Lung Adenocarcinoma Cells Respond Differently to Mechanical Stress in 3D Versus 2D Environments

Lung Adenocarcinoma Cells Respond Differently to Mechanical Stress in 3D Versus 2D Environments | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lung Adenocarcinoma Cells Respond Differently to Mechanical Stress in 3D Versus 2D Environments Naoya Kitamura, Mayumi Iwatake, Satoshi Mizoguchi, Shadil Wani, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6482124/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract The tumour microenvironment is influenced by mechanical stress, including shear and stretch forces, which regulate cancer cell behaviour. Although two-dimensional (2D) culture models are commonly used in cancer research, they fail to recapitulate complex mechanical cues of native tissues. In this study, we developed an ex vivo three-dimensional (3D) lung cancer model by seeding human lung adenocarcinoma cells into decellularised rat lungs and culturing them in a bioreactor mimicking respiratory motion and blood flow. Comparative analysis between 2D and 3D cultures, with and without simulated respiratory motion, revealed striking differences in cellular behaviour and gene expression. In 3D culture, respiratory motion enhanced cell adhesion, proliferation, and nuclear translocation of β-catenin and YAP, along with upregulation of integrin β1, E-cadherin, and genes related to extracellular matrix and cytokine signalling. In contrast, respiratory motion in 2D culture suppressed proliferation and induced apoptosis, highlighting the importance of extracellular matrix-mediated mechanotransduction. Our findings demonstrate that dimensionality and mechanical stress synergistically affect lung cancer cell dynamics and underscore the need for physiologically relevant 3D models incorporating mechanical cues for accurate cancer research. Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer Biological sciences/Biological techniques/Biological models/Cancer models Biological sciences/Cancer/Cancer microenvironment Biological sciences/Biotechnology/Sequencing/RNA sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cell differentiation and proliferation are influenced by the surrounding environment 1 , 2 . Extracellular mechanical and physical stimuli (mechanical stress), including compression, stretching, cell–cell contact, and shear stress, are converted into intracellular biochemical signals and are crucial in regulating the behaviour of normal and cancer cells 3 – 8 . Mechanotransduction involves complex crosstalk 9 , 10 . Given the potential limitations of simple models in accurately reproducing such complex mechanisms 11 , developing models that closely mimic the in vivo environment is essential when analyzing the dynamics and behaviour of lung cancer cells. Two-dimensional (2D) culture models have long been employed in lung cancer research with or without mechanical stresses. However, the limitations of 2D models in accurately replicating the tumour microenvironment have been widely recognised 12 , 13 . Cells cultured in three-dimensional (3D) structures exhibit distinct cellular morphology, tissue architecture, and protein expression compared to those cultured under 2D conditions 11 , 14 . Thus, the importance of 3D culture in studying cellular dynamics is undeniable. To overcome the shortcomings of 2D models, various 3D culture techniques have been developed, including hydrogels as scaffolds, Matrigel-based organoid cultures, and microfluidic devices replicating lung structure and function, such as “Lung-on-a-chip” 15 – 19 . Although these models bridge the gap between 2D cell cultures and animal models 15 , they are limited in fully replicating the lung’s natural 3D structure, the mechanical environment induced by respiratory motion, and the presence of immune cells within the tumour microenvironment 20 . The lung is a unique organ, and its 3D structure is influenced by respiratory motion (stretching stress) and blood flow (shear stress). Notably, the lung undergoes periodic mechanical stresses due to respiration, exhibiting anisotropic and heterogeneous large deformations 21 . Due to its negative pressure environment and 3D scaffold, the lung differs from other parenchymal organs, such as the liver, kidneys, and heart. Therefore, understanding lung cancer pathophysiology accurately requires models replicating these physiological conditions. Lung decellularisation removes cellular components from tissues or organs while preserving the extracellular matrix (ECM) structure 22 . This process retains the vascular network, yielding a complex ECM serving as a 3D scaffold, which has been refined and applied in tissue engineering and regenerative medicine 23 , 24 . In a previous study, a 3D lung cancer model generated using decellularised rat lungs provided a more physiologically relevant cancer microenvironment than 2D cultures, enabling a detailed evaluation of cancer cell characteristics 25 . However, research on 3D lung cancer models utilising decellularisation technique is lacking, and further investigation is needed to elucidate the relationship between mechanical stress and cancer cell regulation. Accordingly, we established an ex vivo 3D lung cancer model by seeding human cancer cells into decellularised rat lungs and culturing them in a tightly controlled bioreactor mimicking the physiological environment. Using the generated model, this study aimed to elucidate the impact of mechanical stress, particularly respiratory motion, on lung cancer cell dynamics. Results 2D Cell Culture Using a Bioreactor In 2D culture, a pressure stimulation unit (PSU; TOKAIHIT, Shizuoka, Japan) was used to alternatively change the air pressure inside a sealed chamber between -5 mmHg and atmospheric pressure (0 mmHg). This process was defined as respiratory motion (RM). The cultures were divided into RM (RM⁺) and non-RM (RM⁻) groups (Fig. 1a). The pressure inside the chamber, where the well plate containing A549 cells was placed and connected to the PSU, was controlled within the range of -5–0 mmHg (Fig. 1b). Compared with the RM⁺ group, RM⁻ group exhibited rapid proliferation (Fig. 1c). After four days of culture, the attached cell count per well was higher in the RM⁻ group compared with the RM⁺ group (5.74 ± 1.97 × 10⁵ vs. 12.90 ± 1.21 × 10⁵; p = 0.0096, n = 3) (Fig. 1d). The number of Ki-67-positive cells (MIB-1 index), an indicator of cell proliferation, was significantly higher in the RM⁻ group (57.43 ± 17.39% vs. 95.75 ± 1.99%, p < 0.0001, n = 3) (Fig. 1e). Conversely, cleaved caspase-3-positive cells, indicative of apoptosis, were detected in 25.07 ± 11.59% of RM⁺ cells but were entirely absent in the RM⁻ group, showing a statistically significant difference between the two groups (p < 0.0001, n = 3) (Fig. 1f). Yes-associated protein (YAP) and β-catenin were not detected in the nuclei of any cells in either group (Fig. 1g, h). 3D Cell Culture Using a Bioreactor The ex vivo 3D lung cancer model was constructed by decellularising rat lungs (Supplementary Fig. 1), followed by recellularisation with A549 cells via airway seeding. The samples were cultured for four days—RM⁺ and RM⁻ conditions were compared—and subsequently harvested (Fig. 2a). A blood pressure unit (BPU; TOKAIHIT, Shizuoka, Japan) was employed for continuous vascular perfusion and pressure monitoring. Only the RM⁺ group was subjected to pressure changes inside the sealed chamber via the PSU, establishing a bioreactor circuit similar to that used in 2D culture (Fig. 2b). After overnight preconditioning, A549 cells were used for recellularisation. As the perfusion rate increased, vascular pressure increased but was controlled to remain below an average of 20 mmHg at all times (Fig. 2c). Due to RM, the lungs inside the chamber underwent repeated expansion and contraction (Fig. 2d and Supplementary Video 1). The baseline fluctuations in vascular pressure observed in RM⁺ samples reflected the pressure changes inside the chamber, indicating respiratory fluctuations. The vascular pressure at each phase (Day 0, 1, 2, and 3–4) tended to be lower in the RM⁺ group, presumably due to the effects of negative pressure; however, no significant difference was found between the two groups (p = 0.72, 0.24, 0.45, and 0.55, respectively) (Fig. 2e). Macroscopic observations of the lungs after four days of 3D culture showed that both lungs in the RM⁺ group were extensively white, and haematoxylin and eosin (HE) staining revealed widespread cell adhesion (Fig. 3a). In contrast, in the RM⁻ group, only localised white areas were observed, primarily in the bilateral upper lobes, and HE staining showed scattered, localised cell adhesion (Fig. 3b). The cell adhesion rate was significantly higher in the RM⁺ group (33.13 ± 4.29% vs. 13.60 ± 8.14%, p = 0.01, n = 4) (Fig. 3c, d). A detailed examination of HE-stained slides revealed minimal cell detachment in the RM⁺ group; widespread cell detachment was observed in the RM⁻ group (Fig. 3e). The MIB-1 index was significantly higher in the RM⁺ group (71.92 ± 18.72% vs. 45.90 ± 15.50%, p < 0.0001) (Fig. 4a). Cleaved caspase-3 was positive only in detached cells in both groups, while adherent cells remained negative. The number of cleaved caspase-3-positive cells did not differ between the two groups (9.77 ± 6.59% vs. 10.37 ± 9.69%, p = 0.746) (Fig. 4b). The proportion of YAP- and β-catenin-positive nuclei was significantly higher in the RM⁺ group (2.51 ± 2.84% vs. 0.46 ± 1.11%, p < 0.0001; 1.78 ± 1.55% vs. 0.20 ± 0.30%, p < 0.0001). In contrast, in the RM⁻ group, a higher proportion of cells exhibited cytoplasmic localisation, indicating the absence of nuclear translocation (Fig. 4c, d). Furthermore, integrin β1 expression was higher in the RM⁺ group (246.8 ± 91.2 vs. 154.3 ± 82.6 per field, p < 0.0001) (Fig. 4e), and E-cadherin fluorescence intensity was increased in RM⁺ cells (32,925 ± 17,586 vs. 21,120 ± 16,774, p = 0.0029) (Fig. 4f). RNA Sequencing A heatmap and volcano plot of differentially expressed genes (DEGs) (p 1 or < −1) in 2D cultures ( n = 2) are shown (Fig. 5a). In the RM⁺ group under 2D conditions, tumour suppressor genes, CDKN1A and NR4A3 , were upregulated and tumour-promoting genes, CA9 , EFNA1 , and SUSD2 , were downregulated. Gene Ontology (GO) analysis revealed no prominent enrichment in the ‘Cellular Component’ or ‘Molecular Function’ categories. Although no statistically significant terms were found in the ‘Biological Process’ category, processes such as “negative regulation of cell growth” and “negative regulation of G1/S transition of mitotic cell cycle”, associated with suppression of cell proliferation and cell cycle progression, were enriched. In 3D cultures ( n = 4), INHBA , LOX , LRRC15 , and CXCL12 , potentially involved in cell proliferation and adhesion, were upregulated (Fig. 5b). GO analysis showed no statistically significant enrichment in the ‘Biological Process’ category. However, “extracellular space”, “protein complex involved in cell–cell adhesion”, and “extracellular region” under the ‘Cellular Component’ category, were enriched. “cytokine activity” was significantly enriched under the ‘Molecular Function’ category. Gene set enrichment analysis (GSEA) identified one significantly enriched gene set in the RM⁺ group (Androgen Response; ES: 0.3, NES: 1.44, p < 0.0001) and three in the RM⁻ group (Peroxisome; ES: −0.33, NES: −1.59, p < 0.0001; Acid Metabolism; ES: −0.41, NES: −1.58, p < 0.0001; P53 Pathway; ES: −0.28, NES: −1.42, p = 0.0059) (Fig. 5c). DEGs upregulated or downregulated in the 2D and 3D models were 66/31 and 63/64, respectively. Among them, SIK1 was a commonly regulated DEG in both models (Fig. 5d). Discussion In the ex vivo 3D lung cancer model, the group subjected to respiratory motion—mimicking human-like breathing—exhibited enhanced cell adhesion and proliferation compared to the group with no motion, accompanied by changes in gene expression and signalling pathways that could explain these phenomena. In contrast, adding respiratory motion in 2D cultures led to suppressed cell proliferation, showing a markedly different gene expression profile in RNA sequencing compared to the 3D model. These findings highlight that cellular behaviour is strongly influenced by the experimental model and external mechanical environment. Our findings emphasise the importance of incorporating respiratory motion and an ECM that supports mechanosensing to investigate lung cancer biology more accurately. Bioreactor Mimicking a Physiological Environment Bioreactors that implement a perfusion–ventilation model enhance nutrient distribution and promote cell proliferation, leading to more uniform cell distribution 26 . The lung adenocarcinoma cells studied herein were naturally exposed to continuous pressure fluctuations, stretch forces associated with respiratory motion, and blood flow within the lung microenvironment 5,27 . Our bioreactor was designed to provide a favourable environment for cell growth along with the application of physiologically relevant mechanical stresses. Specifically, it truly replicated both shear stress derived from capillary blood flow and stretch stress caused by the large, multidirectional deformation of lung tissue 21 . Unlike conventional studies employing volume-controlled systems 25 , our pressure-controlled system manages negative pressure within the chamber and vascular pressure at levels close to that of the human pulmonary artery, facilitating a more realistic simulation of the intrathoracic environment. Furthermore, the baseline fluctuation observed in the pulmonary arterial pressure graph reflects the respiratory variation observed in humans, reinforcing the physiological relevance of the environment produced by this bioreactor. Comparison Between 2D and 3D Models Cell proliferation was suppressed, and apoptosis was induced in the 2D RM⁺ group. RNA sequencing revealed upregulation of tumour suppressor genes, including CDKN1A and NR4A3 28,29 , and downregulation of tumour-promoting genes, including CA9 , EFNA1 , and SUSD2 30–33 . Furthermore, nuclear translocation of β-catenin and YAP was not observed. These findings suggest that, in a 2D environment lacking ECM, negative pressure may act as a stressor, and respiratory motion alone is insufficient to activate intracellular signalling pathways. In contrast, the 3D RM⁺ group exhibited a higher proliferation rate without any apparent induction of apoptosis. RNA sequencing revealed the upregulation of genes involved in cell proliferation and tumour progression, including INHBA , LOX , LRRC15 , and CXCL12 34–38 . GO analysis showed enrichment of genes related to cell–cell adhesion and ECM components. Notably, nuclear translocation of β-catenin and YAP were increased in the RM⁺ group. These results suggest that in a 3D model with ECM support, negative pressure does not act as a stressor; rather, stretch stress transmitted through the ECM triggers signal transduction pathways, particularly those involved in cell adhesion. Based on the RNA sequencing data, the increased cell proliferation in the RM⁺ group may be a secondary effect of enhanced cell adhesion stability rather than a direct consequence of respiratory motion. Supporting this interpretation, GSEA revealed upregulation of the tumour-suppressive p53 gene set in the RM⁻ group, suggesting that the absence of respiratory motion may exert tumour-suppressive effects in lung adenocarcinoma cells. Interestingly, only one gene, SIK1 , was a common DEG between the 2D and 3D models. This finding indicates that even when the same cell line is exposed to the same respiratory motion, gene expression responses differ markedly depending on the model system, including factors such as dimensionality and the presence or absence of physical scaffolding. These results underscore the critical role of ECM in modulating the cellular response to respiratory motion and highlight the profound influences of model choice on cell behaviour. Although previous studies have demonstrated that 3D models more accurately recapitulate the physiological dynamics of cancer cells 14,39–43 , few have directly compared dimensional models under mechanical stress. In this study, we conducted a direct comparison and found that 3D models indeed provide a more physiologically relevant context, supporting their utility in lung cancer research. Effects of Respiratory Motion in the 3D Model In the 3D RM⁺ group, cell adhesion was enhanced, accompanied by increased expression of adhesion-related proteins, including integrin β1 and E-cadherin, and increased nuclear translocation of β-catenin. Integrin β1 is a well-known mechanosensor responsible for ECM adhesion and mechanical signal transduction 44–46 . E-cadherin is crucial in cell–cell adhesion, epithelial–mesenchymal transition (EMT), and tumour suppression 47,48 . β-catenin is involved in both cell adhesion and Wnt signalling; upon nuclear translocation, it binds to transcription factors (TCF/LEF) and promotes gene expression 48 . Cyclic stretch enhances Wnt/β-catenin signalling in A549 cells 49 , and accurately replicating mechanical stretch is crucial for cellular responses 50 . Additionally, CXCL12, upregulated in RM⁺, activates integrins independently of CXCR4 51 . Integrin-mediated activation of β-catenin and YAP has been suggested 52 . In the RM⁺ group, nuclear translocation of YAP was significantly increased, providing strong evidence that cells actively sense mechanical stress 53 . Considering these findings, the 3D expansion of the lung induced by respiratory motion 21,54 likely facilitates ECM-mediated mechanosensing, promoting the expression of adhesion-related proteins to reinforce cell–ECM and cell–cell interactions. This study underscores the necessity of incorporating respiratory motion in 3D models to investigate lung cancer biology accurately. Characteristics of the Ex Vivo 3D Lung Cancer Model and Comparison with Other Models Current experimental strategies for reproducing 3D environments include hydrogel-based culture systems and microfluidic technologies, such as the ‘Lung-on-a-chip’ model 15–18 . Each model possesses distinct advantages and limitations and differs significantly from the ex vivo 3D lung cancer model employed in this study. Hydrogels enable 3D cell culture, offering a more physiologically relevant context for cell–cell and cell–ECM interactions compared to 2D cultures 16 . However, Matrigel composition differs from that of the ECM found in the tumour microenvironment, making it challenging to truly replicate specific tissue- or cell-type-derived ECM components. Furthermore, the physical properties of Matrigel are difficult to control, limiting its suitability for precise analyses of mechanical stress responses. ‘Lung-on-a-chip’ systems utilise microfluidic devices to culture cells in microscale channels that simulate the dynamic environment of the lung 55 . A notable advantage of this model is the ability to establish air–liquid interface cultures, essential for mimicking lung-specific functionality. Nevertheless, the complexity of device fabrication and operation, along with limitations in culture scale, makes this model less suitable for tissue-level investigations 56 . ‘Lung-on-a-chip’ systems have limited capacity for assessing ECM interactions in detail and are not ideal for studying signalling pathways mediated by native tissue-derived ECM. In contrast, our ex vivo 3D model retains the ECM architecture and microenvironment of actual lung tissue, allowing for the observation of cellular adhesion, proliferation, and tissue-level responses. This model can facilitate studies on ECM-mediated signalling changes that are difficult to replicate using conventional 2D culture or artificial matrices such as hydrogels. Observing ECM-specific signalling pathways—particularly those unique to lung tissue—is a key advantage of our model. However, we did not directly compare it with other widely adopted 3D models, such as organoids or ‘Lung-on-a-chip’ systems, so the relative superiority of our 3D model within the broader landscape of 3D culture systems remains to be determined. In summary, our findings revealed substantial differences in gene and protein expressions between 2D and 3D cultures, as well as between conditions with and without respiratory motion. These results demonstrate that cellular behaviour and gene expression on 2D substrates do not fully recapitulate those observed in 3D environments or in vivo . This study highlights the limitations of conventional 2D models in basic research and emphasises the need for 3D culture systems that provide respiratory motion and appropriate scaffolding to support lung adenocarcinoma cell proliferation. Methods Harvest of Rat Heart–Lung Blocks Lungs were obtained from 6- to 10-week-old male Sprague–Dawley (SD) rats (Jackson Laboratory Japan, Inc., Kanagawa, Japan). All animal procedures were approved by the Animal Care and Use Committee of the University of Toyama (approval number: A2023UH-01) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals . Rats were anaesthetised with inhaled isoflurane (DS Pharma Animal Health, Osaka, Japan), followed by intraperitoneal administration of a mixed anaesthetic cocktail (total volume: 25 mL) comprising 1.5 mL of medetomidine hydrochloride (1 mg/mL; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), 4 mL of midazolam (5 mg/mL; Sandoz K.K., Tokyo, Japan), 5 mL of butorphanol tartrate (5 mg/mL; Meiji Animal Health Co., Ltd., Tokyo, Japan), and 14.5 mL of saline. The dose was adjusted to 0.5 mL per 200 g of body weight to ensure adequate analgesia and sedation. A tracheotomy was performed, and a 16G catheter was inserted into the trachea for intubation. The rats were on a ventilator at a tidal volume of 10 mL/kg with a respiratory rate of 90 breaths per minute. After a transverse abdominal incision, the anterior thoracic wall was removed. Anticoagulation was achieved by injecting heparin sodium (1000 U/kg; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) via the inferior vena cava, followed by transection of the cardiac apex. A 