# Cyanine 5-huwentoxin-IV as a novel imaging probe to detect hNa_v1.7 channel overexpressed in non-small cell lung cancer

Romain Baudat, Jérôme Montnach, Evelyne Benoit, Claude Zoukimian, Cathy Carvalhosa, Rémy Béroud, Michel De Waard, Denis Servent

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## To cite this version:

Romain Baudat, Jérôme Montnach, Evelyne Benoit, Claude Zoukimian, Cathy Carvalhosa, et al. Cyanine 5-huwentoxin-IV as a novel imaging probe to detect hNa_v1.7 channel overexpressed in non-small cell lung cancer. Biochemical Pharmacology, 2025, 242, pp.117203. 10.1016/j.bcp.2025.117203. hal-05221560

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# Cyanine 5-huwentoxin-IV as a novel imaging probe to detect hNa<sub>v</sub>1.7 channel overexpressed in non-small cell lung cancer

Romain Baudat<sup>a</sup>, Jérôme Montnach<sup>b</sup>, Evelyne Benoit<sup>a</sup>, Claude Zoukimian<sup>c</sup>, Cathy Carvalhosa<sup>c</sup>, Rémy Béroud<sup>c</sup>, Michel De Waard<sup>b,c,d</sup>, Denis Servent<sup>a,*</sup>

## ARTICLE INFO

Keywords:

Non-small cell lung cancer

Huwentoxin-IV

Na<sub>v</sub>1.7

Fluorescence imaging

Diagnosis

## ABSTRACT

Currently, tools for detecting cancer cells belonging to the non-small cell lung cancer (NSCLC) are lacking. As overexpression of human voltage-gated sodium channel subtype 1.7 (hNa<sub>v</sub>1.7) channels was identified as a promising marker in NSCLC, we investigated whether huwentoxin-IV (HwTx-IV), known to interact preferentially with this sodium channel subtype at nanomolar concentrations, may serve as a practical tool for detecting NSCLC. To achieve this goal, we coupled HwTx-IV to the cyanine 5 fluorophore and evaluated the potential of cyanine 5-HwTx-IV (Cy5-HwTx-IV) as an imaging agent to improve the detection of this cancer type. Increased messager ribonucleic acid (mRNA) expression levels of hNa<sub>v</sub>1.7 were confirmed in the A549 and H460 NSCLC cell lines, but not in the Beas-2B healthy lung cell line. Moreover, electrophysiological recordings validated the presence of functional hNa<sub>v</sub>1.7 in the metastatic H460 cell line, the recorded Na<sup>+</sup> current in this cell line being completely abolished in the presence of nanomolar concentrations of Cy5-HwTx-IV and HwTx-IV. In contrast, functional hNa<sub>v</sub>1.7 was not detected in the non-metastatic A549 and healthy lung Beas-2B cell lines. Finally, confocal laser scanning microscopy allowed localisation of hNa<sub>v</sub>1.7 at the plasma membranes of cells labelled with Cy5-HwTx-IV. In conclusion, this work establishes Cy5-HwTx-IV as a promising tool for NSCLC imaging, thereby enhancing diagnostic precision for this cancer type.

**Abbreviations:** ¹⁸F-FDG, ¹⁹F-fluoro-deoxy-glucose; BSA, bovine serum albumin; cDNA, complementary deoxyribonucleic acid; CHO, Chinese hamster ovary; CTX, chlorotoxin; Cy5-HwTx-IV, Cy5-conjugated huwentoxin-IV; DDCt, Delta-Delta Ct; DIEA, N,N-Diisopropylethylamine; DMF, N,N-dimethylformamid; DMEM, dulbecco's Modified Eagle medium; DRG, dorsal root ganglion; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; ESI-HRMS, electrospray ionization high-resolution mass spectrometry; FA, formic acid; FBS, foetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCTU, O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HEPES, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; Na<sub>v</sub>, voltage-gated sodium channel; hNa<sub>v</sub>1.7, human Na<sub>v</sub> subtype 1.7; HPLC, high performance liquid chromatography; HRMS, high-resolution mass spectrometry; HwTx-IV, huwentoxin-IV; IC<sub>50</sub>, half maximal inhibitory concentration; ICK, inhibitor cystine knot; iPr3SiH, triisopropylsilane; LC, liquid chromatography; mRNA, messager ribonucleic acid; MS, mass spectrometry; NMP, N-methyl-2-pyrrolidone; NSCLC, non-small cell lung cancer; PBS, phosphate-buffered saline; PET, positron emission tomography; QTOF, quadrupole time-of-flight; RAM, rink amid; RP, reversed-phase; RP-HPLC, reversed-phase high performance liquid chromatography; RT-qPCR, real-time quantitative polymerase chain reaction; SCLC, small cell lung cancer; S.D., standard deviation; S.E.M., standard error of the mean; SPECT, single photon emission computed tomography; SPPS, solid-phase peptide synthesis; TEA, tetraethylammonium chloride; TFA, trifluoroacetic acid; UPLC, ultra-performance liquid chromatography; UV, ultraviolet; WGA, wheat germ agglutinin.

* Corresponding author.

**E-mail addresses:** romain.baudat@cea.fr (R. Baudat), jerome.montnach@univ-nantes.fr (J. Montnach), evelyne.benoit@cea.fr (E. Benoit), claude.zoukimian@smartox-biotech.com (C. Zoukimian), cathy.carvalhosa@sb-peptide.com (C. Carvalhosa), remy.beroud@smartox-biotech.com (R. Béroud), michel.dewaard@univ-nantes.fr (M.D. Waard), denis.servent@cea.fr (D. Servent).

