The essential role of cytoskeleton and ciliary system alterations in the development of congenital pulmonary airway malformations

The essential role of cytoskeleton and ciliary system alterations in the development of congenital pulmonary airway malformations | 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 Research Article The essential role of cytoskeleton and ciliary system alterations in the development of congenital pulmonary airway malformations Tianqi Zhu, Xinyao Meng, Qingxuan Hu, Ke Chen, Xiaofeng Xiong, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7036277/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Pediatric Surgery International → Version 1 posted 7 You are reading this latest preprint version Abstract Purpose Congenital pulmonary airway malformation (CPAM) is a developmental lung malformation that seriously endangers children's health. The objective of this study was to investigate the etiology of CPAM by observing changes at the molecular and cellular levels. Methods Patient clinical data were collected and analyzed. Tissue samples were collected from CPAM lesions and marginal normal lung tissue during CPAM surgery. The samples were subjected to H&E staining for pathological analysis. Tissue RNA was isolated for RNA sequencing, and the differentially expressed genes (DEGs) were enriched for Gene Ontology (GO) analysis. The cytoskeletal and cell subtypes were subjected to immunofluorescence staining. Results RNA sequencing of 7 CPAM patients revealed 1252 DEGs, with 1041 upregulated and 211 downregulated. GO analysis revealed that biological processes related to cilium organization and movement were strongly enriched. Protein‒protein interaction (PPI) network analysis highlighted genes such as BMP2, TNF, DNAI1, DNAI2, and DNAH5 as potentially important in CPAM. Immunofluorescence staining revealed abnormalities in the cytoskeleton and a reduction in the number of alveolar epithelial type II (AEC II) cells in CPAM lesions compared with normal lung tissue. Conclusion Our study revealed that CPAM is associated with a significant increase in cytoskeletal elements and a decrease in AEC IIs, along with an abnormal increase in the expression of cilium-related genes. These alterations provide critical insights into the etiology of CPAM and may guide the development of improved diagnostic and therapeutic strategies for this condition. Congenital pulmonary airway malformation cytoskeleton cilia alveolar epithelial cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Congenital pulmonary airway malformation (CPAM) of the lung, also known as congenital cystic adenomatoid malformation (CCAM), is a rare developmental anomaly characterized by the proliferation of distal bronchi [ 1 ] . It accounts for 25% of congenital pulmonary malformations, with the majority of cases occurring in neonates and infants [ 2 ] . The initial categorization of CPAM was based on the predominant characteristics of cyst size and morphology, which were grouped into three categories. Type I is characterized by the presence of single or multiple large cysts (> 2 cm) with pseudostratified columnar epithelium. Type II is defined by the occurrence of multiple smaller cysts (< 1 cm) with cuboidal or columnar epithelia. Type III is associated with a microcystic lesion with a cuboidal epithelium. Two additional categories, Type 0 (solid lesions) and Type IV (large cysts with flattened epithelium), were subsequently incorporated into this classification system. Both Type 0 and Type IV fractures are relatively rare [ 3 ] . A large lesion may cause a respiratory disorder, resulting in a mediastinal shift, infections, edema and even malignant transformation [ 4 , 5 ] . The pathogenesis of CPAM is not fully understood, but it is thought to result from the arrest of lung development at different stages of branching of the bronchopulmonary tree. This can lead to the formation of cysts lined with different types of epithelium, reflecting the stage of development at which arrest occurs [ 6 ] . The proposed mechanism is related to the overgrowth of mesenchymal elements with subsequent suppression of alveolar growth and decreased apoptosis. Potential genes of influence, such as HOXB5, cyclin D1, and PDGF-BB, have been identified; however, the complete mechanism has not yet been elucidated [ 7 ] [ 8 ] . Therefore, the etiology of this disease remains unclear and controversial. The human lung contains two main types of alveolar epithelial cells: alveolar type I epithelial cells (AECs) and alveolar type II epithelial cells (AEC IIs). These cells are responsible for maintaining the normal function of the alveolar epithelium through their distinctive morphological and functional characteristics [ 9 , 10 ] . The results of a recent study revealed an increase in the number of these two cell types in CPAM [ 11 ] . Nevertheless, few studies have been conducted with the objective of either corroborating or refuting the findings of this