), and distal small airway epithelia (alveolar). Persistent inflammation, bacterial colonization, and airway structural changes in CF occur in the lower respiratory tract. Primary HBE cells are therefore the gold standard for studying CF disease pathogenesis and evaluating CFTR functional response (Van Goor et al., 2006; Awatade et al., 2015). HBE cells can be isolated from biopsy samples, lung explants and cadavers. Explanted lung and post-mortem samples from individuals with CF provide high cell yield. However, the extensive damage to the tissue, particularly the epithelial cell layer, plus the presence of chronic microbial colonization present technical challenges to establishing successful ex vivo cultures. Acquisition of airway biopsies or brushings involves highly invasive procedures, while bronchoalveolar lavage (BAL) fluid and induced sputum samples usually do not provide sufficient cell counts to initiate culture. Therefore, supply of CF patient-derived HBE cells are often limited and hard to come by.

Human nasal epithelial (HNE) cells are increasingly used as surrogates for the lower airway epithelium in CF research (de Courcey et al., 2012; Brewington et al., 2018a). HNE cells demonstrate many characteristics common to HBE cells including the ability to form polarized, pseudostratified epithelium mimicking in vivo airways and the expression of ion channels such as CFTR. Their advantage is the lack of need for invasive procurement (Clarke et al., 2013). HNE cells are grown using the same culture media and protocol as for HBE cells.

#### Human Airway Epithelial (HAE) Cell Culture Models

Primary human airway epithelial cells are conventionally cultured as monolayers (2-D cultures). Expansion of epithelial cells is often necessary in the initial passages for biobanking purposes and to generate enough cell numbers needed to differentiate cells under air-liquid interface (ALI) conditions. HBE cells are most "in vivo" like when fully differentiated; they display a striking phenotypic resemblance to the lower airway epithelium. They form pseudostratified epithelium with mucociliary differentiation indicated by the presence of functional beating cilia and mucus secretion. They also exhibit characteristic epithelial barrier functions, including expression of cell junctional proteins and the development of robust transepithelial electrical resistance (TEER) values (Berube et al., 2010). The polarized, differentiated phenotype is critical for in vitro measurement of CFTR function as the protein is primarily expressed at the apical surface of ciliated cells. While ALI cultures accurately represent in vivo phenotypes, their wider use is deterred by limited propagation of cells in culture, attributed to squamous transformation and cellular senescence (Gentzsch et al., 2017). To date, three expansion methods have been used to extend the lifespan of cells and delay cellular senescence beyond that of the standard cultures.

#### Improved Cell Expansion Culture Methods

Conditionally reprogrammed cells (CRCs) are generated by coculturing patient-derived airway epithelial cells with irradiated fibroblast feeder cells. Specialized conditioned media (termed F-Media) which contains Rho-associated protein kinase (ROCK) inhibitor, promotes serial passages of airway epithelial cells and enhances population doubling without compromising the characteristic epithelial cell morphology. Both ROCK inhibitor and the feeder layer are essential in maintaining the stem cell-like phenotype evident in CRCs (Reynolds et al., 2016; Martinovich et al., 2017). Their removal results in differentiated cell lines, with intact barrier function and the ability to polarize and form mucociliary epithelium. CFTR-mediated Cl− transport in these cells is also preserved.

A modified CRCs protocol using BEGM, ROCK inhibitor and an irradiated feeder layer cultured in reduced oxygen concentration (2%) demonstrated some advantage over the standard CRCs. The modified CRCs could support airway epithelial cell growth up to passage 10 with robust formation of pseudostratified epithelium at the extended passage, although a modest decrease in CFTR-dependent Cl− transport was observed. Meanwhile, reduced numbers of ciliated cells and goblet cells were observed in standard CRCs cultures at P10 and robust TEER could not be established (Peters-Hall et al., 2018).

A feeder-free culture protocol that relies on disruption of SMAD signaling pathway through inhibition of dual ligands, transforming growth factor-beta (TGF-β) and bone morphogenic protein (BMP), showed airway epithelial cells could be expanded up to 18–25 passages without loss of proliferative potential (Mou et al., 2016). Combined TGF-β/BMP inhibition led to basal cell hyperplasia with hyper-proliferative p63 cells (basal cell marker) and produced homogenous, tightly packed cells, resembling stem cell morphology. Epithelial cells expanded using dual SMAD inhibition method can undergo mucociliary differentiation up to P12 with preserved TEER although Na+ currents and Cl− conductance declined fairly rapidly after serial passage 6 (Mou et al., 2016).