<footnote><sup>a</sup> Université Paris-Saclay, CEA, Département Médicaments et Technologies pour la Santé (DMTS), Service d'Ingénierie Moléculaire pour la Santé (SIMoS), EMR CNRS 9004 Gif-sur-Yvette, France</footnote>

<footnote><sup>b</sup> Université de Nantes, INSERM, CNRS, l'institut du thorax, Nantes, France</footnote>

<footnote><sup>c</sup> Smartox Biotechnology, Saint-Egrève, France</footnote>

<footnote><sup>d</sup> LabEx "Ion Channels, Science and Therapeutics", Valbonne, France</footnote>
# 1. Introduction

Lung cancer remains the most prevalent and lethal cancer worldwide, with 1.8 million deaths reported in 2020 (World Health Organization 2020). It is broadly categorized into non-small cell lung cancer (NSCLC, 85 % of cases) and small cell lung cancer (SCLC, 15 %). Current diagnostic methods, including clinical examination, X-rays, and positron emission tomography (PET) scans using $^{18}$F-fluoro-deoxy-glucose ($^{18}$F-FDG), are widely used due to their non-invasive nature and rapid results [1]. However, these methods have significant limitations since only 16 % of lung cancers are detected at early stages, and tumours with low glucose uptake, such as certain adenocarcinomas, may go undiagnosed [2]. This highlights the need for innovative imaging probes that target molecular biomarkers specifically overexpressed in NSCLC to improve diagnosis and optimize treatment strategies.

Ion channels, particularly voltage-gated sodium channels (Na$_v$S), are emerging as key players in cancer biology [3,4]. They regulate ion homeostasis and are implicated in tumour progression through mechanisms involving migration, invasion, angiogenesis, apoptosis resistance, and proliferation [3–6]. Among Na$_v$S, human Na$_v$ subtype 1.7 (hNa$_v$1.7) is overexpressed in several cancers, including prostate [7,8], cervical [9], endometrial [10], gastric [11] and medullary thyroid cancers [12], and is specifically detected in NSCLC but not in SCLC cell lines [13]. This channel subtype facilitates sodium influx in the metastatic NSCLC cell line H460 [13] and recruits proteins such as cysteine cathepsins, driving NSCLC cell proliferation, migration, and invasion [6,14,15]. These characteristics make hNa$_v$1.7 a promising biomarker and therapeutic target for NSCLC.

Peptides have garnered significant attention as imaging tools in cancer research due to their high specificity and favorable pharmacokinetic properties [16]. Because of these properties, dozens of radiolabelled peptides are clinically used for diagnosis, such as $^{111}$In-/$^{99m}$Tc-/$^{68}$Ga-octreotide derivatives for single photon emission computed tomography (SPECT) or PET imaging of somatostatin receptor subtype-expressing endocrine tumours [17–20]. Venom-derived peptides may also be exploited as imaging tools, based on their exceptional selectivity for various ion channels and receptors, high structural stability and appropriate biodistribution properties associated with rapid accumulation in target tissues and fast clearance rate. In the form of fluorescent or radioactive derivatives, they have been used to localise and define the functional properties of molecular targets in cells and tissues or to identify tumour location during surgery [16,21,22]. Notable examples include chlorotoxin (CTX), as well as Lqh8/6 [23], both from the venom of Leiurus quinquestriatus scorpion, that bind to several proteins overexpressed in glioblastoma, enabling tumour delinuation and aiding in surgery and radiotherapy [24–29]. Similar advances have been made with fluorescent or radiolabelled venom peptides targeting Na$_v$1.7 for imaging applications, such as (i) ATTO 488- and cyanine 5-conjugated protoxin-II (ATTO488-ProTx-II and Cy5-ProTx-II, respectively) for imaging channel distribution in cells [30], (ii) cyanine 7.5- or BODIPY-conjugated Hsp1a toxin (Cy7.5-Hsp1a and BODIPY-Hsp1a, respectively) for nerve visualization during surgical procedure [31], and (iii) $^{64}$Cu-Hs1a-Bac for assessing nerve damage [32].

In this study, we explored the potential of Cy5-conjugated hwen-toxin-IV (Cy5-HwTx-IV) as an imaging probe for NSCLC detection. Huwentoxin-IV (HwTx-IV), a peptide derived from the venom of Selec-nocosmia huwena spider, is known for its high-affinity binding and inhibitory action on Na$_v$1.7 by trapping the channel's voltage sensor in a closed state [33–36]. We assessed the expression level of hNa$_v$1.7 messager ribonucleic acid (mRNA) in NSCLC cell lines (A549, H460) and a healthy lung cell line (Beas-2B) using real-time quantitative polymerase chain reaction (RT-qPCR), synthesized HwTx-IV and its cyanine 5-conjugate, confirmed their closed high affinities for hNa$_v$1.7 *via* electrophysiology, and localised hNa$_v$1.7 at the plasma membranes of cells labelled with Cy5-HwTx-IV *via* confocal microscopy.

