# Amphiregulin reflects brain metastasis progression and leads to PD-L1 expression in non-small cell lung cancer cells

Dimitri Leite Ferreira, Tiphaine Biojout, Céline Bazille, Maelle Guyot, Lucie Malandain, Laureline Charrier, Jérôme Toutain, Elodie A. Pérès, Samuel Valable, Jeannick Madelaine, et al.

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## To cite this version:

Dimitri Leite Ferreira, Tiphaine Biojout, Céline Bazille, Maelle Guyot, Lucie Malandain, et al. Amphiregulin reflects brain metastasis progression and leads to PD-L1 expression in non-small cell lung cancer cells. Biomedicine & Pharmacotherapy, 2025, 192, pp.118600. 10.1016/j.biopha.2025.118600. hal-05274144

HAL Id: hal-05274144

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Submitted on 23 Sep 2025

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# Amphiregulin reflects brain metastasis progression and leads to PD-L1 expression in non-small cell lung cancer cells

Dimitri Leite Ferreira<sup>a,b,1</sup>, Tiphaine Biojout<sup>a,1</sup>, Céline Bazille<sup>c</sup>, Maelle Guyot<sup>d</sup>,
Lucie Malandain<sup>a</sup>, Laureline Charrier<sup>a</sup>, Jérôme Toutain<sup>a</sup>, Elodie Peres<sup>a,D</sup>, Samuel Valable<sup>a,D</sup>,
Jeannick Madelaine<sup>b</sup>, Jérôme Levallet<sup>a,D</sup>, Guénaëlle Levallet<sup>a,c,*1,D</sup>, Emmanuel Bergot<sup>a,b,1</sup>

## ARTICLE INFO

### Keywords:

* NSCLC
* Brain metastases
* Shear stress
* Hippo pathway
* AREG
* Biomarker

## ABSTRACT

**Background:** The Hippo kinase Nuclear Dbf2-related kinase 2 (NDR2) promotes brain metastasis (BM) in non-small cell lung cancer (NSCLC) by the disrupting Yes-associated protein 1 (YAP-1), suggesting a role in circulating tumor cells and/or brain colonization. The underlying mechanism remains to be clarified.

**Methods:** Human bronchial epithelial tumor cells (A549, H1975, H2030 and the brain-tropic H2030-BrM3 line) with or without NDR2 depletion (via siRNA or shRNA), were exposed to shear stress (up to 40 dyn/cm² for 3 h) using an Ibidi® pump system, in the presence or absence of exogenous Amphiregulin (AREG), and subsequently reseeded or not. Viability, apoptosis, proliferation, and YAP-1-dependent gene expression were analyzed. H2030-BrM3 cells (shControl or shNDR2) were injected intracardially into nude athymic mice (n = 10/group) to evaluate plasma AREG levels correlation with BM. AREG expression was assessed in human NSCLC tumor samples from primary and/or brain metastatic sites.

**Results:** NSCLC cells tolerated shear stress and showed reduced apoptosis after reseeding when expressing NDR2. Shear stress induced a dedifferentiation (via Sox2/9 variation) and increased AREG expression in most NSCLC cell lines. AREG enhanced NSCLC cells survival and proliferation under shear stress. In mice, plasma AREG levels correlated with BM volume. In human NSCLC samples, AREG-positive tumors displayed elevated Programmed Death-Ligand 1 (PD-L1), suggesting immune escape. Exogenous AREG treatment in H2030-BrM3 cells induced PD-L1 expression *in vitro*.

**Conclusion:** AREG supports tumor adaptation to mechanical stress and may drive immune tolerance via PD-L1. Its correlation with BM burden highlights its potential as a biomarker and therapeutic target in NSCLC.

## 1. Introduction

Bronchial tumors, including non-small cell lung cancers (which account for approximately 85 % of all lung cancer cases), are highly metastatic neoplasms capable of spreading through hematogenous and lymphatic routes to distant organs such as the brain, bones, and liver [1]. Most circulating tumor cells (CTCs), which are tumor cells released from a primary tumor into the bloodstream [2,3], rapidly die in the bloodstream [4], due to mechanical shear stress, deformation in microvessels, anoikis, and immune clearance by Natural Killer (NK) cells and CD8+ T

lymphocytes. High shear stress induces necrosis and apoptosis in CTCs [5], while also enhancing NK cell-mediated killing via mechanosensitive pathways [6]. Mechanical forces further sensitize CTCs to immune elimination, highlighting their critical role in limiting metastasis [7]. However, some CTCs manage to survive until extravasation, and for these cells, shear stresses induce phenotypic adaptations by activating signaling pathways and promoting the translation of new proteins [8].

In Non-Small Cell Lung Cancer (NSCLC), the Hippo kinase NDR2 (also known as STK38L) promotes tumor growth and brain metastasis [9,10], and is associated with poorer patient survival (Human Atlas

<footnote><sup>a</sup> Université de Caen Normandie, CNRS, Normandie Université, ISTCT UMR6030, GIP CYCERON, Caen F-14000, France</footnote>

<footnote><sup>b</sup> Centre Hospitalier Universitaire de Caen, Service de Pneumologie et Oncologie Thoracique, Caen 14000, France</footnote>

<footnote><sup>c</sup> Centre Hospitalier Universitaire de Caen, Service d'Anatomie et Cytologie pathologiques, Caen 14000, France</footnote>

<footnote><sup>d</sup> Université de Caen Normandie, Federative Structure 4207 "Normandie Oncologie", Service Unit PLATON, Virtual'His platform, Caen F-14000, France</footnote>

<footnote>* Correspondence to: ISTCT-UMR6030, Avenue Henri Becquerel, Caen 14074, France.</footnote>

<footnote>E-mail address: guenaelle.levallet@unicaen.fr (G. Levallet).</footnote>

<footnote n="1">¹ Equal contribution</footnote>
protein). This is achieved through the deregulation of YAP-1, GEFH-1/RHOB, and c-jun activities [9,10]. Notably, YAP-1 nuclear expression is higher in NSCLC with brain metastasis compared to primary NSCLC tumors [10]. The observation that NDR2/YAP-1 promotes brain metastasis in NSCLC suggests that NDR2 supports the survival of CTCs. Interestingly, YAP-1/TAZ are either activated or inhibited by shear stress depending on its nature: they are inhibited under steady, laminar flow but activated under disturbed or low, turbulent flow [11–14] (for review: [15]).

YAP-1 is a transcriptional cofactor that binds to DNA-binding transcription factors, such as TEADs [16], to regulate gene expression and subsequently controlling cell proliferation, survival, and differentiation [16]. Among YAP-1's target genes is amphiregulin (AREG) [17].