#### Three-Dimensional Airway Spheroids and Organoids

Two-D cultures lack a third dimension, the scaffolding extracellular matrix (ECM), which establishes intercellular cues or network in the in vivo airway epithelium. Therefore, 3-D cell cultures are a major area of development, where cells are cultured in a matrix (such as matrigel) or are cultured in such a manner that they develop ECM-like scaffolds between them, thus mimicking the in vivo phenotype more faithfully. While cell-derived ECMs such as Matrigel have been most commonly used for development of organoids, their undefined components introduce inconsistency in replicating the native 3-D culture environment (Czerwinski and Spence, 2017). To overcome this challenge, new biomaterial systems, such as polymers and hydrogels are being developed [reviewed in (Dye et al., 2017)].

Different methods for generating organoids (or spheroids) from human lung cells have been described to date, each producing organoids with distinct definitive structure and cellular compositions. Barkauskas and colleagues have elegantly reviewed the different lung organoids (airway organoids inclusive) established from varying lung progenitor cell populations, including basal cells in the proximal airways, secretory club Clara cells in bronchioles and alveolar type II cells in the alveoli, as well as those from embryonic and iPSCs (Barkauskas et al., 2017). Given CFTR is primarily expressed at the apical surface of ciliated cells and recently discovered in the pulmonary ionocytes in tracheal epithelium (Montoro et al., 2018; Plasschaert et al., 2018), only airway organoids displaying proximal differentiation are discussed here in light of their relevance for measuring CFTR functional response (**Table 2**).

#### Three-Dimensional Airway Explant Spheroid (ECM—Free)

Airway spheroid cultures were first described in 1991 when nasal epithelial multicellular spheroids were generated from non-dissociated nasal epithelial sheets (Bridges et al., 1991). These explant spheroids were maintained in culture media (no matrigel) and were reported to form rapidly, within 2–5 days. Explant spheroids have an apical-membrane-out orientation; apical ciliated cells face the media bath and the basolateral membrane lines the fluid-filled lumen (Bridges et al., 1991). Pedersen and colleagues later found amiloride-sensitive Na+ transport drives changes in the lumen size, suggesting nasal spheroids may be a useful model for ion/fluid transport studies and for investigating diseases associated with defective ion channels such as cystic fibrosis (Pedersen et al., 1999). Nasal spheroids were also responsive to forskolin stimulation (Guimbellot et al., 2017). As the CFTR-expressing apical side of nasal spheroids face outward, forskolin causes the outflow of fluid from the lumen to the exterior bath and the spheroids to shrink in size. This response was blunted in spheroids established from CF patients but could be partially rescued by CFTR modulators (Guimbellot et al., 2017). Notably, successful spheroids have also been established from bronchial brushings, although CFTR function was not tested in the study (Deslee et al., 2007). Interestingly, no evidence of cell proliferation was observed throughout the nasal spheroid formation process and the spheroids plateaued in size once fully formed. This suggests that the spheroids are self-organizing aggregates of terminally differentiated epithelial cells only (Castillon et al., 2002). While spheroids may be ready for ex vivo CFTR measurement rapidly, they are short-lived, lasting for up to 12 weeks only and biobanking for future drug screening is not possible (Guimbellot et al., 2017).

#### Three-Dimensional Spheroid From Airway Basal Progenitor Cells

Airway spheroids, both bronchial and nasal can also be derived from primary airway basal cells embedded in the

#### TABLE 2 | Comparison of three-dimensional organotypic airway epithelial culture methods.

2D, Two-dimensional; ALI, Air-liquid interphase; BAL, Bronchoalveolar lavage; BEGM, Bronchial epithelial cell growth media; ECM, Extracellular matrix; FGF, Fibroblast growth factor; TGF-β, Transforming growth factor-beta.

basement membrane matrix matrigel (Hild and Jaffe, 2016; Brewington et al., 2018c).  Different culture protocols using varying concentrations of matrigel have been reported; (1) cells are resuspended in 100% matrigel and seeded as spherical drops, then overlaid with ALI differentiation media; (2) cells are resuspended in 5% matrigel with ALI differentiation media and then layered over a denser 25–40% matrigel base layer which the cells sink into (Hild and Jaffe, 2016; Tan et al., 2017; Brewington et al., 2018c). Method 1 (100% matrigel) was reported to yield the best success, as the sinking method could result in formation of some spheroids with cell-apex-out morphology and disorganized cellular aggregates (Brewington et al., 2018b), likely due to incomplete embedding of cells in the denser matrigel base layer e.g., growing on top of TABLE 3 | Comparison of human cell models relevant for CF therapeutic application.