16G catheter was inserted into the pulmonary artery via the right ventricle, and the lungs were perfused with 50 mL of phosphate-buffered saline (PBS) containing heparin sodium (50 U/mL) and sodium nitroprusside dihydrate (SNP; Sigma-Aldrich, St. Louis, MO, USA; 10 μg/mL) to flush out the blood. The lungs were excised en bloc together with the heart and trachea. Decellularisation of the Lung Tissue Cannulas were inserted into the trachea, pulmonary artery, and left ventricle (pulmonary vein), and lungs were decellularised following a previously reported method 25,57 . In brief, the pulmonary vasculature was perfused via the pulmonary artery with PBS containing calcium and magnesium (PBS⁺) supplemented with heparin sodium, SNP, antibiotics, and 0.0035% Triton X-100 (Nacalai Tesque, Kyoto, Japan). Subsequently, 20 units of Benzonase (25 U/μL; Enzynomics, Daejeon, South Korea) in Benz Buffer (50 mM Tris-HCl, 0.1 mg/mL BSA, 1 mM MgCl₂) was administered via the airway. Next, sodium deoxycholate (SDC; Nacalai Tesque, Kyoto, Japan) at concentrations of 0.01%, 0.05%, and 0.1% in PBS without calcium and magnesium (PBS⁻) was perfused through the vasculature, followed by airway administration of 20 units of Benzonase in Benz Buffer. After vascular perfusion with 0.5% Triton X-100, the lungs were flushed with PBS⁻ and perfused with PBS⁻ containing penicillin–streptomycin, amphotericin B, and gentamicin (Supplementary Fig. 1a). Decellularised lungs were temporarily stored in PBS⁻ containing these antibiotics (Supplementary Fig. 1b). Culture and Preparation of Human Lung Cancer Cells The human lung adenocarcinoma cell line A549 was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan; https://cellbank.nibiohn.go.jp). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, high glucose, 4.5 g/L; Nacalai Tesque, Kyoto, Japan), supplemented with 10% foetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin–streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin; Nacalai Tesque, Kyoto, Japan), 1% amphotericin B (250 μg/mL; FUJIFILM, Tokyo, Japan), and 0.5% gentamicin (10 mg/mL; Gibco, Waltham, MA, USA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. For passaging or harvesting, cells were detached using 0.05% trypsin–EDTA solution (Nacalai Tesque, Kyoto, Japan) at 37 °C for 5–7 minutes. Bioreactor Setup for the 3D Culture Decellularised lungs with cannulas in place were placed into the chamber, with only the trachea and pulmonary artery connected to the tubing. The cannula inserted into the left ventricle (representing the pulmonary vein) was left open to the culture medium inside the chamber. The medium collected from the pulmonary vein side—from within the chamber—was circulated into the pulmonary artery via the BPU, enabling continuous intravascular perfusion and pressure monitoring (Fig. 2b). The tubing connected to the airway was linked to a reservoir bottle containing 60 mL of culture medium, allowing for passive medium movement in response to changes in lung volume. The chamber was connected to the PSU, which continuously regulated and monitored air pressure to maintain the desired pressure settings (Fig. 2b, c). Intrachamber pressure measurements were taken once every 300 seconds (Fig. 2c). Bioreactor Setup for the 2D Culture Cells were seeded onto plastic well plates to evaluate the effect of respiratory motion—represented by pressure fluctuations within the chamber—on A549 cells cultured in a conventional 2D environment. Two 6-well plates (SPL Life Sciences, Pocheon, South Korea) were prepared, with 3 × 10⁵ A549 cells seeded per well. One plate was placed in a 2D culture chamber and connected to the PSU, referred to as the RM⁺ group, while the other plate was not connected to the PSU and served as the RM⁻ group (Fig. 1a). Both plates were incubated under standard conditions (37 ℃, 5% CO₂) for four days. After incubation, cells were collected for further analysis. Intrachamber pressure measurements were taken once every 300 seconds (Fig. 1b). Seeding and Perfusion Culture of Lung Cancer Cells Two decellularised lungs and corresponding chambers were prepared to compare conditions with and without respiratory motion. On the evening of Day 0, preconditioning of the decellularised lungs was initiated to promote peripheral vascular expansion. Specifically, each lung was placed in a chamber filled with 200 mL of DMEM and connected to vascular perfusion at a flow rate of 14 mL/min in an incubator at 37 °C with 5% CO₂. During this period, neither chamber was subjected to respiratory motion. On Day 1, A549 lung cancer cells were seeded (recellularised) via the airway. While applying a negative pressure of -10 mmHg using the PSU to expand the lung in the sealed chamber, 4–5 × 10⁷ A549 cells suspended in 10 mL of DMEM were injected through the airway using a syringe. To prevent the backflow of cells, the airway tubing was clamped, and the lungs were kept stationary for 1 hour in an incubator at 37 °C with 5% CO₂. Subsequently, vascular perfusion was resumed in both chambers at a flow rate of 7 mL/min, and respiratory motion was initiated in only one chamber (RM⁺ group) while the other remained static (RM⁻ group). Respiratory motion was simulated by cyclically modulating the intrachamber pressure between -5 mmHg and atmospheric pressure using the PSU, inducing expansion and contraction of the lung tissue, respectively (Fig. 2d). The vascular perfusion rate was gradually increased every 24 hours: 7 mL/min → 14 mL/min → 21 mL/min. After 72 hours of culture, the lungs were harvested for further analysis. Four independent sets of RM⁺ and RM⁻ groups were prepared for comparative analysis. Histology, Immunohistochemistry, and Immunofluorescence For 2D cultures, cells were collected using 0.05% trypsin–EDTA solution and put into Millicell EZ SLIDE 8-well glass slides (Merck KGaA, Darmstadt, Germany) for subsequent staining. For 3D cultures, the entire left lung was fixed in 10% neutral buffered formalin (Nacalai Tesque, Kyoto, Japan), sliced longitudinally into 4–5 sections, embedded in paraffin, and sectioned at a thickness of 5 μm. HE staining was performed following standard protocols. For immunohistochemistry, paraffin sections were deparaffinised with xylene and ethanol, followed by washing with PBS. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) at temperatures exceeding 100 °C for 10 minutes. The membrane was permeabilised using 0.1% Triton X-100 for 15 minutes, and non-specific binding was blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) for 15 minutes. Primary antibodies were diluted in SignalStain Antibody Diluent (Cell Signaling Technology, Danvers, MA, USA), and samples were incubated overnight at 4 °C. After PBS washing, samples were incubated with secondary antibodies at room temperature (approximately 21 °C) for 1 hour and mounted using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Inc., Hercules, CA, USA). The primary antibodies used in this study included anti-Ki-67 (418071; Nichirei Biosciences, Tokyo, Japan), cleaved caspase-3 (25128-1-AP; Proteintech, IL, USA), β-catenin (84805; Cell Signaling Technology, MA, USA), integrin β1 (ab30394; Abcam, Cambridge, United Kingdom), and E-cadherin (20874-1-AP; Proteintech, IL, USA). Secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (Thermo Fisher Scientific, MA, USA) were used as appropriate. Image Acquisition and Intensity Normalisation for Quantification Images were acquired using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) or a confocal laser scanning microscope (LSM780, Carl Zeiss, Jena, Germany). Ten high-power fields per sample were examined at 400× magnification using a 40× objective lens with a 10× eyepiece for quantitative analysis. Image analysis was performed using the ImageJ software (National Institutes of Health, https://imagej.nih.gov/ij/). Images were first converted to greyscale for fluorescence intensity analysis, and background noise was subtracted. Ten regions of interest (ROIs) corresponding to intercellular boundaries were manually selected for each sample. Fluorescence intensity was measured, and comparisons between samples were performed using the corrected integrated density, accounting for background correction. The following formula was used: Corrected IntDen = Raw Integrated Density (RawIntDen) – (Background Mean × ROI Area) Calculation of Cell Adhesion Rate Among all HE-stained sections prepared using the left lung, the proportion of the tissue area showing attached tumour cells was defined as the “cell adhesion rate”. HE-stained slide images were imported into ImageJ software and converted to 8-bit greyscale images. The threshold was adjusted to visualise the attached cells. Based on the histogram displayed by ImageJ, the lower threshold (0%) was set for cell detection, and the upper threshold (100%) was set to include the entire tissue section. The area detected at the 50% threshold, representing attached cells, was divided by the area detected at the 100% threshold, representing the entire tissue section. This ratio was used to calculate the cell adhesion rate as follows (Fig. 3c): Cell adhesion rate (%) = (Area of detected cells at 50% threshold / Area of total section at 100% threshold) × 100 RNA Extraction and RNA Sequencing For 2D cultures, A549 cells were harvested from well plates using 0.05% trypsin–EDTA solution (Nacalai Tesque, Kyoto, Japan). For 3D cultures, a portion of the right lung was finely minced and immediately immersed in RNAlater Stabilisation Solution (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The fixed lung samples were homogenised for at least 30 seconds using a BioMasher II homogeniser (Funakoshi, Tokyo, Japan). Total RNA was extracted using the RNeasy Mini Kit (QIAGEN N.V., Venlo, The Netherlands) following the manufacturer’s protocol. RNA concentration and purity were assessed using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples were sent to Rhelixa (Tokyo, Japan) for RNA sequencing. GSEA was performed using the RNA data obtained from 3D cultures. GSEA version 4.4.0 (https://www.gsea-msigdb.org/gsea/index.jsp) was used, with gene sets retrieved from the Molecular Signatures Database (MSigDB). RNA was extracted from decellularised lungs (without cell seeding) using the same protocol to assess residual rat-derived RNA as a control ( n = 1) (Supplementary Table 1). Statistical Analysis Fisher’s exact test was used for categorical variables. Welch’s t -test was used to compare continuous variables between the two groups. A p-value of less than 0.05 was considered statistically significant. A -log₁₀(p-value) greater than 1.30 was considered statistically significant. All statistical analyses were performed using JMP Pro software (version 16.2.0; JMP Statistical Discovery LLC, Cary, NC, USA). Declarations Funding This study was supported by the Grant-in-Aid for Scientific Research on Research Activity Start-up (Project title: Analysis of Mechanical Stress in Ex Vivo Lung Cancer Models ; Grant number: 23K19530; FY2023–2024) and a donation from Hayashida Finance LLC. Institutional Review Board Statement All animal experiments were approved by the Animal Experimentation Committee of the University of Toyama (approval number: A2023UH-01). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available from the corresponding author upon reasonable request. Acknowledgements The authors gratefully acknowledge the technical support provided by Sanae Hirota (University of Toyama). Author Contributions N.K. performed manuscript writing, harvesting of rat lungs, 2D/3D cell culture, immunofluorescence staining, and data analysis. M.I. assisted with immunofluorescence staining, image preparation, and RNA extraction. S.M. and S.R. supported decellularisation and recellularisation. S.W., K.K., M.H., D.N., R.Y., N.K., and T.O., under the supervision of Y.M., carried out decellularisation and recellularisation procedures and contributed to sample preservation. K.S. provided technical guidance for the harvesting of rat lungs. N.K. also developed the bioreactor and provided technical support. H.H. and K.H. prepared pathological slides. All authors reviewed and edited the manuscript. T.T. conceived, designed, directed, and supervised the study. Competing Interests We have no conflicts of interest to disclose. References Nakaji‐Hirabayashi, T. et al. Enhanced proliferation and differentiation of human mesenchymal stem cells in the gravity‐controlled environment. Artif. Organs 46 , 1760–1770 (2022). Spill, F., Reynolds, D. S., Kamm, R. D. & Zaman, M. H. Impact of the physical microenvironment on tumor progression and metastasis. Curr. Opin. Biotechnol. 40 , 41–48 (2016). 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Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 SupplementaryInformation.docx Supplementary information SupplementaryMovie1.mp4 Supplementary Movie 1 Cite Share Download PDF Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6482124","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":447423391,"identity":"3ac5e84a-ee3f-4941-a8b1-f75cf8df4c90","order_by":0,"name":"Naoya 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One was placed in a 2D culture chamber connected to a Pressure Stimulation Unit (PSU), with continuous pressure changes between -5 mmHg and atmospheric pressure to simulate respiratory motion (RM⁺ group), while the other was not connected to the PSU and served as the non-RM group (RM⁻). Both plates were cultured in a 37 °C, 5% CO₂ incubator (n = 3).\u003c/p\u003e\n\u003cp\u003e(b) Pressure data within the RM⁺ chamber, showing continuous fluctuations between –5 mmHg and atmospheric pressure (0 mmHg) from Day 1 to Day 4. (c) Under microscopic examination, the RM⁻ group appeared to proliferate more rapidly.\u003c/p\u003e\n\u003cp\u003e(d) Comparison of attached cell counts collected on Day 4. The RM⁻ group exhibited a significantly higher cell count (p = 0.0096).\u003c/p\u003e\n\u003cp\u003e(e) Ki-67 staining. The RM⁻ group exhibited a significantly higher number of Ki-67-positive cells, and the MIB-1 index (percentage of Ki-67-positive cells) was significantly greater than in the RM⁺ group (n = 3).\u003c/p\u003e\n\u003cp\u003e(f) Cleaved caspase-3 staining. Positive cells were observed in the RM⁺ group but were entirely absent in the RM⁻ group, showing a statistically significant difference between the groups (n = 3).\u003c/p\u003e\n\u003cp\u003e(g, h) Yes-associated protein (YAP) or β-catenin did not show nuclear localisation in either group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/b82542bee644fc49fe8631fe.png"},{"id":84562976,"identity":"8ef3c781-4863-46f5-8b6b-aa67e1b53084","added_by":"auto","created_at":"2025-06-13 13:35:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":459915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEx Vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e 3D Lung Cancer Model and Overview of the Bioreactor.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Lungs and heart were harvested \u003cem\u003een bloc\u003c/em\u003e from rats, and decellularisation was performed by perfusing the airway and vasculature with solutions containing Triton X-100, Benzonase, and sodium deoxycholate. A549 cells were subsequently seeded \u003cem\u003evia\u003c/em\u003ethe airway and allowed to adhere (recellularisation) to create an \u003cem\u003eex vivo\u003c/em\u003e 3D lung cancer model. The samples were cultured for four days under respiratory motion (RM⁺ group) or static conditions (RM⁻ group), followed by analysis.\u003c/p\u003e\n\u003cp\u003e(b) Overview of the bioreactor system. The decellularised lung was placed in a chamber filled with culture medium. The airway was connected to an airway reservoir, the pulmonary artery (PA) to the Blood Pressure Unit (BPU), and the cannula in the pulmonary vein (PV) (left ventricle) was left open to the medium inside the chamber. This configuration enabled passive medium exchange \u003cem\u003evia\u003c/em\u003ethe airway and a perfusion circuit was established from PA through the lung to the PV and back to the BPU. The chamber was sealed and connected to a Pressure Stimulation Unit (PSU), which continuously modulated the intrachamber pressure to simulate a negative pressure environment and respiratory motion, mimicking the thoracic cavity.\u003c/p\u003e\n\u003cp\u003e(c) Monitoring data during 3D culture in the RM⁺ and RM⁻ groups. On the evening of Day 0, intravascular perfusion (14 mL/min) was initiated for preconditioning. The perfusion rate was increased every 24 hours (7 → 14 → 21 mL/min). Only the RM⁺ group was subjected to continuous pressure fluctuations between -5 mmHg and atmospheric pressure from the time of recellularisation on Day 1. The vascular pressure waveform showed fine oscillations reflecting respiratory variation once the PSU was activated.\u003c/p\u003e\n\u003cp\u003e(d) Representative images of the lung inside the chamber. The lung expands under negative pressure (yellow arrows) and contracts at atmospheric pressure (white arrows).\u003c/p\u003e\n\u003cp\u003e(e) Comparison of vascular pressures between RM⁺ and RM⁻ groups throughout the procedure revealed no statistically significant differences. ns, not significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/64daf8dbe5aab556e8ea6da1.png"},{"id":84563666,"identity":"c569b06a-c6ce-40b8-973f-cde9872fc2e1","added_by":"auto","created_at":"2025-06-13 13:43:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":719653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell adhesion rate and macroscopic/histological findings in 3D culture.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) Macroscopic and histological appearance of lungs of the respiratory motion (RM⁺) and non-RM (RM⁻) groups. White regions observed macroscopically indicate areas of cell adhesion. In the RM⁺ group, cells were uniformly adhered throughout both lungs (yellow arrows), whereas in the RM⁻ group, adhesion was limited to partial regions (white arrows). These observations were consistent with haematoxylin and eosin (HE) staining.\u003c/p\u003e\n\u003cp\u003e(c) The cell adhesion rate was calculated from HE-stained sections using the ImageJ software. Images were converted to 8-bit greyscale and analysed using a histogram-based threshold method. The lower threshold (0%) was set to begin detecting cells, and the upper threshold (100%) was set to include the entire tissue section. The proportion of the area detected at the 50% threshold was defined as the cell adhesion rate:\u003c/p\u003e\n\u003cp\u003eCell adhesion rate (%) = (Area at 50% threshold / Area at 100% threshold) × 100\u003c/p\u003e\n\u003cp\u003e(d) Comparison of cell adhesion rates. The RM⁺ group showed a significantly higher adhesion rate than the RM⁻ group (p = 0.01, n = 4).\u003c/p\u003e\n\u003cp\u003e(e) High-power views of HE-stained sections. In the RM⁺ group, cells were widely and uniformly adhered with minimal detachment, whereas in the RM⁻ group, cell adhesion was sparse and irregular, showing frequent areas of detachment. Scale bars: (a, b) 5 mm; (4×) 500 μm; (10×) 200 μm; (20×) 100 μm; (40×) 50 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/7e9e21466d6de07a238be22a.png"},{"id":84562978,"identity":"a176294e-4f3e-4bd3-9846-7d5b83aeaa6f","added_by":"auto","created_at":"2025-06-13 13:35:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":961521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry and immunofluorescence in 3D culture.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Ki-67-positive cells were more frequently observed in the RM⁺ group, and the MIB-1 index (percentage of Ki-67-positive cells) was significantly higher than that in the RM⁻ group (n = 4).\u003c/p\u003e\n\u003cp\u003e(b) Cleaved caspase-3 positivity was detected only in detached cells in both groups, with no statistically significant difference (n = 4).\u003c/p\u003e\n\u003cp\u003e(c, d) The proportion of cells showing nuclear localisation of yes-associated protein (YAP) (p \u0026lt; 0.0001) and β-catenin (p \u0026lt; 0.0001) was significantly higher in the RM⁺ group (white arrowheads). In contrast, in the RM⁻ group, cytoplasmic staining without nuclear localisation was frequently observed (yellow arrowheads).\u003c/p\u003e\n\u003cp\u003e(e) Integrin β1 expression was significantly higher in the RM⁺ group (p \u0026lt; 0.0001; n = 4).\u003c/p\u003e\n\u003cp\u003e(f) Fluorescence intensity of E-cadherin was enhanced in the RM⁺ group (p = 0.0029; n = 4).\u003c/p\u003e\n\u003cp\u003eScale bars: (a, b) 50 μm; (c, d) 100 μm, 50 μm; (e, f) 200 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/c03e52449ecc3a076548818f.png"},{"id":84562980,"identity":"7f870777-bf94-4d21-993e-64b1a6e45720","added_by":"auto","created_at":"2025-06-13 13:35:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":571860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResults of RNA sequencing analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) Heatmaps and volcano plots of differentially expressed genes (DEGs) (non-adjusted p \u0026lt; 0.05 and log₂ fold change \u0026gt; 1 or \u0026lt; −1) between the respiratory motion (RM⁺) and non-RM (RM⁻) groups in 2D (\u003cem\u003en\u003c/em\u003e= 2) and 3D (\u003cem\u003en\u003c/em\u003e = 4) culture conditions. Results of Gene Ontology (GO) analysis are shown. In the 2D culture, the RM⁺ group showed upregulation of tumour suppressor genes, such as \u003cem\u003eCDKN1A \u003c/em\u003eand\u003cem\u003eNR4A3\u003c/em\u003e, and downregulation of tumour-promoting genes, including \u003cem\u003eCA9, EFNA1, \u003c/em\u003eand\u003cem\u003e SUSD2\u003c/em\u003e. In 3D culture, the RM⁺ group exhibited upregulation of genes potentially involved in cell proliferation and adhesion, such as \u003cem\u003eINHBA, LOX, LRRC15, \u003c/em\u003eand\u003cem\u003e CXCL1\u003c/em\u003e2. In the 2D culture, no significant enrichment was observed under the Cellular Component or Molecular Function categories. Although not statistically significant, enrichment trends were found under the Biological Process category for terms including “negative regulation of cell growth” and “negative regulation of G1/S transition of mitotic cell cycle”. In the 3D culture, no significant enrichment was observed in the 'Biological Process category. However, “extracellular space”, “protein complex involved in cell–cell adhesion” and “extracellular region” were enriched under the Cellular Component category and “cytokine activity” under the Molecular Function category.