AREG is a type II cytokine and a member of the epidermal growth factor family. It is widely considered an oncogene and a potential biomarker, as it reflects tumor activity and the presence of metastatic cells [18]. AREG is overexpressed in the serum of NSCLC patients who do not respond to Gefitinib treatment and is associated with poor prognosis in NSCLC patients [19]. In pleural mesothelioma, the loss of AREG decreases the 2D migration, invasion, proliferation, and growth of malignant cell lines while increasing apoptosis [20]. Based on these findings, we hypothesize that the NDR2/YAP-1/AREG pathway could support the dissemination of NSCLC cells to the brain. Furthermore, we propose that AREG could serve both as a biomarker for early diagnosis and monitoring of brain metastases, and as a potential therapeutic target to prevent or treat metastasis in NSCLC patients. To test this hypothesis, we assessed the resistance of lung CTCs, depleted or not of NDR2, using a microfluidic model (Ibidi® Pump). Our results show that AREG, induced by NDR2/YAP-1 during exposure to shear stress or in BM, reflects BM progression and promotes PD-L1 expression in NSCLC.

## 2. Materials and methods

### 2.1. Patients

We retrospectively selected a cohort of 45 patients who underwent surgery for either non-metastatic (n = 25) or metastatic NSCLC (n = 20) and for whom both the primary tumor and the resulting brain metastasis (BM) were available, at Caen University Hospital between December 2009 and December 2019. Among the 25 patients with localized NSCLC, 17 were men and 8 were women with an average age of 71 years [54–86 years]. Among the 20 patients with metastatic NSCLC, 15 were men and 5 were women with an average age of 67 years [40–82 years].

All patients provided informed consent, and the study was approved by the institutional ethics committee (North-West-Committee-for-Persons-Protection-III N°DC-2008-588).

### 2.2. Mice brain metastasis model and plasma collection

All animal investigations were performed under the current European directive (2010/63/EU) following the ARRIVE guidelines, in the housing and laboratories approved (F14118001 and G14118001) and with the permission of the regional committee on animal ethics (C2EA-54 CENOMEXA, APAFIS #23280). Nude athymic mice (20–25 g, 8 weeks, male) were maintained in specific pathogen-free housing (ONCOModels platform located in Cyceron Centre). Mice were manipulated under general anesthesia (5 % isoflurane for induction, 2 % for maintenance with a flow rate of 1 l/min of 70 %N2O/30 %O2). Body temperature was monitored and maintained at 37.5 ± 0.5 °C throughout the experiments. H2030-BrM3 cells (1.75*10⁵ cells in 100-µl-PBS supplemented by glutamine (2 mM)) were administered under echograph imaging via intracardiac injection via a fine needle (27 G). Mice were randomly selected, and injected with H2030-BrM3 cells (shControl (shCtr) or shNDR2). The endpoints of the protocol to limit animal pain are described in the project authorization number #23280 (C2EA-54 CENOMEXA). The number of animals (n = 10/lot) was calculated to be

as low as possible while allowing robust measurements (the distribution is Gaussian and parametric tests are applicable).

During anesthesia for the MRI procedure, one mouse from the shCtr group and two mice from the shNDR2 group died. Additionally, in another mouse from the shNDR2 group, a metastasis was observed as early as the first MRI on day 7. The data from this animal were not included in the tumor growth and volume statistics.

At the time of euthanasia (either at the end of the protocol or due to the appearance of clinical warning signs), a blood sample was collected, and plasma was isolated using standard procedures within 30 min of blood collection.

### 2.3. Magnetic resonance imaging (MRI) acquisition and image analysis

The development of the brain metastases was monitored twice a week by anatomical MRI over a 56-day period using magnetic resonance imaging (MRI) on a 7 Tesla magnet (Pharmascan, Bruker, CYCERON biomedical imaging platform, Caen). A cross coil configuration was used (volume/surface coil, Bruker). All experiments were performed under isoflurane anesthesia: 5 % for induction and 2.5 % during imaging acquisition in a 1 L/min mixture of N2O and O2 (70 % and 30 %, respectively). The mouse was placed in a prone position in a brain cradle. Breathing was monitored in real time using a pressure balloon under the abdomen.

A scout imaging based on a FLASH (Fast Low Angle Shot) sequence (TR/TEeff: 100/4 ms; resolution 0.39 × 0.39 × 3 mm3, acquisition time = 12 s) was first used to verify the positioning of the animal and allow acquisition adjustments. T2-weighted imaging by rapid spin echo or RARE8 (Rapid Acquisition Relaxation Enhanced 8) sequence was then acquired with the following parameters: TR/TEeff = 5000/65 ms, number of repetitions = 3, spatial resolution = 0.078 × 0.078, 26 slices of 0.5 mm, acquisition time = 2 min.

Tumor delineation was performed using ImageJ software on all adjacent T2w slices and tumor volume was calculated by multiplying the sum of contiguous tumor surface areas by slice thickness.

All MRI acquisitions and subsequent analyses were performed blind to the experimental groups (shControl or shNDR2).

### 2.4. Cell culture, transfection and treatment

The isogenic HBEC-3 (and HBEC-3-cherry) bronchial epithelial cell lines were a generously provided by Dr. White (UT Southwestern Medical Center, Dallas, TX, USA) and were cultured in keratinocyte serum-free medium (KFSM) complemented with 0.2 ng/mL of human recombinant epidermal growth factor (EGF) and 25 µg/mL of bovine pituitary extract (BPE) supplements (Thermo Fisher Scientific, Rockford, IL, USA). The Human tumoral bronchial epithelial Cells A549 (CCL-185), H1975 (CRL-5908), and H2030 (CRL-5914) cells were purchased from the American Type Culture Collection (ATCC). The H2030-BrM3, i.e. H2030 cells which preferentially metastasize to the brain, were obtained from Dr Joan Massagué (MSKCC, USA). NSCLC cells were grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (4,500 mg/L) supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum. DMEM mediums were complemented by 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine (Gibco, Life Technologies, Grand Island, NY, USA). For the H2030-BrM3, H1299 and A549 cells, shRNA-depleted stable lines for NDR2 were used as previously described [9].

CD4⁺ T lymphocytes (#2W200), CD8⁺ T lymphocytes (#2W300), and NK cells (#2W502) were obtained from Lonza (Bâle, Suisse) and cultured in the supplier-recommended medium (Lymphocyte Growth Medium 3 (LGM3), Lonza, #CC3211). Microglial cells were purchased from CelProgen (#37089–01) and cultured in the appropriate medium, Human Microglial Cell Culture Serum-Free Media (HMM) (#M37089–01S, CelProgen). LGM3 as HMM mediums were complemented by 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM
1-glutamine (Gibco, Life Technologies, Grand Island, NY, USA).

The introduction of siRNA in NSCLC cells was performed using Lipofectamine RNAiMAX in accordance with the manufacturer's instructions at 40 % of cell confluence. RNAi oligonucleotides (Eurogentec®) sequences are listed in Table S1. The mock siRNA was employed as the non-silencing negative control (Eurogentec®). The silencing efficiency of each target was routinely verified by RT-PCR and/or Western Blot.

Cells were seeded and cultured at 37°C, 5 % CO₂ and replenished with fresh media every 3-4 days, until use. Cells were routinely tested for mycoplasma contamination using MycoAlert® Mycoplasma Detection Kits (Lonza, Colmar, France) and recently authenticated by STR profiling (Microsynth AG, Switzerland).