+ + +, High; ++, Moderate; +, Low; 2-D, 2-Dimensional; ALI, Air-liquid interphase; ASL, Airway surface liquid; CBF, Cilia beating frequency; FIS, Forskolin-induced swelling; HBE, Human bronchial epithelial; ICM, Intestinal current measurement; SLA, Steady-state lumen area.

or at the interface of the denser matrigel base layer. For certain downstream applications such as live imaging (e.g., forskolin-induced swelling—see section The Cell Model of the Future), the sinking method confers an advantage because spheroids are suspended in the same plane and permit HTS. We also note that successful differentiated, pseudostratified spheroid formation has been reported from airway basal cells maintained in the standard BEGM media and those derived from conditionally reprogrammed culture (Hild and Jaffe, 2016; Brewington et al., 2018c). Airway basal cell spheroids have no self-renewal capacity and are used for end-point applications.

#### Three-Dimensional Airway Organoid—Long-Term Expanding

Long term expanding human airway organoids were first reported by Sachs et al. (Sachs et al., 2018). These organoids were established from lung biopsies or cells isolated from BAL fluid and then cultured in media containing biochemical cues for self-renewal such as R-spondin, Noggin, fibroblast growth factor (FGF) and TGF-β inhibitors. The established organoids comprised of a polarized, pseudostratified airway epithelium containing basal cells, ciliated cells, mucus-producing goblet cells, and secretory club (Clara) cells. The airway organoids are amenable to passaging by mechanical disruption every other week for at least a year, with no loss in proliferation reported up to P18. In addition, single cell suspensions dissociated from the indefinitely expanding airway organoids yield an improved 2-D ALI culture (Zhou et al., 2018). These cultures displayed full ALI differentiation by day 12–14 as opposed to 21–28 days for conventional cultures grown from 2-D basal cells (Zhou et al., 2018). Together, these findings suggest the possibility of expanding isolated primary lung epithelial cells in 3-D, given cells maintained under conditionally reprogrammed and dual SMAD inhibition culture conditions do not expand beyond P10 without reaching senescence (Mou et al., 2016; Gentzsch et al., 2017). The indefinite expansion of airway organoids also means in theory the availability of "endless" amounts of ready-to-use airway epithelial cells.

#### Human Intestinal Epithelial Cells

While work in CF research has primarily focused on primary airway epithelial cells (the gold standard), patient-derived intestinal epithelial cells also present an invaluable resource in characterizing the relationship between the CFTR gene mutation and the disease phenotype. More recently, they play an important role in the development of HTS strategies to elucidate novel drugs for the treatment of CF.

#### Intestinal Organoids

Compared to human airway tissue, colon tissue damage in CF patients is minimal and the rectal epithelium is accessible in a less invasive manner. The abundant expression of CFTR in the distal colon makes rectal biopsies an attractive cellular source for interrogating CFTR function (Hug et al., 2011). Assessment of intestinal current measurement (ICM) as a readout for CFTR activity has provided strong evidence for the diagnostic and prognostic utility of rectal biopsies (De Jonge et al., 2004; Hirtz et al., 2004; Mall et al., 2004; De Boeck et al., 2006; Taylor et al., 2009; Derichs et al., 2010; Sousa et al., 2012; Clancy et al., 2013; Cohen-Cymberknoh et al., 2013). ICM can be applied to biopsies collected from CF patients which are subsequently exposed to CFTR modulators, to assess the efficacy of treatment in a personalized manner. One concern is the possibility of reduced penetration of modulator drug into the biopsy tissues under ex vivo conditions. Other limitations to the use of rectal biopsies include the small number of biopsies collected (4–8 biopsies) and the need for all biopsies to be tested on collection day i.e., the biopsies cannot be preserved for further testing.

Emergence of intestinal organoids (or mini-guts) overcome these limitations by extending the use of rectal biopsies in cultures. Intestinal organoids can be grown from crypts isolated from freshly isolated rectal biopsies. Crypts are rich in Lgr5+ stem cells which grow and differentiate into selforganizing, multicellular structures (Sato et al., 2009; Jung et al., 2011). These organoids contain all of the distinct intestinal cell types present in the in vivo epithelium (Leushacke and Barker, 2014). The growth and differentiation of Lgr5+ stem cells into eventual closed epithelial structures with an internal lumen requires a fine balance of growth factors (Rspondin, EGF, Noggin, Wnt-3a), inhibitors of TGF-β and BMP signaling and the basement membrane matrix (matrigel). Intestinal organoids can be indefinitely cultured and remain genetically and phenotypically stable upon repeated passaging and long term culture (Ikpa et al., 2014). Capitalizing on the high expression of CFTR in rectal tissues and rapidly expanding stem cells, they make an attractive model for assessment of CFTR functional response in pharmacologic testing. They provide the added advantage to test combination of modulators without established safety profiles as part of pre-clinical evaluation in CF patients with rare CFTR genotype.