# 2. Materials and methods

## 2.1. Chemical synthesis of HwTx-IV and Cy5-HwTx-IV

### 2.1.1. High performance liquid chromatography (HPLC) and mass spectrometry (MS) analyzes

The peptides were analysed by HPLC and liquid chromatography (LC)-electrospray ionization (ESI)-high-resolution mass spectrometry (HRMS). HPLC analyses were carried out on an Agilent system equipped with XSelect Peptide CSH C18 column (130 Å, 2.5 µm, 150 x 4.6 mm) at a flow rate of 1 mL/min and 214 nm. Solvents A and B were 0.1 % trifluoroacetic acid (TFA) in H$_2$O and 0.1 % TFA in acetonitrile. Peptide elution was performed at a flow rate of 1 mL/min with a 5 % buffer B isocratic equilibration of 3 min followed by a 5–60 % gradient of buffer B over 20 min and at 40 °C. The acquisition of the LC-ESI-HRMS data was carried out on an accurate mass quadrupole time-of-flight (QTOF) 6530 mass spectrometer (Agilent) coupled to an Agilent 1290 ultra-performance liquid chromatography (UPLC) system. Solvents A and B were 0.1 % formic acid (FA) in water and 0.1 % FA in acetonitrile. All calculated and found masses were monoisotopic. Purification of the peptides was performed by HPLC on a preparative Agilent 1260 system equipped with a Phenomenex Jupiter column (4 µm Proteo 90 Å, C12, 250 x 4.6 mm) at a flow rate of 20 mL/min or a semi-preparative Agilent 1260 system equipped with an Agilent Poroshell EC-C18 column (120 Å, 4 µm, 250 x 9.4 mm) at a flow rate of 5 mL/min. All chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

### 2.1.2. Syntheses

The linear sequence of HwTx-IV (ECLEIF-KACNPSNDQCCKSSKLVCSRKTRWCKYQI) was assembled by solid-phase peptide synthesis (SPPS) on a Symphony X automated synthesizer (Gyros Protein Technologies, Uppsala, Sweden), using Fmoc/tBu chemistry at a 0.1 mmol scale with TentaGel® Rink amid (RAM) resin as the solid support (Rapp Polymere, Tuebingen, Germany). The elongation was carried out using a 10-fold excess of protected amino acids and O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU) and 20-fold excess of N,N-Diisopropylethylamine (DIEA) in N,N-dimethylformamid (DMF). The side chain protecting groups used were Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Trt), Gln(Trt), Glu(OtBu), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc) and Tyr(tBu). Fmoc deprotection was performed using a 20 % piperidine solution in DMF. Each coupling lasted 15 min and was repeated three times, followed by capping with a 10 % acetic anhydride in N-methyl-2-pyrrolidone (NMP). After the last amino acid, an alkyne moiety was introduced on the N-terminus by a single coupling of 5 equivalents of pentynoic acid with a 5-fold excess of HCTU and 10-fold excess of DIEA in DMF. The crude peptide was released from the resin with TFA/triisopropylsilane (iPr3SiH)/2,20-(ethylenedioxy)diethanethiol/H$_2$O (85:5:5:5) for 3 h, and the peptide was precipitated with ice-cold diethyl ether, recovered by centrifugation and washed twice with diethyl ether. Reversed-phase (RP)-HPLC and MS analyses were performed on the crude linear HwTx-IV-alkyne before refolding. The reversed-phase high performance liquid chromatography (RP-HPLC) retention time was 13.43 min. The ESI-HRMS C$_{179}$H$_{288}$N$_{52}$O$_{52}$S$_6$ calculated was 4189.98 and that found was 4189.99. All peptide synthesis reagents were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

### 2.1.3. Peptide folding

Crude linear HwTx-IV-alkyne was refolded batchwise by adding the peptide solubilized in 20 % acetonitrile at 10 mg/mL in a 0.1 M Tris.HCl buffer at pH 8.2 containing 10 % dimethylsulfoxide, to a final concentration of 0.1 mg/mL of peptide. After 48 h at room temperature, the reaction mixture was acidified with 1 mL of FA, filtered and purified by preparative RP-HPLC using a linear 20–50 % buffer B gradient over 50 min (buffer B: 90 % acetonitrile and 0.1 % TFA in water). Refolding was performed on a typical scale of 100 mg of crude linear peptide, and
typical yield was around 15 % (purity rate > 90 %). The RP-HPLC retention time was 12.07 min. The ESI-HRMS C179H282N52O52S6 calculated was 4183.93 and that found was 4183.94. All chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

### 2.1.4. Cy5 modification

Oxidized HwTx-IV-alkyne (12 mg, 1 equiv.) was dissolved in 0.5 mL of (NMP/tBuOH 9:1)/H₂O/(0.4 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), 50 mM aminoguanidine hydrochloride) 1:1:3, and 3.5 mg of Cy5-N₃ (2 equiv.) dissolved in 50 µL of NMP/tBuOH 9:1 was added, followed by 11.8 mg of CuBr(CH₃)₂S (20 equiv.) and 125 mg of THPTA (100 equiv.) dissolved in 0.25 mL of NMP/tBuOH 9:1. The reaction mixture was agitated for 4 h and 30 min at 40 °C under inert atmosphere, then acidified with TFA, diluted 5 fold with water and purified by semi-preparative RP-HPLC using a linear 25–55 % buffer B gradient for 50 min at 60 °C. Lyophilized Cy5-HwTx-IV (3.0 mg quantified by ultraviolet (UV) measurement, ε = 250000 L·mol⁻¹·cm⁻¹ at 646 nm) was obtained (yield 22 %, purity rate > 95 %). The RP-HPLC retention time was 15.87 min (XSelect Peptide CSH C18, 5–60 % B in 20 min). The ESI-HRMS C214H327N58O53S6 calculated was 4749.30 and that found was 4749.39. Except Cy5-N₃ that was obtained from Lumiprobe (Westminster, MD, USA), all other chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

## 2.2. Cell cultures

Human lung cell lines were obtained from CliniSciences (Santerre, France): A549 from a non-metastatic NSCLC (AddexBio C0016002), H460 from a metastatic NSCLC (AddexBio C0016003) and Beas-2B (AddexBio T0007001). Cells were maintained at 37 °C and 5 % CO₂ in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10 % foetal bovine serum (FBS), penicillin (50 U/mL) and streptomycin (50 µg/mL). Chinese hamster ovary (CHO) cells were cultured at 37°C and 5 % CO₂ in DMEM/F-12 supplemented with 10 % FBS, penicillin (50 U/mL) and streptomycin (50 µg/mL). CHO cells stably expressing the hNav1.7 channel subtype were cultured in DMEM/F12 supplemented with 10 % FBS, glutamine (2 mM), hygromycin B (200 µg/mL), penicillin (10 U/mL) and streptomycin (10 µg/mL) and incubated at 37 °C in a 5 % CO₂ atmosphere. All chemicals were purchased from Gibco (Thermo Fisher Scientific, Villebon-sur-Yvette, France).