research. The cytoskeleton is a complex network of protein filaments that play pivotal roles in maintaining cellular shape, enabling cellular motion, and facilitating intracellular transport. [ 12 ] . Cilia are cellular organelles that project from the cell surface and are involved in a number of critical functions, including the sensing of environmental signals and the facilitation of cellular movement [ 13 , 14 ] . Both the cytoskeleton and cilia are integral structures that facilitate cell movement. A significant body of research has highlighted the interconnection between cilia and CPAM [ 15 – 17 ] . However, the relationship between CPAM and the cytoskeleton remains relatively understudied. In this study, we collected tissue samples from surgical patients, performed experiments utilizing RNA-seq and immunohistochemistry, and explored alterations in cellular structures, such as the cytoskeleton and cilia, during the pathogenesis of CPAM, as well as the impact and role of two types of alveolar epithelial cells in this progression. Materials and methods Study design This is a prospective study of 34 children patients (aged from 4 months to 13 years) who had received a preoperative diagnosis of CPAM and were surgically treated from 2022 to 2024 at Tongji Hospital. All patients were histopathologically confirmed as CPAM after surgery. During hospitalization, the children’s caregivers were informed about the study and they provided written consent. Chest computed tomography (CT) scans were performed before surgery to clarify the classification(Fig. 1 ). The intraoperative findings in all cases were consistent with the preoperative classification. In all cases,, we used aerosolized indocyanine green to visualize the lesion and show its margin [ 18 ] (Fig. 1 ). We obtained samples of the lesion and adjacent normal tissue for subsequent experimental comparison. All adjacent normal tissues were confirmed by histopathology. The sampling was approved by the ethics committee and the patient consented. Tissue samples were immediately preserved after obtained from surgery. In every case, a portion of the tissue sample was fixed with formalin for HE staining, and the remainder was snap-frozen in liquid nitrogen for other experiments. RNA sequencing Among the patients, 7 were selected for RNA sequencing, and the relevant information is presented in Table 1 . Total RNA was extracted from the frozen lung tissues of CPAM patients via TRIzol Reagent (Invitrogen). Four micrograms of total RNA were used for cDNA sequencing library generation via the NEBNext Ultra RNA Library Prep Kit (NEB, USA). The purified products were evaluated with an Agilent Bioanalyzer (Agilent Technologies). Eligible libraries were sequenced on the Illumina HiSeq 2000 platform via 100-bp paired-end reads. The original fastq data were evaluated, and low-quality reads were filtered by the Trimmomatic tool. The filtered clean reads were aligned to the human reference genome (GRCh38) via HISAT2. Only protein-coding genes were retained for analysis, and genes with no mapped reads in at least half of the samples were excluded. Gene expression levels were quantified with raw counts (FPKM) via StringTie. Table 1 Characteristics of patients Gender Age(Years old) Lesion location Male:16 < 1:21 Left lung:12 Female:18 ≥ 1:13 Right lung:20 Both lungs:2 Differential expression analysis Differentially expressed genes (DEGs) between cysts and simple controls were identified via the DESeq2 package in R. DEGs were selected on the basis of the thresholds of both a P value 2. DEGs were visualized via volcano plots. Principal component analysis (PCA) was performed to detect the overall differences between the individual samples via the normalized counts of all the genes. Functional enrichment and protein‒protein interaction network analysis To evaluate the functions of the differentially expressed genes (DEGs) identified in CPAM, Gene Ontology (GO) enrichment analysis was conducted via the clusterProfiler package with the upregulated genes and downregulated genes. Only those GO terms with an FDR value of less than 0.05 were considered to represent a significant event. The PPI data were downloaded from the STRING database to investigate protein interactions among the DEG sets that were enriched in the aforementioned significant GO terms. Only those interactions with a PPI score greater than 500 were retained for further analysis, and the PPI network was visualized via Cytoscape software. Immunohistochemistry The paraffin-embedded sections underwent deparaffinization in xylene and subsequent rehydration through a descending ethanol concentration series. The sections were subsequently subjected to microwave treatment for 16 minutes in Tris-EDTA buffer for antigen retrieval. To quench endogenous peroxidase activity, a solution of 3% hydrogen peroxide was applied at room temperature for 5–10 minutes. The slides were then treated to block nonspecific binding by immersion in a