#### FORSKOLIN-INDUCED SWELLING ASSAY AS A PROXY FOR CFTR FUNCTION

The primary functional assay to assess CFTR activity in organoids, the forskolin-induced swelling (FIS) assay is CFTR dependent. This approach does not measure the net transepithelial ionic transport. Rather, forskolin is used to stimulate intracellular cAMP production which then activates CFTR at the plasma membrane. CFTR activation drives chloride and fluid flux to the organoid lumen (apical membrane facing inwards), causing rapid swelling of organoids with functional CFTR or those derived from non-CF subjects. This swelling effect is significantly reduced or absent in organoids derived from CF subjects who exhibit differing rates of FIS with different classes of CFTR mutations and also between individuals with the same CFTR mutation (Dekkers et al., 2013). There is accumulating evidence that the CFTR modulator-corrected FIS response is predictive of patient-specific clinical response, with close correlation observed between rescued swelling and improvement in lung function (measured by FEV1) and sweat chloride concentration (Dekkers et al., 2016a; Guimbellot et al., 2017; Brewington et al., 2018c). Intestinal organoids can be disrupted to single cells to generate 2-D-monolayers on porous membranes for electrophysiological studies. FIS was shown to positively correlate with forskolin-induced current in subject-matched organoid-derived monolayers, supporting the notion that CFTR-dependent fluid secretion in rectal organoids reflects CFTR-dependent ion transport (Zomer-van Ommen et al., 2018)

The FIS assay has been adapted for use in airway organoids but is much less well-characterized compared to rectal organoids (Guimbellot et al., 2017; Brewington et al., 2018c; McCarthy et al., 2018). Studies performed thus far, have involved cultures from small datasets of patients. Given the lack of standardized culture protocol for airway organoids, forskolin induces either swelling or shrinking of airway organoid depending on the orientation of CFTR-expressing apical epithelium (swelling for apical membrane facing inwards organoids and vice versa). Similar to rectal organoids, forskolin-induced changes in cross sectional area is dependent on the severity of CFTR genotypes, and could be altered with CFTR modulators. However, the changes are far smaller given the lower expression of CFTR in the airways compare to the intestine. It is noteworthy that only a single concentration of forskolin (10µM) has been interrogated in airway organoids FIS, while a total of eight concentrations (0.008–5µM) were used in rectal organoids (Dekkers et al., 2016a; Guimbellot et al., 2017; Brewington et al., 2018c). To detect CFTR rescue with adequate resolution and sensitivity, higher-powered objectives are used resulting in lower throughput application compared to rectal (Dekkers et al., 2016a; Guimbellot et al., 2017; Brewington et al., 2018c). Establishment of a broader assay dynamic range may facilitate higher throughput use of FIS in airway organoids. Although preliminary, favorable correlation between in vitro FIS response to clinical response FEV1, sweat chloride concentration and BMI have been reported (Brewington et al., 2018c; McCarthy et al., 2018).

## THE CELL MODEL OF THE FUTURE

In this Review, we described the rapidly developing field of organoid models specific to CF. Organoids, with their close resemblance to the human organs most affected by CF disease, hold great appeal for translational research. The capacity to adapt these models for assays such as FIS is important for theratyping and for conferring decisions on personalized CFTR pharmacotherapy. This provides an almost immediate application of in vitro research findings in the clinical setting. Nonetheless, it is apparent that each model has respective strengths and limitations (**Table 3**). The question as to which model has the essential features for precision medicine in CF and best predicts the long-term clinical benefits of a drug remains. Is it sufficient to use just one model and if so which cell model is best?

#### REFERENCES

Intestinal organoids are the most developed so far amongst the 3-D model systems and seemingly an easier model to establish. But how representative is the intestinal epithelium to its respiratory counterpart? There are clear physiological differences between the airway mucosal surface and the gut. First, alternative ion channels critical for solute and water transport in the airways such as ENaC and calcium-activated chloride channels (CaCC), are either absent or present in negligible amounts in the gut to be functional (Rajendran et al., 2018). A modified swelling assay showed airway but not rectal organoids swell upon addition of Eact, an activator of the alternative chloride channel TMEM16A (Sachs et al., 2018). Second, there is no mucociliary clearance in the gastrointestinal tract. These differences highlight the need for organoids of bronchial or nasal epithelial origin to provide a closer resemblance to the in vivo airways. It then seems logical that one will need to choose the most appropriate 3-D cell culture model for each specific application.

# AUTHOR CONTRIBUTIONS

SAW, NTA, and SLW wrote the manuscript with critical input from CKH, LKF, AK and AJ.

# FUNDING

SAW, NTA, and SLW are supported by Sydney Children's Hospitals Foundation (SCHF). AK holds a Rothwell Family Fellowship.

**Conflict of Interest Statement:** AK is a scientific advisor for Unizyme Laboratories A/S.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Awatade, Wong, Hewson, Fawcett, Kicic, Jaffe and Waters. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# In vitro Methods for the Development and Analysis of Human Primary Airway Epithelia

Ambra Gianotti† , Livia Delpiano† and Emanuela Caci\*

U.O.C. Genetica Medica, IRCSS Istituto Giannina Gaslini, Genoa, Italy

Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.