For application of shear stress, NSCLC cells (1–6.10⁶) were injected in the perfusion set linked to ibidi µ-Slide 0.4 (ibidi #80166) Luer ibi-Treat. Unidirectional laminar flow (0, 10, 20 or 40 dyn/cm2 from 30 min up to 6 h) was applied using the ibidi pump system (ibidi #10902). For treatment with exogene AREG, the cells were grown in their medium and then incubated or not with recombinant AREG (AREGr, #262-AR-100, R&D system) at 20 ng/mL during the microfluidic test (3 h) or in static condition (up to 48 h). For pharmacological rescue tests, Gefitinib and Osimertinib mesylate (Merck, Saint Quentain Fallavier, France) were added at 2 µM final concentration two hours before AREGr addition.

For time-Lapse Video Microscopy, NSCLC cells were cultured with Lymphocyte CD4 +, CD8 +, Natural Killer or microglia in glass bottom cell culture dishes and monitored by video microscopy in a humidified, CO₂-equilibrated chamber (Incubator i8, Singapore) using an inverted microscope (Leica DMi8, Wetzlar, Germany) equipped with a motorized stage controlled by Metamorph 7.8.13.0 software. The acquired images were then analyzed with the ImageJ software (version 1.50d).

## 2.5. Cell viability

NSCLC cells viability was measured using the NucleoCounter 3000 (Chemometec) and Via-1 viability cassettes (Chemometec), which contain Acridine orange and DAPI. Cell viability is expressed as a percentage of the total cell population analyzed.

## 2.6. Bromodeoxyuridine incorporation analysis

Cells were transfected, left for 24 h, and then labeled with bromodeoxyuridine (BrdU) (1:500 dilution, cell proliferation assay, Millipore) for an additional 24 or 48 h. BrdU incorporation was measured according to the manufacturer's instructions. The colored reaction was quantified using a microplate reader at 450 nm.

## 2.7. Caspase-3/7 assay

Caspase-3/7 activation was assayed using the Caspase-Glo 3/7 Luminescence Assay (Promega Corp., Madison, Wisc., USA), according to the manufacturer's instructions.

## 2.8. AREG measurements on plasma or cells

Amphiregulin release in the plasma of mice was quantified using the amphiregulin Quantikine ELISA kit (R&D system), according to the manufacturer's instructions.

Amphiregulin that was associated with cells (cellular AREG) or that was released in the culture medium (sAREG) was measured using a DuoSet-ELISA kit (R&D system), according to the manufacturer's instructions.

## 2.9. RNA preparation and RT-PCR

Total RNA was extracted from cells using the ReliaPrep™ RNA Cell

Miniprep System (Promega), according to the manufacturer's instructions. Total RNA (250 ng) was reverse-transcribed with random primers and 200 IU of M-MLV reverse transcriptase (37 °C, 90 min), followed by dissociation (70 °C, 5 min) with Mastercycler Eppendorf®. The resulting cDNAs were diluted (1/10) and used as templates. Polymerase chain reaction (PCR) was performed using an Mx3005P QPCR system (Agilent Technology, Les Ulis, France) with 5 pmol of each primer set (Table S2) and iTaq™ Universal Supermixes (Bio-Rad, Marnes La Coquette, France). S16 was used as an internal control. Positive standards and reaction mixtures lacking the reverse transcriptase were routinely employed as controls for each RNA sample. Relative quantification was calculated using the ΔΔCt method.

## 2.10. Antibodies, immunohistochemistry (IHC), immunocytochemistry (IC), immunoblotting and image analysis

The antibodies used in this work are listed in Table S3.

Immunohistochemistry was performed on 3 µm-thick Formalin-Fixed Paraffin-Embedded (FFPE) sections. All subsequent steps were carried out using the Discovery Ultra Ventana immunohistochemistry analyzer. The sections were deparaffinized and antigen retrieval was performed by incubating them with a Tris-EDTA-based buffer (pH 8.2, Ultra CC1 (#950-500, ready-to-use, Ventana Medical Systems, Tucson, AZ, USA) for 64 min at 95°C or with citrate (pH 6) or EDTA (pH 9) solution (Dako) for 20 min at 100 °C. The sections were then incubated with the primary antibodies listed in Table S3 at 1:75-1:300 according primary antibody then revealed HRP-conjugated secondary antibodies were detected using Ventana or Novolink (Leica®) kit. Nuclei were counterstained with Hematoxylin.

For immunocytochemistry, 2.10⁴ cells were seeded and grown on coverslips in 24-well tissue culture trays, washed with PBS and fixed with 4 % paraformaldehyde for 20 min at 37 °C. The cells were blocked with 4 % bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h and stained with primary antibodies (Table S3) at 4 °C overnight. After several washes with PBS, cells were stained with either Alexa-conjugated secondary antibodies (with 488 or 555 Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h at room temperature (RT) and with DAPI (4,6 diamidino-2-phenylindole) (Santa Cruz Biotechnology, Dallas, TX, USA). Digital pictures were captured using a high-throughput confocal microscopy (FluoView FV1000, Olympus). Ki-67 was analyzed using StarDist by averaging nuclear intensity and classifying cells as positive or negative based on a defined threshold (a minimum of 2000 cells were analyzed per condition). Cytochrome c-positive cells were quantified in ImageJ software using the Cell Counter tool (minimum of 250 cells per condition).

For immunoblotting, whole-cell protein extracts were prepared as previously described [10], and proteins were detected by immunoblotting with the primary antibody (Table S3) diluted to 1:1000 in Tween (0.1 %-TBS buffer, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody, then revealed by enhanced chemiluminescence using the Clarity Western ECL Substrate (BioRad). Densitometry results of western blot were analyzed with Image J software. The signal intensity of each band was normalized with GAPDH densitometry values.