\u003c/p\u003e\n\u003cp\u003e(c) Gene set enrichment analysis (GSEA) based on the gene expression profiles in 3D culture revealed significant upregulation of the “androgen response” gene set in the RM⁺ group (p \u0026lt; 0.0001) and “peroxisome” (p \u0026lt; 0.0001), “acid metabolism” (p \u0026lt; 0.0001), and “P53 pathway” (p = 0.0059) gene sets in the RM⁻ group.\u003c/p\u003e\n\u003cp\u003e(d) Venn diagram of DEGs identified in 2D and 3D cultures. Only one gene was commonly upregulated across both models; no other common DEG was identified.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/107b2fff8c459612ad7b9079.png"},{"id":99211842,"identity":"e1fa2ca3-79a2-426c-8735-0f1aa27e9583","added_by":"auto","created_at":"2025-12-30 08:10:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5163736,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/a0f4d9ce-5033-4a05-a32c-49b3fe70f738.pdf"},{"id":84562973,"identity":"facb0224-952d-4de3-a511-49d4cc45ad59","added_by":"auto","created_at":"2025-06-13 13:35:45","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9989,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/5ed686cf6d540cbac5860c4d.xlsx"},{"id":84563669,"identity":"b9da3862-c7b1-4c07-a621-efe35ae4484a","added_by":"auto","created_at":"2025-06-13 13:43:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":706020,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/95262779642f4b3303db2ede.docx"},{"id":84562998,"identity":"7a6ce7f2-ddfa-4bfb-8d75-0819c2288f67","added_by":"auto","created_at":"2025-06-13 13:35:46","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24456707,"visible":true,"origin":"","legend":"Supplementary Movie 1","description":"","filename":"SupplementaryMovie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6482124/v1/96192fc9e4eee1ee03ef8d6f.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Lung Adenocarcinoma Cells Respond Differently to Mechanical Stress in 3D Versus 2D Environments","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCell differentiation and proliferation are influenced by the surrounding environment \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Extracellular mechanical and physical stimuli (mechanical stress), including compression, stretching, cell\u0026ndash;cell contact, and shear stress, are converted into intracellular biochemical signals and are crucial in regulating the behaviour of normal and cancer cells\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Mechanotransduction involves complex crosstalk \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Given the potential limitations of simple models in accurately reproducing such complex mechanisms \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, developing models that closely mimic the \u003cem\u003ein vivo\u003c/em\u003e environment is essential when analyzing the dynamics and behaviour of lung cancer cells.\u003c/p\u003e \u003cp\u003eTwo-dimensional (2D) culture models have long been employed in lung cancer research with or without mechanical stresses. However, the limitations of 2D models in accurately replicating the tumour microenvironment have been widely recognised \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Cells cultured in three-dimensional (3D) structures exhibit distinct cellular morphology, tissue architecture, and protein expression compared to those cultured under 2D conditions \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Thus, the importance of 3D culture in studying cellular dynamics is undeniable. To overcome the shortcomings of 2D models, various 3D culture techniques have been developed, including hydrogels as scaffolds, Matrigel-based organoid cultures, and microfluidic devices replicating lung structure and function, such as \u0026ldquo;Lung-on-a-chip\u0026rdquo; \u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although these models bridge the gap between 2D cell cultures and animal models \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, they are limited in fully replicating the lung\u0026rsquo;s natural 3D structure, the mechanical environment induced by respiratory motion, and the presence of immune cells within the tumour microenvironment \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe lung is a unique organ, and its 3D structure is influenced by respiratory motion (stretching stress) and blood flow (shear stress). Notably, the lung undergoes periodic mechanical stresses due to respiration, exhibiting anisotropic and heterogeneous large deformations \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Due to its negative pressure environment and 3D scaffold, the lung differs from other parenchymal organs, such as the liver, kidneys, and heart. Therefore, understanding lung cancer pathophysiology accurately requires models replicating these physiological conditions.\u003c/p\u003e \u003cp\u003eLung decellularisation removes cellular components from tissues or organs while preserving the extracellular matrix (ECM) structure \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This process retains the vascular network, yielding a complex ECM serving as a 3D scaffold, which has been refined and applied in tissue engineering and regenerative medicine \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In a previous study, a 3D lung cancer model generated using decellularised rat lungs provided a more physiologically relevant cancer microenvironment than 2D cultures, enabling a detailed evaluation of cancer cell characteristics \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, research on 3D lung cancer models utilising decellularisation technique is lacking, and further investigation is needed to elucidate the relationship between mechanical stress and cancer cell regulation.\u003c/p\u003e \u003cp\u003eAccordingly, we established an \u003cem\u003eex vivo\u003c/em\u003e 3D lung cancer model by seeding human cancer cells into decellularised rat lungs and culturing them in a tightly controlled bioreactor mimicking the physiological environment. Using the generated model, this study aimed to elucidate the impact of mechanical stress, particularly respiratory motion, on lung cancer cell dynamics.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e2D Cell Culture Using a Bioreactor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 2D culture, a pressure stimulation unit (PSU; TOKAIHIT, Shizuoka, Japan) was used to alternatively change the air pressure inside a sealed chamber between -5 mmHg and atmospheric pressure (0 mmHg). This process was defined as respiratory motion (RM). The cultures were divided into RM (RM⁺) and non-RM (RM⁻) groups (Fig. 1a). The pressure inside the chamber, where the well plate containing A549 cells was placed and connected to the PSU, was controlled within the range of -5\u0026ndash;0 mmHg (Fig. 1b). Compared with the RM⁺ group, RM⁻ group exhibited rapid proliferation (Fig. 1c). After four days of culture, the attached cell count per well was higher in the RM⁻ group compared with the RM⁺ group (5.74 \u0026plusmn; 1.97 \u0026times; 10⁵ vs. 12.90 \u0026plusmn; 1.21 \u0026times; 10⁵; p = 0.0096, n = 3) (Fig. 1d).\u003c/p\u003e\n\u003cp\u003eThe number of Ki-67-positive cells (MIB-1 index), an indicator of cell proliferation, was significantly higher in the RM⁻ group (57.43 \u0026plusmn; 17.39% vs. 95.75 \u0026plusmn; 1.99%, p \u0026lt; 0.0001, n = 3) (Fig. 1e). Conversely, cleaved caspase-3-positive cells, indicative of apoptosis, were detected in 25.07 \u0026plusmn; 11.59% of RM⁺ cells but were entirely absent in the RM⁻ group, showing a statistically significant difference between the two groups (p \u0026lt; 0.0001, n = 3) (Fig. 1f). Yes-associated protein\u003c/p\u003e\n\u003cp\u003e(YAP) and \u0026beta;-catenin were not detected in the nuclei of any cells in either group (Fig. 1g, h).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D Cell Culture Using a Bioreactor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eex vivo\u003c/em\u003e 3D lung cancer model was constructed by decellularising rat lungs (Supplementary Fig. 1), followed by recellularisation with A549 cells via airway seeding. The samples were cultured for four days\u0026mdash;RM⁺ and RM⁻ conditions were compared\u0026mdash;and subsequently harvested (Fig. 2a). A blood pressure unit (BPU; TOKAIHIT, Shizuoka, Japan) was employed for continuous vascular perfusion and pressure monitoring. Only the RM⁺ group was subjected to pressure changes inside the sealed chamber via the PSU, establishing a bioreactor circuit similar to that used in 2D culture (Fig. 2b).\u003c/p\u003e\n\u003cp\u003eAfter overnight preconditioning, A549 cells were used for recellularisation. As the perfusion rate increased, vascular pressure increased but was controlled to remain below an average of 20 mmHg at all times (Fig. 2c). Due to RM, the lungs inside the chamber underwent repeated expansion and contraction (Fig. 2d and Supplementary Video 1). The baseline fluctuations in vascular pressure observed in RM⁺ samples reflected the pressure changes inside the chamber, indicating respiratory fluctuations.\u003c/p\u003e\n\u003cp\u003eThe vascular pressure at each phase (Day 0, 1, 2, and 3\u0026ndash;4) tended to be lower in the RM⁺ group, presumably due to the effects of negative pressure; however, no significant difference was found between the two groups (p = 0.72, 0.24, 0.45, and 0.55, respectively) (Fig. 2e).\u003c/p\u003e\n\u003cp\u003eMacroscopic observations of the lungs after four days of 3D culture showed that both lungs in the RM⁺ group were extensively white, and haematoxylin and eosin (HE) staining revealed widespread cell adhesion (Fig. 3a). In contrast, in the RM⁻ group, only localised white areas were observed, primarily in the bilateral upper lobes, and HE staining showed scattered, localised cell adhesion (Fig. 3b). The cell adhesion rate was significantly higher in the RM⁺ group (33.13 \u0026plusmn; 4.29% vs. 13.60 \u0026plusmn; 8.14%, p = 0.01, n = 4) (Fig. 3c, d). A detailed examination of HE-stained slides revealed minimal cell detachment in the RM⁺ group; widespread cell detachment was observed in the RM⁻ group (Fig. 3e).\u003c/p\u003e\n\u003cp\u003eThe MIB-1 index was significantly higher in the RM⁺ group (71.92 \u0026plusmn; 18.72% vs. 45.90 \u0026plusmn; 15.50%, p \u0026lt; 0.0001) (Fig. 4a). Cleaved caspase-3 was positive only in detached cells in both groups, while adherent cells remained negative. The number of cleaved caspase-3-positive cells did not differ between the two groups (9.77 \u0026plusmn; 6.59% vs. 10.37 \u0026plusmn; 9.69%, p = 0.746) (Fig. 4b).