Keywords: cystic fibrosis, bronchus, airway epithelial cells culture, short-circuit current recording, transepithelial electrical resistance measurement, periciliary mucus properties

# INTRODUCTION

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene coding for the protein cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a chloride channel expressed at the apical surface of secretory epithelia where it plays an important role in salt and fluid homeostasis (Saint-Criq and Gray, 2017).

The consequences of mutated CFTR are most important in the airways. The resulting defective chloride secretion, coupled to enhanced sodium absorption, impairs normal mucociliary

#### Edited by:

Miquéias Lopes-Pacheco, Universidade de Lisboa, Portugal

#### Reviewed by:

Kavisha Arora, Cincinnati Children's Hospital Medical Center, United States Guido Veit, McGill University, Canada

\*Correspondence:

Emanuela Caci<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Pharmacology of Ion Channels and Channelopathies, a section of the journal Frontiers in Pharmacology

Received: 01 August 2018 Accepted: 28 September 2018 Published: 26 October 2018

#### Citation:

Gianotti A, Delpiano L and Caci E (2018) In vitro Methods for the Development and Analysis of Human Primary Airway Epithelia. Front. Pharmacol. 9:1176. doi: 10.3389/fphar.2018.01176

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clearance due to dehydration of the airway surface and consequently causes uncontrolled inflammation and chronic bacterial infections (Castellani and Assael, 2017).

Recently, bicarbonate has emerged as another important anion that contributes to the genesis of CF lung disease due to its role in mucus release (Quinton, 2008; Borowitz, 2015).

Over the last years, basic research has sustained a great effort to define a strategy to identify novel therapies that correct the basic defects responsible for CFTR loss-of-function.

Studies have demonstrated that pharmacological rescue of the CFTR protein, in the presence of small molecules called correctors and potentiators, can repair folding and gating defects (Pedemonte et al., 2005; Okiyoneda et al., 2013; Van Goor et al., 2009, 2011).

The most successful compounds, ivacaftor and lumacaftor developed by Vertex Pharmaceuticals company, have been approved for treatment of CF patients (Wainwright et al., 2015).

Drug discovery is supported by in vitro cell models that offer the opportunity to study epithelium physiology, the alterations caused by mutated CFTR, and the efficacy of therapies that aim to correct basic CF defects.

In vitro cell models for airway epithelium studies have been mostly based on primary human bronchial epithelial cells (hereafter termed HBEC), obtained from CF lung resection.

HBEC provide the ideal tool since they exhibit several of the morphological and functional defects of airway epithelia. Despite their value, it is difficult to acquire a large amount of cells and they can only be grown for 4–5 passages before reverting to a poorly differentiated phenotype.

Primary nasal epithelial cells (hereafter termed HNEC) have been recently proposed as an alternative method to HBEC culture.

HNEC are easy to collect by nasal brushing and recapitulate the properties of HBEC cultures (McDougall et al., 2008; Mosler et al., 2008). Furthermore, this model is very useful to predict the clinical treatment efficacy in patients (Pranke et al., 2017).

Several research groups have optimized protocols that allow isolation, expansion, and differentiation of primary HBEC and HNEC (Fulcher et al., 2005; Yaghi et al., 2010; Neuberger et al., 2011; Müller et al., 2013; Stokes et al., 2014).

Primary cells have a very limited proliferative capacity in vitro and it is possible to culture for at least five-six passages before noting a slowing down of cell growth. To overcome this problem, culturing of bronchial and nasal epithelial cells under CRC conditions, namely irradiated feeder cells and the RhoA kinase inhibitor Y, enhances cell growth and lifespan while preserving electrophysiological and morphological properties (Gentzsch et al., 2017).

The aim of this article was to present a detailed protocol, optimized in our laboratory, for culture and differentiation of airway epithelia. This method results in large-scale production of isolated HBEC and HNEC and fully differentiated epithelia that exhibit the morphological and functional defects of CF airways. Therefore, these cell models are very useful for improving our knowledge about physiopathology mechanisms involved in CF and to support therapeutics strategies.

Our approach is based on the isolation of airway cells from bronchi or nasal brushings obtained from CF and non-CF subjects. Then, isolated cells are cultured and expanded with a high proliferation rate using a proliferative serum-free medium. This step is followed by epithelial cell differentiation on permeable supports, whose ion transport properties can be evaluated with electrophysiological techniques (Galietta et al., 1998; Scudieri et al., 2012).

For this purpose, we review two powerful methods for ion transport measurements underlining the application, advantages, and limits. These include Ussing chamber and Trans-Epithelial Electrical Resistance (TEER) techniques (Li et al., 2004; Srinivasan et al., 2015).