## 2.11. Statistical analysis

Data were expressed as mean ± SEM of experiments, independently conducted three times for in vitro analysis. Correlation was evaluated by a Pearson coefficient. All statistical analyses were performed using GraphPad Prism 4, a GraphPad Software program (San Diego, CA, USA). The data were analyzed using a two-tailed Student's t-test for single comparison or one-way ANOVA followed by Dunnett's multiple comparison analysis. Differences were considered significant at the p < 0.05 level.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** NDR2 reduces shear stress-induced apoptosis in human bronchial epithelial cells upon reseeding. (A–D) Between 1 and 6 × 10⁶ NSCLC cells transduced with either control (shCtr) or NDR2-silencing (shNDR2) constructs were subjected to shear stress (up to 40 dyn/cm²) for durations ranging from 30 min to 6 h. At the end of the incubation, we measured: (A) the number of recovered cells, (B) their viability using the NucleoCounter 3000 (Chemometec) and Via-1 viability cassettes (Chemometec), (C) NDR2 expression by RT-qPCR, and (D) NDR2 protein levels by Western blot. (E–H) Following shear stress, H2030-BrM3 were reseeded, and (E) cell density was evaluated 24–48 h later; (F) proliferation was assessed via BrdU incorporation assay; (G) apoptosis was quantified by cytochrome c release; and (H) a representative image illustrating cytochrome c release is shown. The scale bar indicates 50 µm. (A–G) Data are presented as mean ± SEM, n = 3. Statistical significance is indicated as *p < 0.05 and **p < 0.01 compared to the shCtr group.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Shear stress (40 dyn/cm²) upregulates the expression of the YAP-1 target gene *Amphiregulin* in various NSCLC cell lines. (A–J) 3 × 10⁶ NSCLC cells transduced with either control (shCtr) or NDR2-silencing (shNDR2) constructs were exposed to shear stress ranging from 0 to 40 dyn/cm² for 3 h. At the end of the incubation, we assessed the P-YAP-1/YAP1 ratio by Western blot in H2030 cells (A) and H2030-BrM3 cells (B, with a representative blot shown alongside the quantification). Cells were then reseeded to further examine YAP-1 subcellular localization by immunofluorescence in A549 cells (C: representative image; D: quantification of nuclear YAP-1 intensity) and in H2030-BrM3 cells (E: representative image; F: quantification of nuclear YAP-1 intensity). (G–J) Total RNA was extracted and the expression of YAP-1-regulated genes — CTGF, ANKRD1, and Amphiregulin (AREG) — was analyzed by RT-qPCR in H2030, H2030-BrM3, A549, and H1975 cells (G). AREG mRNA expression was also assessed in NSCLC cells following exposure to varying levels of shear stress (H), and in A549 (I) and H2030-BrM3 (J) cells, either with or without NDR2 depletion, after shear stress stimulation. (C-D) The scale bar indicates 50 µm. (A–J, except C and E) Data are presented as mean ± SEM, n = 3. Statistical significance is indicated as *p* < 0.05, **p** < 0.01 and ***p** < 0.001 versus the shCtr group.</figcaption></figure>
## 3. Results

### 3.1. NDR2 attenuated apoptosis triggered by shear stress in human bronchial epithelial cells after reseeding

We subjected 1–6.10⁶ NSCLC cells to a shear stress of up to 40 dyn/cm² lasting 30 min to 6 h. We observed that the number of cells recovered after flow decreases progressively over time, as illustrated under the condition of 3 million H2030 cells at 40 dyn/cm² (Fig. 1A). This decline occurred regardless of the initial number of cells (ranging from 1 to 6.10⁶ cells) injected into the microfluidic system. Near 90 % of the retrieved cells collected at the end of the flow, remained viable (Fig. 1B, shown for H2030). We analyzed NDR2 expression (mRNA and protein levels) following NSCLC cells exposure to shear stress. NDR2 mRNA levels nearly doubled depending on the cell line and shear stress force (Fig. 1C). Although this variation had not yet translated into a detectable change in protein expression (Fig. 1D), evidence of an apparent activation (phosphorylation) of NDR1/2 was also observed (Fig. 1D). To understand the role played by NDR2, we then used NSCLC cells depleted of NDR2 by shRNA (shNDR2) or their control (shCtr) (Fig. 1D).

In NSCLC cells subjected to shear stress and then reseeded, cell confluence measured at 24 or 48 h post-seeding, was similar in flow-exposed H2030-BrM3 shCtr cells compared to unexposed ones (Fig. 1E). Cell confluence was lower in H2030-BrM3 shNDR2 cells than in H2030-BrM3 shCtr cells, especially when exposed to a shear stress of 40 dyn/cm² for 3 h (Fig. 1E). No significant confluence change was observed in A549 cells under similar conditions (not shown). Proliferation analysis via BrdU incorporation revealed a significant 52 % reduction (p = 0.0236) in H2030-BrM3 shCtr cells compared to un-stressed controls (Fig. 1F). This reduction was not observed in A549 cells (not shown). Interestingly, under static conditions, shNDR2-H2030-BrM3 (Fig. 1F) as shNDR2-A549 (not shown) cells exhibit lower proliferation than shCtr cells. This difference in BrdU incorporation between shCtr and shNDR2 cells disappeared in cells exposed to 3 h at 40 dyn/cm². NSCLC cells exposed to 40 dyn/cm² during 3 h, displayed twice as many apoptotic cells (cells with diffuse cytoplasmic cytochrome C staining) compared to their non-sheared counterparts (Fig. 1G and H: H2030-BrM3; not shown for A549 cells). NDR2-deficient (shNDR2) H2030-BrM3 or A549 cells showed a trend toward increased apoptosis following arterial flow exposure compared to shCtr cells.

### 3.2. Shear stress (40 dyn/ cm²) increased the expression of the YAP-1 target gene Amphiregulin in NSCLC cells

Given that the pro-metastatic effects of NDR2 rely on YAP-1 ⁹, we investigated how a 3-hour exposure to arterial-type flow (40 dyn/cm²) influences the expression of YAP-1 and its downstream target genes in A549, H2030 and H2030-BrM3 cells, with (shControl) or without NDR2 (shNDR2) expression. YAP-1 mRNA level was not significantly altered by shear stress in either cell line (data not shown), although a slight decrease was observed in NDR2-deficient cells. Total form of YAP-1 decreased when H2030 (Fig. 2A) or H2030-BrM3 (Fig. 2B) cells were exposed to 40 dyn/cm² of shear stress, while the Ser127-phosphorylated form increased markedly. Notably, this increase was only partially attributable to shear stress, as culturing the cells in suspension (using a non-adherent substrate) also led to an increase in the phosphorylated form, although to a lesser extent than under shear stress (Fig. 2A/B).

Further, analysis of intranuclear YAP-1 staining intensity by immunocytochemistry revealed no significant changes in YAP-1 nuclear localization in NSCLC cells (illustrated for A549 Fig. 2C/D, and H2030-BrM3, Fig. 2E/F) 48 h following reseeded next a 40 dyn/cm² shear stress.

We assessed the expression of some YAP-1 target genes—AREG, CTGF and ANKRD1—via RT-qPCR following a 40 dyn/cm² shear stress. AREG mRNA expression significantly increased in all NSCLC cells line (Fig. 2G). We noted that this increase in AREG transcription occurs as

early as 10 dyn/cm² (Fig. 2H). Moreover, AREG-induced by shear stress increase whatever the level of NDR2 (Fig. 2I/2 J) even if this induction was reduced in NDR2-deficient A549 cells (Fig. 2I). A similar, though less pronounced, trend was observed for ANKRD1, suggesting that YAP-1-mediated transcriptional responses under shear stress conditions in NSCLC cells (Fig. 2G).

### 3.3. Amphiregulin supported NSCLC cells survival and proliferation in response to shear stress

We treated the cells with exogenous recombinant AREG (AREGr, 20 ng/mL), a concentration that does not induce desensitization of its receptor EGFR (i.e., internalization, Supplementary figure 1A) but is sufficient to activate the receptor, as confirmed by increased Phospho-ERK1/2 level (Supplementary figure 1B). Additionally, endogenous AREG expression was silenced using siRNA (siAREG) in NSCLC cells (Fig. 3A/B). NSCLC cells were then exposed or not to 40 dyn/cm² shear stress for 3 h to assess whether siAREG effectively prevents shear stress-induced AREG mRNA expression (Fig. 3B).