## 2.3. Real-time quantitative polymerase chain reaction (RT-qPCR)

Total mRNAs from cell lines were extracted with the miRNeasy mini kit (Qiagen, Hilden, Germany). Total mRNAs were taken up in 14 µL of RNAse-free water and their concentrations were then measured by spectrophotometry (Clariostar, BMG Labtech, Champigny-sur-Marne, France). The 260 nm/280 nm and 260 nm/230 nm ratios were used to check the quality of the mRNA after extraction. Each mRNA sample (1 µg) was reversely transcribed into complementary deoxyribonucleic acid (cDNA) using the kit Quantitech Reverse Transcription (Qiagen, Hilden, Germany). RT-PCR was performed with Thermocycler 280 (Thermo Fischer Scientific, Paisley, UK) using the following program: 42 °C for 30 min, followed by an inactivation step at 95 °C for 3 min. Then, the qPCR method was used to quantify the level of mRNA expression, after having designed the primer pairs of *SCN9A* (forward: AGACCTCTCTTTCCATGTAGATTAC, and reverse: TGTAACTGCCTTT CTGTATTGTTG). Data were analyzed by the Delta-Delta Ct (DDCt) method and Light Cycler SW software (Roche, Bâle, Switzerland). The gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping genes, and the values were then normalized to those of Beas-2B. All primers were purchased from

Sigma-Aldrich (Darmstadt, Germany).

## 2.4. Manual patch-clamp recordings

Just before the experiments, cells were dissociated with trypsin (Thermo Fischer Scientific #25200056, Paisley, UK) and deposited on slides coated, one day before, with poly-L-ornithine to facilitate cell adhesion. Macroscopic currents were recorded at room temperature (18–22 °C) in the whole-cell configuration performed by using a MultiClamp 700B integrating patch-clamp amplifier and the pClamp 10.6 software (Molecular Devices, Sunnyvale, CA, USA). The signals, acquired at a 4-kHz sample rate, were filtered at 2 kHz with a low-pass Bessel filter and digitized with the aid of a computer equipped with an analog-to-digital converter (Digidata-1440A model; Molecular Devices, Sunnyvale, CA, USA). Patch pipettes were pulled from KWIK-Fil 1.5/086 borosilicate glass capillaries (Sutter Instrument CO. #BF150-86-15, Novato, CA, USA) and fire polished to get resistances of 2–3 MΩ in the extracellular solution. The intracellular solution contained (in mM): 90 CsCl, 40 CsMeSO₃, 10 NaCl, 2 MgCl₂, 2 ethylene glycol-bis(β-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA), 10 HEPES and 4 Na₂ATP (pH 7.32, osmolarity 292 mOsm). The extracellular solution contained (in mM): 134 NaCl, 3 KCl, 1 CaCl₂, 1 MgCl₂, 10 tetraethylammonium chloride (TEA), 0.1 CdCl₂, 10 HEPES and 10 glucose (pH 7.35, osmolarity 302 mOsm). All solutions were filtered with a non-pyrogenic vacuum filter and stored at 4 °C until use. The cells were maintained at a holding potential of –60 mV, and transiently 1 s at –100 mV before eliciting currents at a frequency of 0.5 Hz by 50-ms test-pulses to 0 mV. The amplitude of the leakage current was not compensated. The current-voltage relationships were obtained by varying the test-pulses voltage from –60 to 50 or 60 mV in 5 mV increments. Lyophilized synthetic HwTx-IV and Cy5-HwTx-IV were dissolved in phosphate-buffered saline (PBS 1X) solution supplemented with 0.5 % bovine serum albumin (BSA) to give 6.1 mM and 20 µM stock solutions, respectively. All chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Just prior the experiments, successive dilutions were performed in the adequate standard physiological medium to give the HwTx-IV and Cy5-HwTx-IV final concentrations indicated in the text. A fast solution application system allowed to change the solution (standard physiological medium supplemented with a given toxin concentration or not) around the recorded cell within a few seconds. Percentages of current inhibition were measured at steady-state of blockage.