Moreover, we describe a cell culture protocol to achieve a fully differentiated mucociliary airway epithelium to study the properties of periciliary mucus considering its important involvement in the CF pathology. Indeed, the reduction of fluid secretion in CF alters the composition of the airway surface fluid (ASL) and induces the production of mucus with rheological properties making it inadequate for normal physiology (Gianotti et al., 2016).

Finally, our culture protocol allows morphological and functional characteristics of the airway epithelium to be reproduced in vivo, supporting the study of CF physiopathology and therapeutic strategies.

# MATERIALS AND EQUIPMENTS

#### Cell Culture

TABLE 1 | Proliferative serum-free medium.

TABLE 2 | Differentiation serum-supplemented medium.

• Differentiation serum-free medium: See **Table 3** and **Supplementary Materials**. Sterilize with a filter and store at +4 ◦C for no longer than 3 weeks. This medium cannot be frozen, freezing seems to destroy ability to induce differentiation and the appearance of electrical resistance. Protect from light with aluminum foil.

#### Electrophysiological Equipment

TABLE 3 | Differentiation serum-free medium.

#S7139), UTP (Sigma #U6625 ), ATP (Sigma #A9187), PPQ-102 (Sigma #219677).

## STEPWISE PROCEDURES

fphar-09-01176 October 24, 2018 Time: 15:1 # 4

#### Primary HBEC and HNEC Culture

In this step, we describe a cell culture protocol which can provide, from a relatively little amount of sample, a large number of polarized bronchial and nasal epithelia useful for the study of airway physiopathology and predicting in vivo drug efficacy.

This method is based on two distinct phases:

	- serum-supplemented medium condition: cells differentiate into fully polarized monolayers of airway epithelium in order to perform electrophysiological experiments.
	- serum-free medium condition: cells form a fully differentiated mucociliary airway epithelium useful for the study of periciliary fluid properties of airways (Hajj et al., 2007).

#### Isolation and Expansion of Primary HBEC Derived From CF and Non-CF Donors Following Lung Transplantation

FIGURE 1 | Detached epithelial cells isolated from bronchus. Bronchus is placed in a 100-mm Petri-dish and epithelial cells are collected by flushing the bronchial lumen vigorously with physiological saline solution.

To overcome fungal contamination, treat the medium with 2.5 µg/ml of amphotericin B for at least the first 7 days of culture.

#### Isolation and Expansion of Primary HNEC Derived from CF and Non-CF Donors Following Nasal Brushing

1. Collect HNEC by nasal brushing of both nostrils. The nasal brushing is performed without local anesthesia after nasal washings with physiological saline solution, in order to remove the mucus (two washes a day in the previous week and a wash immediately before nasal cells collection).

#### Generation of Differentiated Nasal and Bronchial Epithelia With Serum-Supplemented Medium

1. Culture and detach HBEC and HNEC on the fourth passage when the cell density reaches 90% confluence.

#### Generation of Differentiated Nasal and Bronchial Epithelia With Serum-Free Medium

#### HBEC and HNEC Monolayers Application

Well differentiated HBEC and HNEC epithelia are useful to support the following studies:

#### Important Remarks

FIGURE 2 | Epithelial cell culture density in the proliferative phase. Epithelial cells with a good morphology and a high proliferation rate (A). Epithelial cells with an increasing number of large squamous cells and decreased cell growth (B).

	- Detach cells at the proper growth density.
	- Seed the proper amount of cells on a permeable support.
	- Replace differentiation medium every day.

# Recording Transepithelial Ion Transport

The airway epithelium balances Na+ absorption and Cl− secretion to carefully regulate the properties of the ASL in order to ensure efficient mucociliary clearance.

Here we present electrophysiological approaches to perform transepithelial ion transport measurements. Moreover, we highlight the applications, the strengths, and limitations of the techniques.

#### Short-Circuit Current Recordings Using the Ussing Chamber Technique

The Ussing chamber provides a system to measure the transport of ions across epithelial tissues.

Although it can be complicated to perform, it is considered the gold standard for electrophysiological experiments with the aim of studying epithelial transport.

In the most used configuration, the short-circuit current (Isc) technique, the transepithelial potential difference is clamped at zero with a voltage clamp amplifier and the resulting current is recorded.

The Ussing system consists of two hemi-chambers separated by the monolayer of epithelial cells grown on Snapwell supports, a perfusion system, an amplifier, and a data acquisition system.

Both hemichambers are fill with a Ringer's solution, continuously bubbled with a gas mixture containing 5% CO2 −95% air that maintains the physiological pH. The temperature of the system is kept at 37◦C. The voltage-clamp is connected to the apical and basolateral chambers with Ag/AgCl electrodes and agar bridges (two for voltage and two for current). The short-circuit current is recorded with a PowerLab 4/25 analog-to-digital converter connected to a computer.