NSCLC cells transfected with either siNeg or siAREG were exposed or not to 40 dyn/cm² shear stress for 3 h, in the presence or absence of recombinant AREG (20 ng/mL), and then reseeded for 24 h. We next evaluated proliferation (Ki67 intensity measure, Fig. 3C/D) and apoptosis (Caspase 3/7 Glo measure, Fig. 3E) following these treatments. H2030-BrM3 cells exhibited higher baseline proliferation than H2030, consistent with their metastatic potential. Silencing AREG in H2030 cells decreased proliferation, while addition of AREGr increased the number of Ki67-positive cells (Fig. 3C/D). In H2030-BrM3 cells, AREG silencing reduced Ki67-positive cells by approximately half. Surprisingly, AREGr did not increase proliferation in H2030-BrM3 cells, regardless of whether AREG was silenced (siAREG) or not (siNeg) (Fig. 3C/D).

AREGr did not significantly affect caspase activity in H2030 or H2030-BrM3 cells (Fig. 3E). Interestingly, in AREG-depleted H2030-BrM3, caspase activity was reduced and was not restored by AREGr (Fig. 3E). Together, these findings suggest that endogenous AREG plays a critical role in the regulation of proliferation and apoptosis in NSCLC cells.

### 3.4. Shear stress potentially triggered dedifferentiation in NSCLC cells

We observed that exposing A549, H2030, and H2030-BrM3 cells to shear stress (40 dyn/cm² for 3 h) altered their morphology: once returned to static conditions, these cells appeared more rounded compared to their non-flow-exposed counterparts (not shown). We analyzed the expression of epithelial and mesenchymal markers to determine whether the observed morphological changes were attributable to an epithelial-to-mesenchymal transition (EMT). Only Snail expression was found to be induced by shear stress (Supplementary figure 2A). Indeed, in H2030-BrM3 cells, neither shear stress nor silencing of NDR2 or AREG significantly affected the mRNA expression of epithelial–mesenchymal transition (EMT) markers such as N-cadherin (Supplementary figure 2B), E-cadherin (Supplementary figure 2C), or vimentin (Supplementary figure 2D).

The expression of the dedifferentiation markers SOX9 and SOX2 was also evaluated. We report a decrease in SOX9 (Supplementary figure 2E) and SOX2 (Supplementary figure 2F) mRNA levels following either NDR2 silencing or exposure to shear stress, with no additive effect when both conditions were combined. A similar reduction in SOX2 and SOX9 transcripts upon 3-hour exposure to 40 dyn/cm² shear stress was also observed in the H1975 cell line (Supplementary figure 2E-F). In contrast, in HBEC-3, a non-tumorigenic immortalized cell line, shear stress reduced SOX2 expression but paradoxically increased SOX9 levels (Supplementary figure 2E-F). SOX2 variations are also found at the protein level in the A549 cell line (Supplementary figure 2G).
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Amphiregulin enhances the survival and proliferative capacity of NSCLC cell lines following shear stress. (A–E) $3 \times 10^6$ NSCLC cells depleted (siAREG) or not for AREG (siNeg) were exposed to shear stress ranging (40 dyn/cm$^2$) for 3 h, in presence or not of recombinant AREG (20 ng/mL) during the test. (A) Morphology of H2030 and H2030-BrM3 cells, with or without AREG depletion, observed by phase-contrast microscopy (objective magnification $\times 10$). The scale bar indicates 50 µm. (B) Silencing of AREG (siAREG) or control (siNeg) in H2030 (green bars) and H2030-BrM3 (blue bars) was confirmed by RT-qPCR. At the end of the shear-stress exposure, H2030 and H2030-BrM3 cells were reseeded for 24 h, then: (C–D) Ki67 expression was assessed by immunocytochemistry (C: representative image, the scale bar indicates 500 µm.; D: quantification of Ki67 intensity), and (E) caspase 3/7 activity was measured using the Caspase-Glo® 3/7 assay. (B, D, E) Data are presented as mean $\pm$ SEM, n = 3. Statistical significance is indicated as *$p$ < 0.05 and **$p$ < 0.01 versus the shCtr group.</figcaption></figure>

**3.5. Plasma levels of AREG released by xenografted H2030-BrM3 cells was positively correlated with brain metastasis volume in a late brain metastasis mouse model**

We employed a mouse model of brain metastases (BM) using intra-cardiac injection of H2030-BrM3 cells (shControl (shCtr) or shNDR2) into Nude athymic mice (n = 10 per group). RT-qPCR and western blot confirmed the silencing of NDR2 expression in H2030-BrM3 cells injected into mice (Fig. 4A). The first BM was detected by MRI at day 24 in the shCtr group, and at day 38 for shNDR2 group (Fig. 4B). A significant difference (Log-rank (Mantel-Cox) test p = 0.0357) in median onset time of brain metastases was noticed with 42 days for shCtr vs 56 days for shNDR2 group. At the end of protocol, a significant reduction of

BM number was observed per mice in shNDR2 group compared to the shCtr (1.00 $\pm$ 0.36 vs 2.70 $\pm$ 0.51 respectively) (Fig. 4C). By day 46, the average volume per BM reached 3.57 $\pm$ 1.02 mm$^3$ in the shCtr group, compared to only 1.09 $\pm$ 0,25 mm$^3$ in the shNDR2 group (Fig. 4D) and the rate of BM growth was greater in shCtr group (1.48 $\pm$ 0.25 mm3/day) than in shNDR2 group (0.38 $\pm$ 0.06 mm$^3$/day, p = 0.003, Fig. 4E) as illustrated on MRI pictures in Fig. 4G. In line, the control group displays a poorer overall survival compared to the shNDR2 group (Fig. 4F). Notably, immunohistochemistry against NDR2 or AREG performed on BM samples at the end of collection (i.e., on average 48.37 $\pm$ 2.59 days and 53.00 $\pm$ 3.00 after intracardiac cell implantation for shCtr and shNDR2 group respectively) revealed at this stage, comparable protein expression levels in brain metastases arising from both shCtr and
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Plasmatic AREG levels correlate with brain metastases (BM) in a nude athymic mice BM model involving intracardiac injection of H2030-BrM3 cells (shCtr or shNDR2). (A) Silencing of NDR2 (shNDR2) or control (shCtr) in H2030-BrM3 was confirmed by RTqPCR and western blot before intracardiac injection to Nude mice (a single cell preparation was aliquoted for each batch/group of 10 animals). (B) Brain metastasis onset, (C) number per mouse, (D) volume per BM at J46 and (E) growth rate of BM monitoring using T2w MRI imaging for all mice every 3 days from 7 days post-injection (D7) to 56 days. Mean ± SEM, (shCtr n = 9; shNDR2 n = 7) \*p < 0.05 and \*\*p < 0.01 vs shCtr group. (F) Overall survival and (G) representative T2w-MRI images of the BM growth from representative shCtr and shNDR2 mice. (# Mice who died during anesthesia for the MRI procedure.). (H) Representative immunohistochemically (IHC) analysis of AREG and NDR2 staining in brain metastases from shCtr and shNDR2 mice (the scale bar indicates 50 µm) and (I) IHC quantification by a H-Score (percent of tumoral cells stained 0–100 %) x intensity of the stain (0–3)). (J) Plasmatic AREG levels were measured in nude athymic mice with brain metastases (BM) following intracardiac injection of H2030-BrM3 cells transduced with either shControl (shCtr) or shNDR2. AREG concentrations are expressed in pg/mL of plasma per animal (Identification number of each animal is shown on the X axis). The curves superimposed on the bar graphs (representing plasmatic AREG levels) correspond to BM volumes, expressed in mm³.</figcaption></figure>
shNDR2 cell injections (IHC illustrations: Fig. 4H; and quantification: Fig. 4I).