## 2.5. Automated patch-clamp recordings

Just before the experiments, cells were detached with trypsin, and floating single cells were diluted (~300,000 cells/mL) in an extracellular solution containing (in mM): 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 5 glucose and 10 HEPES (pH 7.4, osmolarity 298 mOsm) that was used throughout the experiments. Cy5-HwTx-IV analogue has been investigated on CHO cells stably expressing hNaV1.7 channel using the automated patch-clamp system from Nanion Technologies (SyncroPatch 384PE; München, Germany). Chips with single-hole and high-resistance (6–7 MΩ) were used. Voltage pulses and whole-cell recordings were achieved using the PatchControl384 v1.5.2 software (Nanion Technologies, München, Germany) and the Biomek v1.0 interface (Beckman Coulter, Fullerton, CA, USA). Prior to recordings, dissociated cells were shaken at 200 rpm in a cell hotel reservoir at 10 °C. After cell catching, sealing, whole-cell formation, liquid application, recording, and data acquisition were all performed sequentially and automatically. The intracellular solution contained (in mM): 110 CsF, 10 CsCl, 10 NaCl, 1 MgCl₂, 1 CaCl₂, 10 EGTA and 10 HEPES (pH 7.2, osmolarity 280 mOsm).
All solutions were sourced from Nanion Technologies (München, Germany). Whole-cell experiments were done at –100 mV holding potential (ensuring that channels remained predominantly in the resting state, rather than the inactivated state) and at room temperature (18–22 °C), while currents were triggered at –10 mV and sampled at 20 kHz. Stimulation frequency was set at 0.2 Hz. Each HwTx-IV and Cy5-HwTx-IV analogue were prepared at various concentrations in the extracellular solution, itself supplemented with 0.3 % BSA (Sigma-Aldrich, Saint-Quentin Fallavier, France), also added in the control solution. Compound solutions were diluted 3 times in the patch-clamp recording well by adding 30 µL to 60 µL external solution, to reach the final reported concentration and the test volume of 90 µL. The percentages of current inhibition were measured at the end of a 15-min application time to ensure steady-state block, particularly at low concentrations, even if inhibition occurred more rapidly at higher concentrations. It is worth noting that only cells exhibiting stable leak currents and series resistance were used in this study.

## 2.6. Confocal laser scanning microscopic imaging

The experiments were performed on Beas-2B, A549, H460 and CHO stably expressing or not hNav1.7. About 10 000 cells were seeded on circular slides deposited in well plates with 1 mL of culture medium in each well, at 37 °C overnight. Circular slides were then put in a home-made dark humidified box, under different conditions: cells were first

pre-incubated with 50 µL of 2 µM HwTx-IV (giving a final concentration of 500 nM) or PBS supplemented with BSA (0.5%) for 15 min, and next incubated with 50 µL of Cy5-HwTx-IV (50 nM) for 15 min. Then, slides were washed 3 times with PBS and BSA. To label the plasma membranes of cells, WGA-488 (iFluor® 488-Wheat Germ Agglutinin Conjugate, CliniSciences, Nanterre, France) were added during 10 min. Three rapid washed were done with PBS and BSA. After cautious drying of slides, Hoescht-405 in the mounting medium (CliniSciences, Nanterre, France) were added on glasses to carefully put one given slide on the droplet. Finally, the slides and lamella were fixed by staying at room temperature in the dark box. Images were acquired using a confocal Zeiss LSM700 microscope (Zeiss, Oberkochen, Germany) and three different laser wavelengths (405, 488 and 595 nm), and analysed using ImageJ (v.1.54i). The mean fluorescence intensity was measured on each cell and averaged.

## 2.7. Data and statistical analyses

Data are expressed as mean ± standard deviation (S.D.) or standard error of the mean (S.E.M.) of *n* different experiments. All statistical analyses, performed using GraphPad (v.9.5.1, Boston, MA, USA), are described within the text. Differences were considered statistically significant at *P* ≤ 0.05.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Chemical synthesis of Cy5-HwTx-IV. (A) Primary structure of HwTx-IV-alkyne indicating the disulfide bridge pattern of HwTx-IV and the chemical structure of alkyne group, coupled through peptide chemistry to the N-terminus of HwTx-IV. The chemical structure of Cy5 is also shown in red, and the blue linker contains the azide function allowing the click chemistry. (B) RP-HPLC elution profile of crude HwTx-IV-alkyne. (C) RP-HPLC elution profile of pure Cy5-HwTx-IV after click chemistry. (D) Mass spectrometry profile of Cy5-HwTx-IV at [M + 4H]⁵⁺. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
# 3. Results

## 3.1. Chemical synthesis of Cy5-HwTx-IV

To synthesize Cy5-HwTx-IV, we first chemically produced HwTx-IV with an extra N-terminal alkyne moiety (pentynoic acid). According to this method, the folding/oxidation of HwTx-IV-alkyne occurred first without grafting Cy5, which is probably too bulky for proper folding. This folding/oxidation occurred smoothly, as seen on the elution profile of the crude peptide (Fig. 1A). Next, click chemistry was used to couple Cy5 to HwTx-IV-alkyne. The final product in its purified form, shown in Fig. 1B, was considerably more hydrophobic as seen from the comparison of the reversed-phase HPLC (RP-HPLC) elution profiles, which is consistent with the hydrophobic nature of Cy5. The [M + 4H]⁵⁺ monoisotopic mass of Cy5-HwTx-IV was 950.6843 (Fig. 1C), which corresponds to the theoretical exact mass 4749.39 Da.

## 3.2. Electrophysiological characterization of HwTx-IV and Cy5-HwTx-IV on hNa<sub>v</sub>1.7 expressed in CHO cells

For Cy5-HwTx-IV to be a valid probe, it must retain its efficacy in recognizing the hNa<sub>v</sub>1.7 biomarker at the plasma membrane of NSCLC cells. Grafting a fluorescent tag at one end of a natural peptide always implies a risk of steric hindrance and loss of pharmacophore integrity.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Comparison of the potencies of HwTx-IV and Cy5-HwTx-IV in blocking hNa<sub>v</sub>1.7 expressed in a CHO cell line. (A) Representative current examples illustrating hNa<sub>v</sub>1.7 current block by 10 and 100 nM HwTx-IV (top panel) and Cy5-HwTx-IV (lower panel). Note that the efficacies of block are quite similar and take place with identical kinetics. (B) Normalized average concentration-response curves for both the synthetic HwTx-IV (n = 45 cells) and Cy5-HwTx-IV (n = 45 cells). Sigmoid Hill fit of the data provides IC₅₀ values of 9.9 ± 1.1 nM (HwTx-IV) and 20.4 ± 1.1 nM (Cy5-HwTx-IV).</figcaption></figure>