A typical experiment is conducted as follows:

#### Ussing Chamber Technique Application

Using ex vivo airway epithelia, this technique gives a representation of the functional characteristics and processes involved in the physiological environment.

3. Evaluation of therapeutic interventions.

#### Important Remarks

fphar-09-01176 October 24, 2018 Time: 15:1 # 8

Agar salt bridges represent another source of contamination. We suggest that the bridges be replaced at the end of each experiment.

#### Electrical Epithelial Properties Measured With the Trans-Epithelial Electrical Resistance (TEER) – Potential Difference (PD) Technique

TEER/PD is a rapid and simple method to quantify ion transport by measuring the electrical resistance and potential difference across the epithelium. TEER/PD is dependent on the expression/activity of channels situated on both apical and basolateral membranes as well as on the properties of tight junctions that control paracellular ion transport.

TEER/PD measurements are performed using the EVOM2 system and placing Ag/AgCl "chopstick" electrodes on both sides of an epithelial monolayer cultured on HTS Transwell permeable supports.

The method is simply based on the separate measurement of TEER and PD values for each epithelium, under resting conditions and following the application of agonists and inhibitors. The equivalent short-circuit current is then calculated from TEER and PD.

Considering the simplicity of the measurement and the data reproducibility compared with the Ussing chamber method, TEER/PD measurements are a good benchmark tool for high-throughput drug discovery screening in CF epithelia.

#### Important Remarks

1. In the TEER method, CFTR-inh172 does not significantly reduce conductance. We have tested different inhibitors and we have observed that PPQ-102 is the best one for completely blocking CFTR activity (Tradtrantip et al., 2009).

#### Fluid Absorption Rate Measurements

In CF airways defective ion transport causes a simultaneous reduction of fluid, chloride, and bicarbonate secretion. The altered properties of mucus may be studied in order to understand the pathological mechanisms and to evaluate treatments for recovering hydration of the airways.

Epithelia with serum-free medium reproduces ASL periciliary characteristics with mucus and fluid secretion.

In this section, we present a protocol that allows the measurement of fluid absorption rate.

Vf = (Wf-Wi) × 000/N. Where Wf = tare and collected fluid weight; Wi = tare weight; N = number of pool samples.

8. Calculate fluid transport rate: Jw = (Vi−Vf)/A × t. Where Vi = initial apical volume (130 µl); Vf = final apical volume; A = area of the epithelium (for Snapwell insert: 1.13 cm2 ); t = time interval.

#### Important Remarks

# ANTICIPATED RESULTS

To overcome the scarcity of biological material available for primary culture, our group has developed a protocol to extract primary airway epithelial cells from lung resections or nasal brushing and subsequently culture and amplify these cells, maintaining most of the characteristics of the airway epithelium in vivo.

FIGURE 3 | Collection of fluid and mucus secreted from differentiated epithelia. Periciliar fluid collection from surface epithelia after D-PBS incubation.

non-CF bronchial epithelia differentiated with serum-supplemented medium (A) or with serum-free medium (B). Cilia are labeled in magenta (anti-tubulin antibody), the goblet cells in red (anti-MUC5AC antibody), the apical membrane in green (anti-TMEM16A antibody) and the nucleus in blue (Hoechst dye). Images kindly provided by Dr. Paolo Scudieri (TIGEM, Pozzuoli, Italy).

The procedure starts by using a proliferative medium that promotes cell growth at a low density and a high proliferation rate in order to obtain a large number of polarized bronchial and nasal epithelia useful for the study of CF physiopathology.

This is followed by a differentiation phase. We have presented two protocols that both require seeding cells on permeable supports and the "ALI" condition, but they differ depending on the final aim of the designed experiments.

The serum-free medium protocol provides a fully

differentiated epithelial monolayer that is useful for evaluating morphological and periciliary fluid properties in primary airway epithelia. However, a critical point of this protocol is the prolonged culture time and the cost of medium supplements.

Instead, we suggest that the serum-complemented medium be applied to the protocol for functional electrophysiologic assays, because it still provides a differentiated monolayer with transepithelial ion transport properties similar to in vivo conditions, but in a shorter time and with lower costs compared to the serum-free medium protocol. Therefore, this medium is optimal for functional electrophysiologic assays.

To compare the two protocols for evaluating epithelial morphological properties, we have used immunofluorescence combined with confocal microscopy. This technique provides high-resolution imaging of protein localization, expression patterns, and different cell types that characterize the airway of mature epithelium.

Cultured bronchial epithelia are analyzed by immunofluorescence for acetylated tubulin and MUC5AC to identify ciliated and goblet cells, respectively. To distinguish the apical from the basolateral membrane we have used an antibody against the ZO-1 tight junction marker.