Plasma AREG levels were successfully measured in 8 (shCtr) or 7 (shNDR2) out of 10 animals per group (some mice died outside of the scheduled monitoring periods). Of note, the assay specifically detects human AREG and not mouse one. The average plasma AREG concentration was 10.99 ± 3.97 pg/mL in the shCtr group versus 6.80 ± 2.80 pg/mL in the shNDR2 group. Since plasma AREG levels appeared to correlate with tumor burden, we investigated this relationship and found a correlation between AREG plasma level and BM volume (Pearson correlation coefficient: r = 0.896, p < 0.0001, Fig. 4J). Of note, mouse #08 (which has been excluded, as mentioned above) exhibited an unusual high AREG level (132.25 pg/mL) along with a very large tumor (38.78 mm³).

### 3.6. Tumoral AREG expression tended to correlate with PD-L1 levels in brain metastases from NSCLC patients

We assessed AREG expression in 25 patients with localized NSCLC and 20 patients with metastatic NSCLC (Fig. 5). We observed differences

in the subcellular localization of AREG (Fig. 5A): in localized NSCLC, AREG was detected in both the cytoplasm and at the cell membrane. In contrast, in primary metastatic NSCLC tumors, AREG was confined to the cell membrane or nuclear, while in brain metastases, AREG was found both in the cytoplasm and at the cell membrane.

AREG expression was lower in primary localized NSCLC tumors (H-Score: 4 ± 11.5, n = 25) than in primary metastatic NSCLC tumors (H-Score: 23.2 ± 12.3, n = 25). Specifically, 36.8% (7/19) of metastatic patients showed AREG expression while only 8.0% (2/25) of localized patients expressed AREG (Fig. 5B). Furthermore, AREG expression was higher in brain metastases (BM) compared to primary tumors from the same metastatic NSCLC patients, with 35.0% (7/20) of patients showing AREG expression (H-score: 30.5 ± 12.0) (Fig. 5B). Notably, four patients expressed AREG in both their primary tumor and BM.

We investigated whether brain metastases of bronchial origin exhibited differential expression of MST3 (the major regulator of NDR2, [21]), NDR2, or YAP-1 according to their AREG expression status. Our results demonstrate that only NDR2 showed a tendency (p = 0.0657) to slightly decrease in AREG-positive samples compared to AREG-negative ones (Fig. 5C).

<figure><img src="image_7.png" /><figcaption>AREG / Hematoxylin</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** AREG-positive brain metastases from NSCLC patients display higher PD-L1 expression in tumor cells than AREG-negative counterparts. AREG expression was evaluated by immunohistochemistry in 45 patients who underwent surgery for non-metastatic NSCLC (n = 25) or metastatic NSCLC (n = 20), for whom both the primary tumor and corresponding brain metastasis (BM) samples were available at Caen University Hospital between December 2009 and December 2019. (A) Representative images illustrating the possible subcellular localization patterns of AREG. The scale bar indicates 25 µm. (B) Quantification of AREG by immunohistochemistry using an H-score: (% of tumor cells stained, 0–100%) × staining intensity (0–3). Data are presented as mean ± SEM; p-values are shown above the histograms. (C–F) Histological characterization of brain metastases (BM) based on the presence (with) or absence (wo/without) of AREG expression, including: (C) Expression of major AREG interactors (MST3, NDR2, YAP-1), (D) Expression of immune cell lineage markers (CD163, CD20, CD3, CD16), (E) Neo-angiogenesis marker (CD34), (F) Immunosuppressive marker (PD-L1). Data are presented as mean ± SEM; p-values are indicated above the histograms.</figcaption></figure>
AREG has been described as possessing immunosuppressive [12,22] and angiogenic [23–26] properties. Accordingly, we analyzed the density of cells per mm² in NSCLC’s BM, focusing on the expression of clusters of differentiation (CD) specific to hematopoietic lineages—namely: CD163 (monocyte/macrophage/microglia lineage), CD20 (B lymphocytes), CD3 (T lymphocytes), CD16 (Natural Killer cells) (Fig. 5D), as well as neovascular markers (CD34) (Fig. 5E) and the immune checkpoint protein PD-L1 (Fig. 5F). These histological analyses were conducted based on whether the BM expressed AREG or not. None comparison reached statistical significance—likely due to a lack of statistical power (Fig. 5C/F). However, our analysis indicated that AREG-expressing samples tended to exhibit a similar infiltration of CD163 + and CD3 + cells (respectively: p = 0.82 and p = 0.99, Fig. 5D) as CD34 expression (p = 0.52, Fig. 5E), alongside a reduced presence of CD20 + cells (p = 0.38, Fig. 5D) but an increase of CD16 + cells (p = 0.34, Fig. 5D) compared to AREG-negative samples. Interestingly, tumor cells within AREG-positive samples appeared to display elevated levels of PD-L1 (p = 0.14, Fig. 5F) expression relative to those in AREG-negative samples.

### 3.7. Exogenous AREG induced PD-L1 expression in NSCLC cells

We next incubated H2030, H2030-BrM3, A549 or H1975, either expressing AREG (siNeg) or silenced for AREG (siAREG), with recombinant AREG (AREGr) at 20 ng/mL for 48 h (Fig. 6: H2030-BrM3; Supplementary figure 3: H2030 and H1975). We observed that AREG knockdown (siAREG) reduced PD-L1 mRNA levels by nearly twofold (Fig. 6A) and decreased PD-L1 protein expression by approximately 90% (Fig. 6B). Conversely, treatment with AREGr increased PD-L1 mRNA expression by about 50% (Fig. 6A) and nearly doubled PD-L1 protein levels (Fig. 6B). This upregulation of PD-L1 expression required both the presence of NDR2, as AREGr failed to induce PD-L1 protein upregulation in its absence (Fig. 6B), and EGFR signaling, as Osimertinib or Gefitinib (EGFR inhibitors) prevented PD-L1 protein upregulation (Fig. 6C/D). Similar results were obtained with H2030 and A549 cell lines (Supplementary figure 3). Notably, inhibition of EGFR with Osimertinib or Gefitinib (2 µM) added to the cell medium 2 h prior to exogenous AREG treatment, also prevents the upregulation of two YAP1 transcriptional targets: AREG and CCNE1 (Supplementary figure 4).