To test the functionality of Cy5-HwTx-IV, we evaluated its potency in blocking Na⁺ currents from hNa<sub>v</sub>1.7 channels stably expressed in CHO cell line. Representative recordings are illustrated in Fig. 2A, showing that both HwTx-IV and Cy5-HwTx-IV were able to block hNa<sub>v</sub>1.7 currents either partially (10 nM) or fully (100 nM). The block occurred with a slow onset and was complete within 15 min at 100 nM for both compounds. Notably, the extent of block at 10 nM was slightly smaller for Cy5-HwTx-IV (24.3 ± 3.9%, n = 6) than for HwTx-IV (45.6 ± 4.8%, n = 5), suggesting that the potency of this analogue was only minimally affected. This finding was further confirmed by plotting the average concentration-response curves for both peptides. As shown in Fig. 2B, Cy5-HwTx-IV was only marginally less effective than HwTx-IV in blocking hNa<sub>v</sub>1.7 currents [2-fold reduction in potency, based on the IC₅₀ (half maximal inhibitory concentration) values of 9.9 nM (HwTx-IV) and 20.4 nM (Cy5-HwTx-IV)]. Finally, the percentage of current decrease, measured at the end of 15-min application of solvent alone, was 1.8 ± 4.7% (n = 11), demonstrating that the currents remained quite stable over time in the absence of toxin. Altogether, these results indicate that Cy5-HwTx-IV retains its high affinity and can reliably be used for detecting hNa<sub>v</sub>1.7 channel expression at the plasma membranes of cancer cells.

## 3.3. Expression and function of hNa<sub>v</sub>1.7 in healthy lung and NSCLC cell lines

### 3.3.1. Expression of mRNA from SCN9A, the gene encoding hNa<sub>v</sub>1.7

First, we assessed by RT-qPCR the mRNA expression of SCN9A, the gene encoding hNa<sub>v</sub>1.7, in three different cell lines: A549, a non-metastatic NSCLC cell line, H460, a metastatic NSCLC cell line and Beas-2B, a healthy lung cell line. For this purpose, total mRNAs from these three cell lines were extracted, reversely transcribed into cDNAs and then quantified using qPCR. In each case, five measurements were performed from five consecutive cell passages.

The results show that the expression of hNa<sub>v</sub>1.7 mRNA was increased by 4.10- and 8.65-fold in the A549 and H460 cell lines, respectively, compared with the Beas-2B cell line, although only the later was statistically significant (Fig. 3). This strongly suggests that hNa<sub>v</sub>1.7 was significantly and specifically overexpressed in H460, which is the metastatic condition of NSCLC that implies high proliferation and migration

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Histogram of expression level of mRNA of hNa<sub>v</sub>1.7 in Beas-2B (healthy lung cell line), A549 (non-metastatic NSCLC cell line) and H460 (metastatic NSCLC cell line), expressed relatively to that of the Beas-2B cell line. Mean ± S. E.M. of 5 experiments. **; *P* = 0.0047 using the Friedmann test with Dunn's multiple comparison test.</figcaption></figure>
rates.

### 3.3.1.1. Electrophysiological detection of Na<sub>v</sub>s.
Next, electrophysiological recordings were performed to determine if Na<sub>v</sub>s are functionally expressed in healthy lung and in the two NSCLC cell lines. The results reveal that no Na<sup>+</sup> current could be recorded from the healthy lung cell line Beas-2B, regardless of the test-pulse voltage used (Fig. 4A). Similar results were obtained from the non-metastasis NSCLC cell line A549 (Fig. 4B). In contrast, the metastatic NSCLC cell line H460 exhibited an inward Na<sup>+</sup> current with a threshold of activation at approximately -35 mV and a reversal potential of about 50 mV (Fig. 4C). The maximum amplitude of this current, determined at 0 mV, was -157.9 ± 54.3 pA (n = 10), e.g., 9.4 ± 3.2 pA/pF, if one takes into account an average cell capacitance of 16.8 ± 5.6 pF, n = 18). Therefore, no Na<sub>v</sub> subtypes could be detected in A549 and Beas-2B cell lines, whereas the expression of

functional these subtypes is reinforced in the H460 cell line.

### 3.3.1.2. Effects of HwTx-IV and Cy5-HwTx-IV to define the pharmacological properties of Na<sub>v</sub>s expressed in H460 cells.
To verify that the Na<sup>+</sup> current recorded in the H460 cell line is supported by hNa<sub>v</sub>1.7, we studied the effects of HwTx-IV and Cy5-HwTx-IV on this current. The results show a complete abolition of the current in the presence of 100 nM of either peptide, regardless of the test-pulse used (Fig. 5). Under control conditions, the maximum current amplitude, determined at 0 mV, was -157.9 ± 60.0 pA (n = 18). This amplitude was decreased to -3.7 ± 6.5 pA (n = 5), e.g., 97.6% of current inhibition, and to -8.8 ± 7.5 pA (n = 5), e.g., 94.0% of current inhibition, by 100 nM HwTx-IV (Fig. 5A and C) and Cy5-HwTx-IV (Fig. 5B and C), respectively. Therefore, at this concentration, both peptides exerted an almost complete blocking effect on the inward Na<sup>+</sup> current of the H460 cell line, most

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Electrophysiological detection of Na<sub>v</sub>s in the healthy lung Beas-2B (A) and the NSCLC cell lines A549 (B) and H460 (C). On the left, representative Na<sup>+</sup> current traces recorded at -50, -40, -30, -20, -15, -10, -5, 0 (red traces), 5, 10 and 20 mV of test-pulses. On the right, current-voltage curves. Mean ± S.E.M. of 6–10 different cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Electrophysiological characterization of HwTx-IV and Cy5-HwTx-IV effects on the NSCLC cell line H460. (A) and (B) Representative current traces recorded at –50, –40, –30, –20, –15, –10, –5, 0 (red traces), 5, 10 and 20 mV of test-pulses, before (left) and after (right) exposure of cells to 100 nM HwTx-IV (A) or Cy5-HwTx-IV (B). (C) Current-voltage curves established before (black circles) and after the application of 100 nM HwTx-IV (blue circles) or Cy5-HwTx-IV (red circles). Mean ± S.E.M. of 4–10 different cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

likely mediated through hNaV1.7.