mixture of 5% bovine serum albumin (BSA) and 0.1% Triton X-100 in phosphate-buffered saline (PBS) for a period of 1 hour at 37°C. Next, the primary antibody was added to the slides. The slides were incubated with the primary antibody overnight at 4°C, after which they were treated with a secondary horseradish peroxidase-conjugated anti-mouse IgG antibody for an additional hour. The chromogenic reaction was developed by applying a 3,3'-diaminobenzidine (DAB) substrate kit from Servicebio to the slides, allowing visualization of color. Examination under a high-powered microscope involved scrutinizing five randomly selected fields from each affected tissue segment. Consistent imaging conditions were maintained by capturing all images with the same microscope and camera setup. The optical density and mean optical density of positively stained cell structures were quantified via ImageJ software (version 1.43u). The following dyes and primary antibodies were used for staining: phalloidin (dilution 1:100, Abbkine, Wuhan, P.R.C.), DAPI (Servicebio, Wuhan, P.R.C.), AQP5 (dilution 1:100, Rabbit, ABclonal, Wuhan, P.R.C.), SFTPC (dilution 1:100, Rabbit, ABclonal, Wuhan, P.R.C.), Claudin-2 (dilution 1:500, Rabbit, Abcam, Cambridge, UK), E-cadherin (dilution 1:300, Rabbit, Proteintech, Rosemont, USA) and ZO1 (dilution 1:1000, Rabbit, Proteintech, Rosemont, USA). Histopathology The tissue samples were fixed in 10% neutral buffered formalin, decalcified, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) via standard histological methods. Microscopy images were taken via a Zeiss Axioscope A1 microscope. Statistical analyses All the data are presented as the means ± SDs from three or more independent experiments. Two-tailed Student's unpaired t tests were used to assess the statistical significance of the data. All the statistical analyses were performed via SPSS 16.0. Statistical significance was set at probability values of p < 0.05. Results Clinical features and pathological changes in CPAM patients A total of 34 patients with CPAM were recruited for the study (Table 1 ). All 34 patients underwent successful thoracoscopic wedge resection, lobectomy, or segmentectomy, with no cases requiring conversion to thoracotomy. The mean operation time was 100.3 ± 24.6 minutes, with a mean intraoperative blood loss of 30.5 ± 25.6 millilitres. The mean postoperative chest closed drainage time was 48.5 ± 24.3 hours, with a mean postoperative hospital stay of 6.6 ± 3.2 days. All patients were successfully treated and discharged, with no deaths. No adverse reactions, including allergic reactions, respiratory depression, or respiratory failure, were observed. Postoperative complications included one case of postoperative pneumothorax and three cases of subcutaneous emphysema. The pneumothorax spontaneously healed with prolonged indwelling chest closed drainage tube, while the subcutaneous emphysema did not receive special treatment. All patients were followed up in an outpatient setting for a period of three to six months following surgery, with a 100% follow-up rate. The follow-up content included a chest CT scan and a pulmonary ventilation function test (tidal breathing pulmonary function test). A reexamination of the chest CT scan revealed the absence of residual lesions or recurrence (Fig. 1 ). Transcriptome sequencing revealed significant abnormalities in the cytoskeleton We selected 7 patients out of 34 for RNA sequencing analysis (Table 2 ), including 5 males and 2 females, with surgical ages ranging from 7 months to 3 years. Their lesions were located in the right lower lobe in 4 cases, the left lower lobe in 1 case, the right middle lobe in 1 case and the left upper lobe in 1 case. Table 2 Characteristics of patients selected for RNA sequencing No. Gender Age Lesion Type Surgery 1 Male 1y3m Right lower lobe Type II Right lower lung wedge resection 2 Male 8y Right lower lobe Type III Right lower lobectomy 3 Male 7 m Right lower lobe Type I Right lower lobectomy 4 Female 3y Left lower lobe Type II Left lower lobectomy 5 Male 7 m Right lower lobe Type II Right lower lung segment resection 6 Female 8 m Right middle lobe Type III Right middle lobectomy 7 Male 9 m Left upper lobe Type I Left upper lung segment resection A total of 380 million reads were generated from the 14 transcriptome sequencing datasets. The mean proportion of reads mapped to the human reference genome was 97%. Principal component analysis(PCA) of the gene expression profile revealed that CPAM samples exhibited a clear separation from the control group. A consolidated analysis of all CPAM samples was conducted to facilitate comparison with the control group. This revealed the identification of 1252 differentially expressed genes (DEGs) between CPAM areas and the paired control areas. Of these, 1041 were found to be upregulated, while 211 were downregulated. Interestingly, a large number of DEGs are related to cell cilia, and these genes are marked in Fig. 2 . Genetic abnormalities leading to ciliary dysfunction play an important role in CPAM GO analysis revealed that the upregulated genes were significantly enriched in a number of biological processes, including cilium organization (FDR = 1.80×10 − 49 ) and cilium movement (FDR = 8.49×10 − 45 ). The GO analysis revealed that the upregulated genes were significantly enriched in several biological processes, including epithelial cilium movement involved in extracellular fluid movement (FDR = 4.94×10 − 23 ), phagocytosis and recognition (FDR = 5.14×10 − 21 ), and the humoral immune response mediated by circulating immunoglobulin (FDR = 5.85×10 − 21 ) (Fig. 2 ). The downregulated genes were found to be significantly enriched in a number of biological processes, including positive regulation of response to external stimulus (FDR = 8.38×10 − 8 ), positive regulation of defense response or cytokine production (FDR = 1.28×10 − 7 , FDR = 1.64×10 − 7 ), leukocyte migration (FDR = 6.61×10 − 7 ), and regulation of the inflammatory response (FDR = 8.99×10 − 7 ) (FDR = 1.12×10 − 4 ) (Fig. 2 ). GO enrichment, which is based on molecular function, also revealed that the differentially expressed genes (DEGs) were predominantly involved in microtubule motor and immune factor activities. The genes were found to be significantly associated with the components of the cilium and granule epithelial cells. The GSEA yielded analogous results, with gene sets enriched in ciliary movement, ciliary development, ciliopathies, and axoneme assembly. A total of 747 differentially expressed genes (DEGs) were filtered into the protein‒protein interaction (PPI) network, with a PPI score ranging from 500–999. The network consisted of 492 nodes and 765 edges (Fig. 3 ). Among the identified genes, BMP2, TNF, DNAI1, DNAI2, and DNAH5 presented relatively high degrees and betweenness centrality, indicating strong interactions with other genes. Immunofluorescence staining revealed the presence of cytoskeletal abnormalities and a reduction in the number of AEC IIs in the CPAM The immunofluorescence staining procedure involved the use of DAPI, phalloidin, and antibodies, as illustrated in Figs. 4 A and B. The subsequent quantification of the fluorescence intensity was conducted using Image J. To ensure the accuracy of the normalization process, the fluorescence intensity of the target protein was divided by the fluorescence intensity of the nucleus (stained with DAPI), thereby yielding the relative mean fluorescence intensity. A two-sample t-test was employed to compare the relative mean fluorescence intensity of CPAM lesion tissue with that of normal lung tissue, and the results are presented in Fig. 4 C. A comparison of CPAM and normal lung tissue revealed a significantly greater mean fluorescence intensity in CPAM lesions when they were stained with phalloidin and a lower ratio when they were stained with the SFTPC antibody. No significant differences were detected when the AQP5, claudin-2, E-cadherin, or ZO-1 antibodies were used. The present study found that AQP5 is generally considered to be a marker for AEC I, while SFTPC is a marker for AEC II [ 19 , 20 ] . Phalloidin has been demonstrated to stain the cytoskeleton, while claudin-2, E-cadherin, and ZO-1 have been shown to play important roles in cell junctions [ 21 – 25 ] . Immunofluorescence staining revealed the presence of cytoskeletal abnormalities and a reduction in the number of AEC IIs in the CPAM. Discussion CPAM is a congenital pulmonary malformation that poses a significant risk to the health and well-being of children [ 2 ] . The pathological features of CPAM are characterized by the presence of adenomatous hyperplasia and cyst formation in bronchial-like structures. The cysts are lined by a variety of epithelial cells, including pseudostratified ciliated columnar epithelium and low cuboidal epithelium [ 3 ] . The pathogenesis of CPAM remains incompletely understood. However, it is hypothesized that this condition results from the arrest of lung development at various stages of branching of the bronchopulmonary tree. This can result in the formation of cysts lined with different types of epithelium, which is reflective of the stage of development at which arrest occurs [ 6 ] . However, the underlying cellular and molecular mechanisms remain to be discovered. RNA-seq analysis revealed a significant number of differentially expressed genes (DEGs) between CPAM and normal lung tissues. For these genes, we employed both Gene Ontology (GO) and gene set enrichment analysis (GSEA) for functional enrichment analyses. Notably, the enrichment of pathways related to the ciliated epithelium suggests its potential roles in the pathogenesis of CPAM. This finding is somewhat consistent with existing research results [ 15 – 17 ] . In our PPI network, BMP2, TNF, DNAI1, DNAI2, and DNAH5 exhibited relatively high degrees and betweenness centrality, which were confirmed to play important roles in the regulation