The derived staining shows that epithelium differentiated with serum-supplemented medium is pseudo-stratified

and CFTR activation and inhibition is obtained by adding CPT-cAMP and CFTRinh-172, respectively. The Ca2+- dependent chloride channel is

epithelia. Representative traces from short circuit current (Isc) recordings of homozygous F508del-CFTR epithelia treated with vehicle (A) and lumacaftor (B). The ENaC channel is blocked with apical amiloride and CFTR activation and inhibition is obtained by adding CPT-cAMP plus ivacaftor and CFTRinh-172, respectively. (C) Graph bars summarizing CFTR-mediated currents from Ussing chamber recordings of HBEC derived from 4 non-CF donors (n = 22) and from 4 homozygous F508del-CFTR patients (n = 41). Mean values ± SEM are shown, ∗ and ∗∗ indicate p < 0.05 or p < 0.001, respectively.

with a relatively small number of ciliated and goblet cells (**Figure 4A**).

On the contrary, when using the serum-free medium protocol we obtained a higher pseudo-stratified epithelium characterized by a higher number of ciliated and goblet cells (**Figure 4B**). This result demonstrates that the latter culture condition provides most of the morphological characteristics of the airway epithelium in vivo, thus we suggest that it be used to analyse the expression and localization of selected proteins and airway cells.

In the CF lung, the decrease in CFTR-mediated chloride and fluid secretion are believed to cause the dehydration of the airway surface by altering the properties of periciliary mucus. To gather significant information, we have applied the serum-free medium protocol, that reproduces ASL periciliary characteristics with mucus and fluid secretion, to analyse the rate of fluid absorption of non-CF and CF airway mucus recovered from cultured HBEC.

Time course experiments confirmed that the different activities of CFTR in non-CF and F508del-CFTR epithelia resulted in significant differences in the net absorption rate after 24 and 48 h; reflecting the different ASL characteristics.

stimulated with UTP.

While non-CF epithelia absorbed after 24 and 48 h 1.67 ± 0.15 µl/h/cm2 (n = 5) and 1.06 ± 0.06 µl/h/cm2 (n = 7), respectively, in F508del-CFTR epithelia net absorpion rate was 2.25 ± 0.19 µl/h/cm2 (n = 5) and 1.58 ± 0.07 µl/h/cm2 (n = 7) (**Figure 5**).

Moreover, during recovery we observed that the CF fluid is more viscous and difficult to pipette compared to non-CF.

The development and validation of CF-HBEC aims to support drug discovery efforts to correct basic CF defects. For this purpose, electrophysiological approaches allow transepithelial ion transport to be measured in order to assess the efficacy of pre-clinical CFTR modulators in primary human airway cultures.

We have used the Ussing chamber technique to measure short-circuit current as an indication of net ion transport across bronchial and nasal epithelia monolayers maintained in serum-supplemented medium.

**Figure 6A** shows a representative trace experiment of short-circuit currents of differentiated non-CF bronchial epithelia. The basal current was inhibited by amiloride, indicating the presence of epithelial Na+ channel (3.1 ± 0.6 µA/cm2 ; n = 25). To assess the amount of CFTR-dependent Isc, cells are blocked with CFTRinh-172 after maximum CFTR activation using CPT-cAMP. The resulting chloride current is strongly sensitive to CFTRinh-172 (10.08 ± 0.04 µA/cm2 ; n = 31). Application of UTP generates a very fast calcium-activated chloride current (3.8 ± 1 µA/cm2 ; n = 29).

Primary human nasal monolayers recapitulate the transepithelial ion transport properties of bronchial cells, confirming the fact that this cell model can predict clinical treatment efficacy in patients (**Figure 6B**).

To further support the conclusion that our culture protocol allows F508del-CFTR-HBEC to reflect the ion transport defects in airway epithelia in vivo, we used cells from patients that were homozygous for this mutation; which causes a severe defect in CFTR function. As expected the response to CPT-cAMP and ivacaftor is markedly reduced in negative control cells (1.04 ± 0.023 µA/cm2 ; n = 37) (**Figure 7A**). However, we have noted that lumacaftor treatment for 24 h induced a significant increase in chloride transport, as indicated by the current blocked by CFTRinh-172 (3.67 ± 0.07 µA/cm2 ; n = 41) (**Figure 7B**).

**Figure 7C** summarizes the CFTR-mediated currents from Ussing chamber recordings of HBEC derived from 4 non-CF donors and 4 homozygous F508del-CFTR patients. Transepithelial current measurements showed that there was a significant difference in the CFTR-mediated Isc in cultured F508del-HBE cells compared to non-CF HBE cells, and incubation of CF epithelium with lumacaftor led to a significant improvement in CFTR-dependent chloride secretion.