\u003c/p\u003e\n\u003cp\u003eThe proportion of YAP- and \u0026beta;-catenin-positive nuclei was significantly higher in the RM⁺ group (2.51 \u0026plusmn; 2.84% vs. 0.46 \u0026plusmn; 1.11%, p \u0026lt; 0.0001; 1.78 \u0026plusmn; 1.55% vs. 0.20 \u0026plusmn; 0.30%, p \u0026lt; 0.0001). In contrast, in the RM⁻ group, a higher proportion of cells exhibited cytoplasmic localisation, indicating the absence of nuclear translocation (Fig. 4c, d).\u003c/p\u003e\n\u003cp\u003eFurthermore, integrin \u0026beta;1 expression was higher in the RM⁺ group (246.8 \u0026plusmn; 91.2 vs. 154.3 \u0026plusmn; 82.6 per field, p \u0026lt; 0.0001) (Fig. 4e), and E-cadherin fluorescence intensity was increased in RM⁺ cells (32,925 \u0026plusmn; 17,586 vs. 21,120 \u0026plusmn; 16,774, p = 0.0029) (Fig. 4f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA heatmap and volcano plot of differentially expressed genes (DEGs) (p \u0026lt; 0.05 and log₂ fold change \u0026gt; 1 or \u0026lt; \u0026minus;1) in 2D cultures (\u003cem\u003en\u003c/em\u003e = 2) are shown (Fig. 5a). In the RM⁺ group under 2D conditions, tumour suppressor genes, \u003cem\u003eCDKN1A\u003c/em\u003e and \u003cem\u003eNR4A3\u003c/em\u003e, were upregulated and tumour-promoting genes, \u003cem\u003eCA9\u003c/em\u003e, \u003cem\u003eEFNA1\u003c/em\u003e, and \u003cem\u003eSUSD2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ewere downregulated. Gene Ontology (GO) analysis revealed no prominent enrichment in the \u0026lsquo;Cellular Component\u0026rsquo; or \u0026lsquo;Molecular Function\u0026rsquo; categories. Although no statistically significant terms were found in the \u0026lsquo;Biological Process\u0026rsquo; category, processes such as \u0026ldquo;negative regulation of cell growth\u0026rdquo; and \u0026ldquo;negative regulation of G1/S transition of mitotic cell cycle\u0026rdquo;, associated with suppression of cell proliferation and cell cycle progression, were enriched.\u003c/p\u003e\n\u003cp\u003eIn 3D cultures (\u003cem\u003en\u003c/em\u003e = 4), \u003cem\u003eINHBA\u003c/em\u003e, \u003cem\u003eLOX\u003c/em\u003e, \u003cem\u003eLRRC15\u003c/em\u003e, and \u003cem\u003eCXCL12\u003c/em\u003e, potentially involved in cell proliferation and adhesion, were upregulated (Fig. 5b). GO analysis showed no statistically significant enrichment in the \u0026lsquo;Biological Process\u0026rsquo; category. However, \u0026ldquo;extracellular space\u0026rdquo;, \u0026ldquo;protein complex involved in cell\u0026ndash;cell adhesion\u0026rdquo;, and \u0026ldquo;extracellular region\u0026rdquo; under the \u0026lsquo;Cellular Component\u0026rsquo; category, were enriched. \u0026ldquo;cytokine activity\u0026rdquo; was significantly enriched under the \u0026lsquo;Molecular Function\u0026rsquo; category. Gene set enrichment analysis (GSEA) identified one significantly enriched gene set in the RM⁺ group (Androgen Response; ES: 0.3, NES: 1.44, p \u0026lt; 0.0001) and three in the RM⁻ group (Peroxisome; ES: \u0026minus;0.33, NES: \u0026minus;1.59, p \u0026lt; 0.0001; Acid Metabolism; ES: \u0026minus;0.41, NES: \u0026minus;1.58, p \u0026lt; 0.0001; P53 Pathway; ES: \u0026minus;0.28, NES: \u0026minus;1.42, p = 0.0059) (Fig. 5c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDEGs upregulated or downregulated in the 2D and 3D models were 66/31 and 63/64, respectively. Among them, \u003cem\u003eSIK1\u003c/em\u003e was a commonly regulated DEG in both models (Fig. 5d).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the \u003cem\u003eex vivo\u003c/em\u003e 3D lung cancer model, the group subjected to respiratory motion\u0026mdash;mimicking human-like breathing\u0026mdash;exhibited enhanced cell adhesion and proliferation compared to the group with no motion, accompanied by changes in gene expression and signalling pathways that could explain these phenomena. In contrast, adding respiratory motion in 2D cultures led to suppressed cell proliferation, showing a markedly different gene expression profile in RNA sequencing compared to the 3D model. These findings highlight that cellular behaviour is strongly influenced by the experimental model and external mechanical environment. Our findings emphasise the importance of incorporating respiratory motion and an ECM that supports mechanosensing to investigate lung cancer biology more accurately.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioreactor Mimicking a Physiological Environment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioreactors that implement a perfusion\u0026ndash;ventilation model enhance nutrient distribution and promote cell proliferation, leading to more uniform cell distribution \u003csup\u003e26\u003c/sup\u003e. The lung adenocarcinoma cells studied herein were naturally exposed to continuous pressure fluctuations, stretch forces associated with respiratory motion, and blood flow within the lung microenvironment \u003csup\u003e5,27\u003c/sup\u003e. Our bioreactor was designed to provide a favourable environment for cell growth along with the application of physiologically relevant mechanical stresses. Specifically, it truly replicated both shear stress derived from capillary blood flow and stretch stress caused by the large, multidirectional deformation of lung tissue \u003csup\u003e21\u003c/sup\u003e. Unlike conventional studies employing volume-controlled systems \u003csup\u003e25\u003c/sup\u003e, our pressure-controlled system manages negative pressure within the chamber and vascular pressure at levels close to that of the human pulmonary artery, facilitating a more realistic simulation of the intrathoracic environment. Furthermore, the baseline fluctuation observed in the pulmonary arterial pressure graph reflects the respiratory variation observed in humans, reinforcing the physiological relevance of the environment produced by this bioreactor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison Between 2D and 3D Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was suppressed, and apoptosis was induced in the 2D RM⁺ group. RNA sequencing revealed upregulation of tumour suppressor genes, including \u003cem\u003eCDKN1A\u003c/em\u003e and \u003cem\u003eNR4A3\u003c/em\u003e \u003csup\u003e28,29\u003c/sup\u003e, and downregulation of tumour-promoting genes, including \u003cem\u003eCA9\u003c/em\u003e, \u003cem\u003eEFNA1\u003c/em\u003e, and \u003cem\u003eSUSD2\u003c/em\u003e \u003csup\u003e30\u0026ndash;33\u003c/sup\u003e. Furthermore, nuclear translocation of \u0026beta;-catenin and YAP was not observed. These findings suggest that, in a 2D environment lacking ECM, negative pressure may act as a stressor, and respiratory motion alone is insufficient to activate intracellular signalling pathways.\u003c/p\u003e\n\u003cp\u003eIn contrast, the 3D RM⁺ group exhibited a higher proliferation rate without any apparent induction of apoptosis. RNA sequencing revealed the upregulation of genes involved in cell proliferation and tumour progression, including \u003cem\u003eINHBA\u003c/em\u003e, \u003cem\u003eLOX\u003c/em\u003e, \u003cem\u003eLRRC15\u003c/em\u003e, and \u003cem\u003eCXCL12\u003c/em\u003e \u003csup\u003e34\u0026ndash;38\u003c/sup\u003e. GO analysis showed enrichment of genes related to cell\u0026ndash;cell adhesion and ECM components. Notably, nuclear translocation of \u0026beta;-catenin and YAP were increased in the RM⁺ group. These results suggest that in a 3D model with ECM support, negative pressure does not act as a stressor; rather, stretch stress transmitted through the ECM triggers signal transduction pathways, particularly those involved in cell adhesion. Based on the RNA sequencing data, the increased cell proliferation in the RM⁺ group may be a secondary effect of enhanced cell adhesion stability rather than a direct consequence of respiratory motion. Supporting this interpretation, GSEA revealed upregulation of the tumour-suppressive p53 gene set in the RM⁻ group, suggesting that the absence of respiratory motion may exert tumour-suppressive effects in lung adenocarcinoma cells.\u003c/p\u003e\n\u003cp\u003eInterestingly, only one gene, \u003cem\u003eSIK1\u003c/em\u003e, was a common DEG between the 2D and 3D models. This finding indicates that even when the same cell line is exposed to the same respiratory motion, gene expression responses differ markedly depending on the model system, including factors such as dimensionality and the presence or absence of physical scaffolding. These results underscore the critical role of ECM in modulating the cellular response to respiratory motion and highlight the profound influences of model choice on cell behaviour. Although previous studies have demonstrated that 3D models more accurately recapitulate the physiological dynamics of cancer cells \u003csup\u003e14,39\u0026ndash;43\u003c/sup\u003e, few have directly compared dimensional models under mechanical stress. In this study, we conducted a direct comparison and found that 3D models indeed provide a more physiologically relevant context, supporting their utility in lung cancer research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Respiratory Motion in the 3D Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the 3D RM⁺ group, cell adhesion was enhanced, accompanied by increased expression of adhesion-related proteins, including integrin \u0026beta;1 and E-cadherin, and increased nuclear translocation of \u0026beta;-catenin. Integrin \u0026beta;1 is a well-known mechanosensor responsible for ECM adhesion and mechanical signal transduction \u003csup\u003e44\u0026ndash;46\u003c/sup\u003e. E-cadherin is crucial in cell\u0026ndash;cell adhesion, epithelial\u0026ndash;mesenchymal transition (EMT), and tumour suppression \u003csup\u003e47,48\u003c/sup\u003e. \u0026beta;-catenin is involved in both cell adhesion and Wnt signalling; upon nuclear translocation, it binds to transcription factors (TCF/LEF) and promotes gene expression \u003csup\u003e48\u003c/sup\u003e. Cyclic stretch enhances Wnt/\u0026beta;-catenin signalling in A549 cells \u003csup\u003e49\u003c/sup\u003e, and accurately replicating mechanical stretch is crucial for cellular responses \u003csup\u003e50\u003c/sup\u003e. Additionally, CXCL12, upregulated in RM⁺, activates integrins independently of CXCR4 \u003csup\u003e51\u003c/sup\u003e. Integrin-mediated activation of \u0026beta;-catenin and YAP has been suggested \u003csup\u003e52\u003c/sup\u003e. In the RM⁺ group, nuclear translocation of YAP was significantly increased, providing strong evidence that cells actively sense mechanical stress \u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eConsidering these findings, the 3D expansion of the lung induced by respiratory motion \u003csup\u003e21,54\u003c/sup\u003e likely facilitates ECM-mediated mechanosensing, promoting the expression of adhesion-related proteins to reinforce cell\u0026ndash;ECM and cell\u0026ndash;cell interactions. This study underscores the necessity of incorporating respiratory motion in 3D models to investigate lung cancer biology accurately.