Finally, we incubated H2030-BrM3 cells expressing either control shRNA (shCtrl) or NDR2-targeting shRNA (shNDR2) in the presence of T lymphocytes or NK cells (Supplementary figure 5A and movies: CD4⁺ T cells—movie 1 with H2030-shCtrl and movie 2 with H2030-shNDR2; CD8⁺ T cells—movie 3 with H2030-shCtrl and movie 4 with H2030-shNDR2; NK cells—movie 5 with H2030-shCtrl and movie 6 with H2030-shNDR2), or seeded on a microglial cell monolayer (Supplementary figure 5B and movie 7 with H2030-shCtrl and movie 8 with H2030-shNDR2). These exploratory results suggest that H2030-BrM3 cells behave similarly regardless of NDR2 expression in the presence of these immune cells. However, we observed that CD4⁺ T cells induce death of H2030-BrM3 (movies 1 and 2), and notably, CD4⁺ T lymphocytes show increased interaction (more lymphocytes per H2030-BrM3 and longer contact duration) with H2030-BrM3 shNDR2 cells compared to shCtrl.

Supplementary material related to this article can be found online at doi:10.1016/j.biopha.2025.118600.

## 4. Discussion

Currently, there is a lack of sensitive and specific tools for the early detection and monitoring of metastasis formation in patients with non-small cell lung cancer (NSCLC), despite metastases developing in over 50% of patients during disease progression—a major factor contributing to poor prognosis and reduced survival [27,28]. This unmet clinical need highlights the importance of identifying reliable biomarkers

and imaging modalities to improve early diagnosis and therapeutic intervention [29]. A hallmark of NSCLC patients with BM is the disruption of the Hippo signaling pathway, which leads to uncontrolled NDR2 activity [9,10]. NDR2 controls the Hippo pathway’s effectors, particularly YAP-1, by promoting its nuclear translocation through the inactivation of RHOB [9]. Once in the nucleus, YAP-1 induces the transcription of multiple genes, which represents a key event in metastasis formation [9,10]. Among the genes induced by the YAP-1 activity is *amphiregulin* (AREG) [17].

NSCLC cells tolerated shear stress, which is consistent with previous findings [30]. However, unlike H2030 cells, the reduction in confluence and BrdU incorporation induced by shear stress was not observed in A549 cells, potentially due to the presence of an *STK11* gene mutation in A549, which leads to resistance to anoikis and enables cells to survive even when detached from the extracellular matrix [31,32]. Shear stress (40 dyn/cm² for 3 h), early upregulated NDR2 mRNA in NSCLC cells, which subsequently showed reduced apoptosis after reseeding. These effects are consistent with the established roles of NDR2 in regulating cell proliferation and apoptosis. NDR kinases contribute to the progression of the G1 [33,34] and S [35] phases of the cell cycle and regulate cell death via YAP-1 [36]. NDR2 could therefore enhance the survival and proliferation of NSCLC circulating tumor cells (CTCs), promoting metastasis by helping them resist apoptosis. This hypothesis is supported by data reporting delayed or reduced brain metastasis formation in Swiss nude mice after intracardiac implantation of H2030-BrM3 shNDR2 cells, compared to control cells (present data and [10]).

We explored the role of YAP-1, a substrate of NDR2, and its target genes. Although previous studies have shown that low shear stress can activate YAP-1, leading to a rapid and transient expression of *CTGF*, *CYR61* or *ANKRD1* [37], our study did not evidence significant increase in *CTGF*, *CYR61* or *ANKRD1* expression (via RT-qPCR), nor detect YAP-1 nuclear translocation (via immunocytochemistry).

The mechanical stimuli generated by shear stress can differentially regulate the nuclear translocation of YAP-1 [12,14,37,38]. The ibidi Pump System maintains unidirectional flow via air-pressurized medium circulation; however, reservoir filling and 90° µ slide connections can induce turbulence, exposing cells to variable shear stresses similar to those experienced by circulating tumor cells in vascular branches. Moreover, the upregulation of guanine nucleotide exchange factor (GEF)-H1, a microtubule-associated Rho GEF, is a critical step in shear stress-induced YAP translocation. GEF-H1 mediates the nuclear activation of YAP by transmitting biomechanical signals from F-actin and Rho-dependent signaling [38,39] but also in response to NDR2 activation [9]. We also reported that YAP Ser127-phosphorylated form increased when cells were exposed to 40 dyn/cm² of shear stress. However, phosphorylation at Ser127 does not abolish all YAP-1 activity consistent with other studies [40,41]. Indeed, we confirmed that YAP-1 is likely activated after exposure to shear stress in NSCLC cells, as indicated by the increased expression of *amphiregulin* (AREG). Taken together, these data suggest a temporally limited or functionally partial activation of YAP-1 in NSCLC cells exposed to 40 dyn/cm² of shear stress. To explain the preferential transcription of AREG in this context, where NSCLC cells are exposed to shear stress, one may speculate that YAP-1 changes its co-transcriptional partner depending on the cellular context to prioritize the transcription of genes essential for adaptation to such stress. This is supported by the work of Jang et al., which reports that RAC-LATS1/2 signaling regulates YAP activity by switching its binding partners between TEAD4 and RUNX3 [42]. Another explanation could be that this activation also involves c-Jun. Indeed, i) AREG expression is induced through c-Jun and YAP-mediated enhancer activation during mitochondrial stress [43], ii) NDR2 promotes tumor growth and brain metastasis by deregulating YAP-1, GEF-H1/RhoB, and c-Jun activities [9,10], and iii) c-Jun expression is upregulated in response to shear stress and pressure [44]. This point warrants further investigation.
<figure><img src="image_9.png" /><figcaption>**Fig. 6. AREG Stimulation Drives PD-L1 Upregulation in H2030-BrM3 cells in an EGFR-dependent manner.** (A–D) H2030-BrM3 cells, either transfected with control siRNA (siNeg) or silenced for AREG (siAREG), were treated or not with recombinant AREG (AREG) at 20 ng/mL for 24 or 48 h (A-B), Gefitinib or Osimertinib (2 µM) was added two hours before AREGr addition in (C) and (D). PD-L1 expression was assessed at the mRNA level by RT-qPCR (A, C) 24 h after AREG addition and at the protein level by Western blot (B, D) 48 h after AREG addition. Data are presented as mean ± SEM, n = 3. Statistical significance is indicated as \*p < 0.05 and \*\*p < 0.01 versus the siNeg group (A-B) or the “Ctrl wo AREG” group (C-D).</figcaption></figure>
AREG, an EGFR ligand, is overexpressed in many lung cancers and is associated with poor prognosis, especially in NSCLC patients treated with Gefitinib [19]. AREG promotes tumor growth by supporting angiogenesis through VEGF-A production [23] and contributes to immune tolerance in the tumor microenvironment, where regulatory CD4 + T cells can express and respond to AREG [22]. NDR2 could therefore indirectly facilitate NSCLC metastasis by activating AREG's roles in neovascularization and immune tolerance.