### 3.4. Healthy lung and NSCLC cell imaging

Finally, confocal laser scanning microscopy was used to localise Cy5-HwTx-IV staining (in red), indicative of hNaV1.7 expression at the cellular level on healthy lung and NSCLC cell lines. For this purpose, we employed Hoesht-405 to specifically label the cell nucleus (in blue) and WGA-488 to specifically label the cell plasma membranes (in green). As shown in a representative experiment (Fig. 6A), Cy5-HwTx-IV (50 nM) staining was observed in the NSCLC cell lines A549 and H460, as well as in CHO cells stably expressing hNaV1.7 (CHO/hNaV1.7 cells, positive control cells). In contrast, no staining was detected in the healthy lung cell line Beas-2B and in CHO cells that did not express hNaV1.7 (CHO cells, negative control cells). Furthermore, the fluorescence intensity profiles of the three different stainings revealed colocalisation of Cy5-HwTx-IV and WGA-488 in H460, A549 and CHO/hNaV1.7 cells, strongly suggesting a plasma membrane localisation of hNaV1.7 in these cells. In contrast, the fluorescence intensity profiles of Beas-2B and CHO cells confirmed the absence of Cy5-HwTx-IV staining in these cells.

The quantification of the mean fluorescence intensity of Cy5-HwTx-IV in the different cell lines is shown in Fig. 6B. The very low mean fluorescence intensities of 2.69 and 5.23 a.u. in Beas-2B and CHO cells, respectively, further confirmed the absence of Cy5-HwTx-IV staining in these cells. In contrast, the NSCLC cell lines A549 and H460, as well as the CHO/hNaV1.7 cells, showed high mean fluorescence intensities, e.g., 30.86, 45.06 and 43.10 a.u., respectively. These values are not significantly different between them (*P* > 0.649). The specificity of Cy5-HwTx-IV (50 nM) staining was validated by pre-incubating cells with an excess of non-labelled HwTx-IV (500 nM). Under these conditions, a significant decrease of Cy5-HwTx-IV staining was detected for A549 (3.99 a.u.), H460 (5.78 a.u.) and CHO/hNaV1.7 (3.91 a.u.) cell lines (Fig. 6B), confirming the specificity of Cy5-HwTx-IV binding to hNaV1.7. As expected, no effect of the excess of HwTx-IV on Cy5-HwTx-IV staining was observed in Beas-2B (2.43 a.u.) and CHO cell lines (3.89 a.u.), where the mean fluorescence intensities remained very low. Altogether, these data strongly suggest that hNaV1.7 was localised at, or near, the plasma membranes of A549, H460 and CHO/hNaV1.7 cells but not at those of Beas-2B and CHO cells.

### 4. Discussion

Growing evidence suggests that ion channels, particularly hNaV1.7, influence various stages of lung cancer progression, notably the invasion process through their role in sodium homeostasis. Several cancer types, including NSCLC, have been reported to overexpress hNaV1.7, making

this channel a promising biomarker for cancer diagnosis [37,38].

Simultaneously, hNaV1.7-selective toxins, such as ProTx-II, Hs1a, Tsp1a or Hsp1a, have been developed as novel imaging agents to localise this channel subtype for disease diagnostics and intraoperative imaging [22]. These toxins have already been exploited as nerve-imaging agents in the peripheral nervous system, enhancing nerve contrast during surgery and reducing the risk of nerve damage [31,32,39]. However, despite their high affinity, selectivity and stability, animal toxins targeting hNaV1.7 have not yet been explored in the context of cancer diagnosis.

In this study, we evaluated Cy5-HwTx-IV as a selective imaging probe for detecting hNaV1.7 overexpression in NSCLC. We selected HwTx-IV, one of the most extensively studied NaV1.7-interacting toxins [33–35], known to inhibit NaV1.7 activation by interacting with the S3-S4 segments of the second voltage sensor domain (VSDII) of the channel [36,40]. Hence, HwTx-IV-alkyne was synthesized via solid-phase peptide synthesis and conjugated at its N-terminal region with fluorescent Cy5-N3 using click chemistry. The N-terminal labelling was chosen to preserve the C-terminal region, which is crucial for the functional properties of HwTx-IV [41,42].

Electrophysiological studies in CHO cells stably expressing hNaV1.7 confirmed that Cy5-HwTx-IV retains its ability to bind to hNaV1.7, although with a two-fold reduction in affinity compared with the native peptide. Notably, this finding is consistent with prior studies demonstrating that inhibitor cystine knot (ICK) peptides from spider venoms (e. g., HwTx-IV in this study, Hs1a, Tsp1a, ProTx-II and Hsp1a in previous reports) can accommodate large fluorophores without markedly affecting their interaction with hNaV1.7 [30–32,39]. Furthermore, some gating-modifier toxins, like ProTx-I or ProTx-II, can bind to both hNaV1.7 and the lipid bilayer to modulate channel function [43–45]. These interactions are likely to compromise the specificity of hNaV1.7 detection in cancer diagnostics, reinforcing HwTx-IV as an optimal imaging agent since it is not engaged in these tri-molecular interactions [43–45]. RT-PCR analysis revealed high hNaV1.7 mRNA expression levels in H460 cells, compared to other NSCLC cell lines. This finding aligns with data from Campbell et al. (2013) which identified hNaV1.7 as the only significantly expressed NaV α-subunit in H460 cells. Thus, although HwTx-IV is not strictly selective for the hNaV1.7 subtype, its action in H460 cells is likely restricted to this specific channel subtype.