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacteristics of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEx Vivo\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e3D Lung Cancer Model and Comparison with Other Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCurrent experimental strategies for reproducing 3D environments include hydrogel-based culture systems and microfluidic technologies, such as the \u0026lsquo;Lung-on-a-chip\u0026rsquo; model \u003csup\u003e15\u0026ndash;18\u003c/sup\u003e. Each model possesses distinct advantages and limitations and differs significantly from the \u003cem\u003eex vivo\u003c/em\u003e 3D lung cancer model employed in this study.\u003c/p\u003e\n\u003cp\u003eHydrogels enable 3D cell culture, offering a more physiologically relevant context for cell\u0026ndash;cell and cell\u0026ndash;ECM interactions compared to 2D cultures \u003csup\u003e16\u003c/sup\u003e. However, Matrigel composition differs from that of the ECM found in the tumour microenvironment, making it challenging to truly replicate specific tissue- or cell-type-derived ECM components. Furthermore, the physical properties of Matrigel are difficult to control, limiting its suitability for precise analyses of mechanical stress responses.\u003c/p\u003e\n\u003cp\u003e\u0026lsquo;Lung-on-a-chip\u0026rsquo; systems utilise microfluidic devices to culture cells in microscale channels that simulate the dynamic environment of the lung\u003csup\u003e55\u003c/sup\u003e. A notable advantage of this model is the ability to establish air\u0026ndash;liquid interface cultures, essential for mimicking lung-specific functionality. Nevertheless, the complexity of device fabrication and operation, along with limitations in culture scale, makes this model less suitable for tissue-level investigations \u003csup\u003e56\u003c/sup\u003e. \u0026lsquo;Lung-on-a-chip\u0026rsquo; systems have limited capacity for assessing ECM interactions in detail and are not ideal for studying signalling pathways mediated by native tissue-derived ECM.\u003c/p\u003e\n\u003cp\u003eIn contrast, our \u003cem\u003eex vivo\u003c/em\u003e 3D model retains the ECM architecture and microenvironment of actual lung tissue, allowing for the observation of cellular adhesion, proliferation, and tissue-level responses. This model can facilitate studies on ECM-mediated signalling changes that are difficult to replicate using conventional 2D culture or artificial matrices such as hydrogels. Observing ECM-specific signalling pathways\u0026mdash;particularly those unique to lung tissue\u0026mdash;is a key advantage of our model. However, we did not directly compare it with other widely adopted 3D models, such as organoids or \u0026lsquo;Lung-on-a-chip\u0026rsquo; systems, so the relative superiority of our 3D model within the broader landscape of 3D culture systems remains to be determined.\u003c/p\u003e\n\u003cp\u003eIn summary, our findings revealed substantial differences in gene and protein expressions between 2D and 3D cultures, as well as between conditions with and without respiratory motion. These results demonstrate that cellular behaviour and gene expression on 2D substrates do not fully recapitulate those observed in 3D environments or \u003cem\u003ein vivo\u003c/em\u003e. This study highlights the limitations of conventional 2D models in basic research and emphasises the need for 3D culture systems that provide respiratory motion and appropriate scaffolding to support lung adenocarcinoma cell proliferation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eHarvest of Rat Heart\u0026ndash;Lung Blocks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLungs were obtained from 6- to 10-week-old male Sprague\u0026ndash;Dawley (SD) rats (Jackson Laboratory Japan, Inc., Kanagawa, Japan). All animal procedures were approved by the Animal Care and Use Committee of the University of Toyama (approval number: A2023UH-01) and conducted in accordance with the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eRats were anaesthetised with inhaled isoflurane (DS Pharma Animal Health, Osaka, Japan), followed by intraperitoneal administration of a mixed anaesthetic cocktail (total volume: 25 mL) comprising 1.5 mL of medetomidine hydrochloride (1 mg/mL; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), 4 mL of midazolam (5 mg/mL; Sandoz K.K., Tokyo, Japan), 5 mL of butorphanol tartrate (5 mg/mL; Meiji Animal Health Co., Ltd., Tokyo, Japan), and 14.5 mL of saline. The dose was adjusted to 0.5 mL per 200 g of body weight to ensure adequate analgesia and sedation.\u003c/p\u003e\n\u003cp\u003eA tracheotomy was performed, and a 16G catheter was inserted into the trachea for intubation. The rats were on a ventilator at a tidal volume of 10 mL/kg with a respiratory rate of 90 breaths per minute. After a transverse abdominal incision, the anterior thoracic wall was removed. Anticoagulation was achieved by injecting heparin sodium (1000 U/kg; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) via the inferior vena cava, followed by transection of the cardiac apex.\u003c/p\u003e\n\u003cp\u003eA 16G catheter was inserted into the pulmonary artery via the right ventricle, and the lungs were perfused with 50 mL of phosphate-buffered saline (PBS) containing heparin sodium (50 U/mL) and sodium nitroprusside dihydrate (SNP; Sigma-Aldrich, St. Louis, MO, USA; 10 \u0026mu;g/mL) to flush out the blood. The lungs were excised \u003cem\u003een bloc\u003c/em\u003e together with the heart and trachea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDecellularisation of the Lung Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCannulas were inserted into the trachea, pulmonary artery, and left ventricle (pulmonary vein), and lungs were decellularised following a previously reported method \u003csup\u003e25,57\u003c/sup\u003e. In brief, the pulmonary vasculature was perfused \u003cem\u003evia\u003c/em\u003e the pulmonary artery with PBS containing calcium and magnesium (PBS⁺) supplemented with heparin sodium, SNP, antibiotics, and 0.0035% Triton X-100 (Nacalai Tesque, Kyoto, Japan). Subsequently, 20 units of Benzonase (25 U/\u0026mu;L; Enzynomics, Daejeon, South Korea) in Benz Buffer (50 mM Tris-HCl, 0.1 mg/mL BSA, 1 mM MgCl₂) was administered via the airway.\u003c/p\u003e\n\u003cp\u003eNext, sodium deoxycholate (SDC; Nacalai Tesque, Kyoto, Japan) at concentrations of 0.01%, 0.05%, and 0.1% in PBS without calcium and magnesium (PBS⁻) was perfused through the vasculature, followed by airway administration of 20 units of Benzonase in Benz Buffer. After vascular perfusion with 0.5% Triton X-100, the lungs were flushed with PBS⁻ and perfused with PBS⁻ containing penicillin\u0026ndash;streptomycin, amphotericin B, and gentamicin (Supplementary Fig. 1a). Decellularised lungs were temporarily stored in PBS⁻ containing these antibiotics (Supplementary Fig. 1b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture and Preparation of Human Lung Cancer Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human lung adenocarcinoma cell line A549 was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan; https://cellbank.nibiohn.go.jp). Cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, high glucose, 4.5 g/L; Nacalai Tesque, Kyoto, Japan), supplemented with 10% foetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin\u0026ndash;streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin; Nacalai Tesque, Kyoto, Japan), 1% amphotericin B (250 \u0026mu;g/mL; FUJIFILM, Tokyo, Japan), and 0.5% gentamicin (10 mg/mL; Gibco, Waltham, MA, USA). Cells were maintained at 37 \u0026deg;C in a humidified atmosphere containing 5% CO₂. For passaging or harvesting, cells were detached using 0.05% trypsin\u0026ndash;EDTA solution (Nacalai Tesque, Kyoto, Japan) at 37 \u0026deg;C for 5\u0026ndash;7 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioreactor Setup for the 3D Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDecellularised lungs with cannulas in place were placed into the chamber, with only the trachea and pulmonary artery connected to the tubing. The cannula inserted into the left ventricle (representing the pulmonary vein) was left open to the culture medium inside the chamber. The medium collected from the pulmonary vein side\u0026mdash;from within the chamber\u0026mdash;was circulated into the pulmonary artery via the BPU, enabling continuous intravascular perfusion and pressure monitoring (Fig. 2b).\u003c/p\u003e\n\u003cp\u003eThe tubing connected to the airway was linked to a reservoir bottle containing 60 mL of culture medium, allowing for passive medium movement in response to changes in lung volume. The chamber was connected to the PSU, which continuously regulated and monitored air pressure to maintain the desired pressure settings (Fig. 2b, c). Intrachamber pressure measurements were taken once every 300 seconds (Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioreactor Setup for the 2D Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded onto plastic well plates to evaluate the effect of respiratory motion\u0026mdash;represented by pressure fluctuations within the chamber\u0026mdash;on A549 cells cultured in a conventional 2D environment. Two 6-well plates (SPL Life Sciences, Pocheon, South Korea) were prepared, with 3 \u0026times; 10⁵ A549 cells seeded per well. One plate was placed in a 2D culture chamber and connected to the PSU, referred to as the RM⁺ group, while the other plate was not connected to the PSU and served as the RM⁻ group (Fig. 1a).\u003c/p\u003e\n\u003cp\u003eBoth plates were incubated under standard conditions (37 ℃, 5% CO₂) for four days. After incubation, cells were collected for further analysis. Intrachamber pressure measurements were taken once every 300 seconds (Fig. 1b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeeding and Perfusion Culture of Lung Cancer Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo decellularised lungs and corresponding chambers were prepared to compare conditions with and without respiratory motion. On the evening of Day 0, preconditioning of the decellularised lungs was initiated to promote peripheral vascular expansion. Specifically, each lung was placed in a chamber filled with 200 mL of DMEM and connected to vascular perfusion at a flow rate of 14 mL/min in an incubator at 37 \u0026deg;C with 5% CO₂. During this period, neither chamber was subjected to respiratory motion.\u003c/p\u003e\n\u003cp\u003eOn Day 1, A549 lung cancer cells were seeded (recellularised) \u003cem\u003evia\u003c/em\u003e the airway. While applying a negative pressure of -10 mmHg using the PSU to expand the lung in the sealed chamber, 4\u0026ndash;5 \u0026times; 10⁷ A549 cells suspended in 10 mL of DMEM were injected through the airway using a syringe. To prevent the backflow of cells, the airway tubing was clamped, and the lungs were kept stationary for 1 hour in an incubator at 37 \u0026deg;C with 5% CO₂.