We next demonstrated that silencing AREG prevents the significant increase in AREG expression induced by shear stress. We also observed decreased apoptosis following flow exposure, which contrasts with the expected high shear stress effects on circulating tumor cells [45]. The cellular response to shear stress varies by cell type, as shown by the almost negligible cell death in HCT116 colorectal carcinoma cells after 20 h of flow exposure [45]. Shear stress has also been shown to induce autophagy, which increases the migratory and invasive capacities of cells, as seen in HeLa cells. [46] These findings, combined with the observed reduction in caspase activity, highlight complex mechanisms of flow-induced cell survival and metastasis formation. It was surprising that a supposed survival factor led to a lower caspase readout. However, to note, AREG plays an ambivalent role in cell survival and apoptosis, promoting survival via EGFR signaling in some contexts [47,48], while contributing to pro-apoptotic responses or drug resistance in others [49], depending on cellular and environmental conditions.

Then, we found that Ki-67 staining showed increased proliferation in H2030-BrM3 cells compared to H2030-WT cells, consistent with their metastatic nature. The addition of exogenous AREG increased proliferation, while AREG silencing reduced it. These results suggest that AREG impacts the proliferation of both bronchial tumor cells with brain metastases and primary bronchial tumor cells. However, we did not observe significant differences in staining between control cells and those exposed to flow, contrary to previous report [30], who attributed unchanged viability to increased proliferation. Nonetheless, they found decreased proliferation with higher flow velocities, indicating the need for further optimization of the flow protocol to better mimic physiological conditions. Additionally, studies suggest that shear stress can lead to G2/M cell cycle arrest, which may explain the lack of proliferation observed [50]. Further studies are required to refine the flow protocol for more accurate modeling of metastasis processes.

We observed that both shear stress (40 dyn/cm²) and NDR2 silencing reduce SOX2 and SOX9 expression in tumor cell lines (H2030-BrM3, H1975) and, to a lesser extent, in the non-tumorigenic HBEC-3 line. This downregulation of SOX2, a key stemness marker, is consistent with previous findings in A549 cells [51] and may reflect an adaptive mechanism by which circulating tumor cells (CTCs) maintain their tumor phenotype and avoid differentiation, notably into endothelial-like cells, as SOX2 is involved in endothelial transdifferentiation [52]. The differential regulation of SOX9, particularly its upregulation in HBEC-3, suggests that the response to mechanical forces is context-dependent. Overall, these findings support the idea that shear stress and NDR2 influence tumor cell identity by modulating transcription factors involved in plasticity and adaptation.

Using a mouse model of BM, involving intracardiac injection of H2030-BrM3 cells (shControl or shNDR2) into nude athymic mice, we observed that plasma levels of AREG secreted by xenografted H2030-BrM3 cells positively correlated with brain metastasis volume in late-stage disease. Some studies previously suggest that larger or more advanced tumors tend to secrete more AREG, but in hepatocellular carcinoma [53] or in colorectal cancer [54]. Such data make circulating AREG a potential biomarker of tumor size and progression. We reported that the average plasma AREG concentration was 10.99 ± 3.97 pg/mL in the shControl group versus 6.80 ± 2.80 pg/mL in the shNDR2 group. This range of circulating plasma AREG levels is consistent with a study published by others, who stratified lung cancer patients based on a threshold of 10 pg/mL [55]. The fact that these levels are relatively low is also consistent with the very short half-life of AREG mRNA, which is

approximately 5 h [56].

We then observed the tumoral AREG expressed in human brain metastases is nearly significantly correlated with PD-L1 levels in the same cells, suggesting that AREG may regulate PD-L1 expression. However, a larger validation cohort in the future would strengthen these conclusions. To our knowledge, this hypothesis has previously been described primarily in the context of senescent cells [57]. AREG, being a ligand of EGFR, could promote PD-L1 expression by engaging multiple pathways, including MAPK/ERK [58], PI3K/AKT/mTOR [59], STAT3 [60], ER stress responses [61], or hypoxia-induced HIF-1α signaling [62]. We thus treated NSCLC cell lines expressing or not AREG *in vitro* with exogenous AREG for up to 48 h and we confirmed that AREG influences the expression of PD-L1, supporting the role of AREG as a protector of tumor cells against the immune system. The observation that CD4+ T lymphocytes show increased interaction (more lymphocytes per tumor cell and longer contact duration) with H2030-BrM3 shNDR2 cells compared to shCtrl supports the hypothesis that the presence of NDR2, possibly through its regulation of AREG, contributes to immune evasion. These preliminary findings need to be confirmed with additional functional analyses (cytotoxicity, lymphocyte activation, expression of activation or inhibitory markers).

## 5. Conclusion

Here we demonstrated that AREG not only supports tumor cell adaptation and proliferation in mechanically dynamic environments but may also facilitate immune escape through PD-L1 upregulation. The exact mechanistic link between shear stress, NDR2/YAP signaling, and AREG expression remains to be fully elucidated. Finally, plasmatic AREG being correlated to brain metastasis suggests that AREG could serve as a valuable biomarker for monitoring NSCLC metastatic progression and represents a promising therapeutic target.

## CRediT authorship contribution statement

**Elodie Peres:** Writing – review & editing, Validation, Methodology, Formal analysis. **Jérôme Toutain:** Writing – review & editing, Validation, Methodology, Formal analysis. **Laureline Charrier:** Writing – review & editing, Methodology, Formal analysis, Data curation. **Lucie Malandain:** Writing – review & editing, Validation, Methodology, Formal analysis, Data curation. **Jérôme Levallet:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Formal analysis, Data curation, Conceptualization. **Jeannick Madelaine:** Writing – review & editing, Validation, Funding acquisition. **Samuel Valable:** Writing – review & editing, Validation, Methodology, Formal analysis. **Maelle Guyot:** Writing – review & editing, Validation, Methodology. **Céline Bazille:** Writing – review & editing, Visualization, Validation, Methodology, Formal analysis. **Tiphaine Biojout:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Emmanuel Bergot:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

**Dimitri Leite Ferreira:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Guénaëlle Levallet:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Funding

Tiphaine Biojout benefits from a doctoral grant from the Normandy Region (RIN 100 % PhD student, 2022). Research grants were from the
AIR (Association des Insuffisants respiratoires) to D Leite Ferreira (2024). Research grants were from the ADP (Association pour le développement de la Pneumologie) to J Madelaine (2024). This research was also funded by the Centre National de la Recherche Scientifique (CNRS) and the Université Caen-Normandie (UNICAEN).

## Declaration of Competing Interest

The authors declare no conflicts of interest related to the data presented in this article.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2025.118600.

## Data availability

Data will be made available on request.

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