Electrophysiological experiments revealed also that hNaV1.7 was functionally expressed in highly metastatic H460 cells, while no Na⁺ current was detected in the non-invasive A549 and non-tumoral Beas-2B cell lines. This correlates well with previous reports where H460 cells exhibited the strongest sodium current among NSCLC cell lines, with activation-voltage properties similar to those described in this study [13,37]. Moreover, the complete inhibition of Na⁺ currents in H460
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Healthy lung and cancer cell imaging. (A) Representative images of the indicated cell lines labelled with three types of fluorescent probes (left) associated with their fluorescence intensity profiles (right). Hoechst-405 specifically labelled cell nucleus, WGA-488 specifically labelled cell plasmatic membrane, and Cy5-HwTx-IV (50 nM, in red) specifically labelled hNaV1.7. (B) Histogram of mean fluorescence intensity of Cy5-HwTx-IV in the different cell lines, under the indicated conditions. Mean ± S.E.M. of 5–8 different cells. ***: *P* = 0.0008, ****: *P* < 0.0001, using the two-way ANOVA test with Šídák's multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
cells by 100 nM HwTx-IV or Cy5-HwTx-IV confirms the functional ac-
tivity of hNaV.1.7 in these cells and supports the hypothesis that its
expression contributes to the invasive properties of metastatic NSCLC
cells [37]. It is worth noting that the Na⁺ currents recorded from H460
cells, similar to those previously observed in dorsal root ganglion (DRG)
neurons [46], display markedly slower kinetics compared to the
hNaV.1.7-mediated currents recorded from CHO cells. The most plausible
explanation is that the activation and inactivation kinetics of native NaV
channels in H460 cells and DRG neurons are modulated by additional
factors (auxiliary subunits being prime candidates [46]) that are absent
in recombinant systems expressing only the hNaV.1.7 α-subunit.

Confocal microscopy imaging of Cy5-HwTx-IV-labelled cells further
demonstrated the presence of hNaV.1.7 in H460 cells, with fluorescence
localised at the plasma membrane. Importantly, the fluorescence in-
tensity was comparable with that of the CHO-hNaV.1.7 positive control,
despite using only 50 nM of fluorescent peptide, a concentration close to
its IC50. This is particularly relevant for future *in vivo* studies and po-
tential clinical applications. Notably, prior studies required micromolar
concentrations of fluorescent peptides, over 500-fold the IC50, to achieve
similar labelling [30,31,47]. As expected, no significant fluorescence
was observed in Beas-2B cells or the negative control CHO cells lacking
hNaV.1.7 expression. However, unexpected fluorescence was detected in
A549 cells, despite their low hNaV.1.7 mRNA levels, comparable with
Beas-2B, and the absence of Na⁺ currents in both cell lines. A plausible
explanation is that hNaV.1.7 in A549 cells is a non-functional protein that
still retains the Cy5-HwTx-IV binding site. This hypothesis aligns with
previous reports suggesting that A549 cells may express a truncated or
misglycosylated form of hNaV.1.7, leading to a loss of function [13].

4.1. Conclusion

This study demonstrates that Cy5-HwTx-IV is a promising imaging
probe for detecting hNaV.1.7 in NSCLC. Its ability to label plasma
membrane-localised hNaV.1.7 at nanomolar concentrations, combined
with its high specificity and functional validation, highlights its poten-
tial for preclinical and clinical applications. Future investigations should
explore *in vivo* imaging studies and evaluate if hNaV.1.7 expression could
serve as a reliable biomarker for metastatic NSCLC detection and
aggressiveness assessment. Additionally, the overexpression of hNaV.1.7
in biopsies samples from lung, prostate and ovarian cancers, as well as in
other cancer types [8–12], strongly suggests that HwTx-IV, in a
radioactive-derived form, could have broader applications, including
PET imaging.

**5. Funding sources**

We acknowledge a 'FOCUS Biomarker' grant from the French
Alternative Energies and Atomic Energy Commission (grant number
AFOCUS080201) and financial support from the French Agence Natio-
nale de la Recherche to the laboratory of excellence "Ion channels, sci-
ence and therapeutics" (grant number ANR-11-LABX-0015).

CRediT authorship contribution statement

**Romain Baudat:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis. **Jérôme Montnach:** Writing – review & editing, Investigation, Formal analysis. **Evelyne Benoit:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Methodology, Data curation, Conceptualization. **Claude Zoukimian:** Writing – review & editing, Investigation, Formal analysis. **Cathy Carvalhosa:** Investigation, Formal analysis. **Rémy Béroud:** Writing – review & editing, Validation, Resources, Project administration, Methodology, Funding acquisition, Data curation. **Michel De Waard:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation. **Denis Servent:** Writing – review & editing,

Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
M. De Waard and R. Béroud are founders of Smartox Biotechnology,
while C. Zoukimian and Cathy Carvalhosa are employees of the com-
pany. The company played no role in the acquisition of data, in their
interpretation, in the writing of the manuscript, or in the decision to
publish the results. The other authors declare no conflict of interest.

Data availability

All data are included in the present work and are available from the
corresponding author (denis.servent@cea.fr) upon reasonable request.

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