\u003c/p\u003e\n\u003cp\u003eSubsequently, vascular perfusion was resumed in both chambers at a flow rate of 7 mL/min, and respiratory motion was initiated in only one chamber (RM⁺ group) while the other remained static (RM⁻ group). Respiratory motion was simulated by cyclically modulating the intrachamber pressure between -5 mmHg and atmospheric pressure using the PSU, inducing expansion and contraction of the lung tissue, respectively (Fig. 2d). The vascular perfusion rate was gradually increased every 24 hours: 7 mL/min \u0026rarr; 14 mL/min \u0026rarr; 21 mL/min. After 72 hours of culture, the lungs were harvested for further analysis. Four independent sets of RM⁺ and RM⁻ groups were prepared for comparative analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology, Immunohistochemistry, and Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 2D cultures, cells were collected using 0.05% trypsin\u0026ndash;EDTA solution and put into Millicell EZ SLIDE 8-well glass slides (Merck KGaA, Darmstadt, Germany) for subsequent staining.\u003c/p\u003e\n\u003cp\u003eFor 3D cultures, the entire left lung was fixed in 10% neutral buffered formalin (Nacalai Tesque, Kyoto, Japan), sliced longitudinally into 4\u0026ndash;5 sections, embedded in paraffin, and sectioned at a thickness of 5 \u0026mu;m. HE staining was performed following standard protocols.\u003c/p\u003e\n\u003cp\u003eFor immunohistochemistry, paraffin sections were deparaffinised with xylene and ethanol, followed by washing with PBS. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) at temperatures exceeding 100 \u0026deg;C for 10 minutes. The membrane was permeabilised using 0.1% Triton X-100 for 15 minutes, and non-specific binding was blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) for 15 minutes. Primary antibodies were diluted in SignalStain Antibody Diluent (Cell Signaling Technology, Danvers, MA, USA), and samples were incubated overnight at 4 \u0026deg;C. After PBS washing, samples were incubated with secondary antibodies at room temperature (approximately 21 \u0026deg;C) for 1 hour and mounted using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Inc., Hercules, CA, USA).\u003c/p\u003e\n\u003cp\u003eThe primary antibodies used in this study included anti-Ki-67 (418071; Nichirei Biosciences, Tokyo, Japan), cleaved caspase-3 (25128-1-AP; Proteintech, IL, USA), \u0026beta;-catenin (84805; Cell Signaling Technology, MA, USA), integrin \u0026beta;1 (ab30394; Abcam, Cambridge, United Kingdom), and E-cadherin (20874-1-AP; Proteintech, IL, USA). Secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (Thermo Fisher Scientific, MA, USA) were used as appropriate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage Acquisition and Intensity Normalisation for Quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages were acquired using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) or a confocal laser scanning microscope (LSM780, Carl Zeiss, Jena, Germany). Ten high-power fields per sample were examined at 400\u0026times; magnification using a 40\u0026times; objective lens with a 10\u0026times; eyepiece for quantitative analysis. Image analysis was performed using the ImageJ software (National Institutes of Health, https://imagej.nih.gov/ij/).\u003c/p\u003e\n\u003cp\u003eImages were first converted to greyscale for fluorescence intensity analysis, and background noise was subtracted. Ten regions of interest (ROIs) corresponding to intercellular boundaries were manually selected for each sample. Fluorescence intensity was measured, and comparisons between samples were performed using the corrected integrated density, accounting for background correction. The following formula was used:\u003c/p\u003e\n\u003cp\u003eCorrected IntDen = Raw Integrated Density (RawIntDen) \u0026ndash; (Background Mean \u0026times; ROI Area)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalculation of Cell Adhesion Rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong all HE-stained sections prepared using the left lung, the proportion of the tissue area showing attached tumour cells was defined as the \u0026ldquo;cell adhesion rate\u0026rdquo;. HE-stained slide images were imported into ImageJ software and converted to 8-bit greyscale images. The threshold was adjusted to visualise the attached cells. Based on the histogram displayed by ImageJ, the lower threshold (0%) was set for cell detection, and the upper threshold (100%) was set to include the entire tissue section.\u003c/p\u003e\n\u003cp\u003eThe area detected at the 50% threshold, representing attached cells, was divided by the area detected at the 100% threshold, representing the entire tissue section. This ratio was used to calculate the cell adhesion rate as follows (Fig. 3c):\u003c/p\u003e\n\u003cp\u003eCell adhesion rate (%) = (Area of detected cells at 50% threshold / Area of total section at 100% threshold) \u0026times; 100\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Extraction and RNA Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 2D cultures, A549 cells were harvested from well plates using 0.05% trypsin\u0026ndash;EDTA solution (Nacalai Tesque, Kyoto, Japan). For 3D cultures, a portion of the right lung was finely minced and immediately immersed in RNAlater Stabilisation Solution (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer\u0026rsquo;s instructions. The fixed lung samples were homogenised for at least 30 seconds using a BioMasher II homogeniser (Funakoshi, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the RNeasy Mini Kit (QIAGEN N.V., Venlo, The Netherlands) following the manufacturer\u0026rsquo;s protocol. RNA concentration and purity were assessed using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples were sent to Rhelixa (Tokyo, Japan) for RNA sequencing.\u003c/p\u003e\n\u003cp\u003eGSEA was performed using the RNA data obtained from 3D cultures. GSEA version 4.4.0 (https://www.gsea-msigdb.org/gsea/index.jsp) was used, with gene sets retrieved from the Molecular Signatures Database (MSigDB).\u003c/p\u003e\n\u003cp\u003eRNA was extracted from decellularised lungs (without cell seeding) using the same protocol to assess residual rat-derived RNA as a control (\u003cem\u003en\u003c/em\u003e = 1) (Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFisher\u0026rsquo;s exact test was used for categorical variables. Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to compare continuous variables between the two groups. A p-value of less than 0.05 was considered statistically significant. A -log₁₀(p-value) greater than 1.30 was considered statistically significant. All statistical analyses were performed using JMP Pro software (version 16.2.0; JMP Statistical Discovery LLC, Cary, NC, USA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Grant-in-Aid for Scientific Research on Research Activity Start-up (Project title: \u003cem\u003eAnalysis of Mechanical Stress in Ex Vivo Lung Cancer Models\u003c/em\u003e; Grant number: 23K19530; FY2023\u0026ndash;2024) and a donation from Hayashida Finance LLC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Experimentation Committee of the University of Toyama (approval number: A2023UH-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the technical support provided by Sanae Hirota (University of Toyama).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.K. performed manuscript writing, harvesting of rat lungs, 2D/3D cell culture, immunofluorescence staining, and data analysis. M.I. assisted with immunofluorescence staining, image preparation, and RNA extraction. S.M. and S.R. supported decellularisation and recellularisation. S.W., K.K., M.H., D.N., R.Y., N.K., and T.O., under the supervision of Y.M., carried out decellularisation and recellularisation procedures and contributed to sample preservation. K.S. provided technical guidance for the harvesting of rat lungs. N.K. also developed the bioreactor and provided technical support. H.H. and K.H. prepared pathological slides. All authors reviewed and edited the manuscript. T.T. conceived, designed, directed, and supervised the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have no conflicts of interest to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNakaji‐Hirabayashi, T. et al. 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Rep.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 8447 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6482124/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6482124/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe tumour microenvironment is influenced by mechanical stress, including shear and stretch forces, which regulate cancer cell behaviour. Although two-dimensional (2D) culture models are commonly used in cancer research, they fail to recapitulate complex mechanical cues of native tissues. In this study, we developed an \u003cem\u003eex vivo\u003c/em\u003e three-dimensional (3D) lung cancer model by seeding human lung adenocarcinoma cells into decellularised rat lungs and culturing them in a bioreactor mimicking respiratory motion and blood flow. Comparative analysis between 2D and 3D cultures, with and without simulated respiratory motion, revealed striking differences in cellular behaviour and gene expression. In 3D culture, respiratory motion enhanced cell adhesion, proliferation, and nuclear translocation of β-catenin and YAP, along with upregulation of integrin β1, E-cadherin, and genes related to extracellular matrix and cytokine signalling. In contrast, respiratory motion in 2D culture suppressed proliferation and induced apoptosis, highlighting the importance of extracellular matrix-mediated mechanotransduction. Our findings demonstrate that dimensionality and mechanical stress synergistically affect lung cancer cell dynamics and underscore the need for physiologically relevant 3D models incorporating mechanical cues for accurate cancer research.\u003c/p\u003e","manuscriptTitle":"Lung Adenocarcinoma Cells Respond Differently to Mechanical Stress in 3D Versus 2D Environments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 13:35:41","doi":"10.21203/rs.3.rs-6482124/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"33804089-2c1e-48d4-8383-cda62b239891","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47623913,"name":"Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer"},{"id":47623914,"name":"Biological sciences/Biological techniques/Biological models/Cancer models"},{"id":47623915,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":47623916,"name":"Biological sciences/Biotechnology/Sequencing/RNA sequencing"}],"tags":[],"updatedAt":"2025-12-30T08:09:40+00:00","versionOfRecord":{"articleIdentity":"rs-6482124","link":"https://doi.org/10.1038/s42003-025-09179-1","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-12-11 05:00:00","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-06-13 13:35:41","video":"","vorDoi":"10.1038/s42003-025-09179-1","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09179-1","workflowStages":[]},"version":"v1","identity":"rs-6482124","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6482124","identity":"rs-6482124","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}