and driving of cilia movement [ 26 – 29 ] . Notably, some of these proteins also significantly influence the development and progression of lung cancer [ 30 , 31 ] , which may help explain the link between CPAM and lung cancer [ 4 , 5 ] . Nevertheless, further clinical research is needed to substantiate this perspective. Phalloidin is a fungal toxin that has become an essential tool in cell biology because of its high affinity for filamentous actin (F-actin) [ 22 ] . In immunohistochemistry, phalloidin and its fluorescently labeled derivatives are widely used for the visualization of F-actin in cells and tissues, providing valuable insights into cytoskeletal organization [ 21 , 22 ] . Aquaporin-5 (AQP5) is a water channel protein predominantly found in the lung, salivary gland and lacrimal gland. In the lung, it is specifically expressed in alveolar type I cells (AEC I) [ 19 ] . Surfactant protein C (SFTPC) is expressed specifically in type II alveolar epithelial cells (AEC II), making it a useful marker for these cells in immunohistochemical studies [ 20 ] . Claudin-2 is a protein that belongs to the claudin family of integral membrane proteins found in tight junctions [ 23 ] . E-cadherin, a transmembrane glycoprotein, is essential for the structural and functional maintenance of adherens junctions in epithelial cells [ 24 ] . Zonula occludens-1 (ZO-1) is a tight junction protein that plays a key role in maintaining the structural integrity and barrier function of epithelial cells [ 25 ] . The results of immunohistochemistry demonstrated that CPAM tissue samples presented greater levels of cytoskeleton fluorescent markers than normal lung tissue samples did. This finding indicates an increase in cytoskeleton structure during the occurrence and development of CPAM, which may represent a crucial biological process in the onset and progression of CPAM. In addition, AEC II decreased in CPAM lesion tissue, whereas the amount of AEC I did not change significantly. Claudin-2 and ZO-1, which are associated with cell tight junctions, did not change significantly, and E-cadherin, which is associated with cell adherens junctions, did not change significantly. It is widely acknowledged that alveoli are responsible for gas exchange in the respiratory system. They are composed of two main types of alveolar epithelial cells: alveolar type I epithelial cells (AECs), which constitute the majority (approximately 96%) of the alveolar surface area, and alveolar type II epithelial cells (AEC IIs), which constitute the majority (approximately 60%) of the alveolar cells [ 9 , 10 ] . AECs are notably thin, which allows for efficient gas exchange between the alveolar air space and the pulmonary capillaries [ 10 ] . Their distinctive flattened morphology enables them to cover a vast surface area with minimal thickness, thereby reducing the diffusion distance for gases [ 32 ] . AEC IIs are cuboidal in shape and perform a vital function in the production and secretion of pulmonary surfactant, which is indispensable for reducing surface tension within the alveoli and preventing their collapse [ 33 ] . Additionally, AEC II cells serve as progenitor cells for AEC I and are involved in regeneration of the alveolar epithelium following injury [ 9 , 33 ] . Both cell types are vital for maintaining the integrity and function of the alveolar epithelium. The cytoskeleton is a complex network of protein filaments that play pivotal roles in maintaining cellular shape, enabling cellular motion, and facilitating intracellular transport. It comprises three principal types of protein filaments: actin filaments, microtubules, and intermediate filaments [ 12 ] . The cytoskeleton plays a pivotal role in maintaining cellular structure, intracellular transport, signal transduction and mechanotransduction [ 34 ] . Cilia are cellular organelles that project from the cell surface and are involved in a number of critical functions, including the sensing of environmental signals and the facilitation of cellular movement [ 13 , 14 ] . They can be broadly classified into two categories: motile cilia and primary (nonmotile) cilia [ 35 ] . Both the cytoskeleton and cilia are integral structures that facilitate cell movement. These results suggest that the cells in CPAM lesions have undergone significant alterations in comparison with normal lung tissue cells. On the one hand, an increase in the cytoskeleton has been observed, and on the other hand, the expression of multiple genes related to cilia has been found to be elevated. Lesion tissue has lost the physiological function of normal lung tissue cilia, which move debris and mucus out of the airways by beating in a coordinated and continuous manner [ 35 ] . Therefore, we tend to assume that these changes in the expression of cilium-related genes lead to changes in ciliary ultrastructure and motility, thus resulting in functional failure of ciliary movement and clearance. Severe mucus accumulation in the airway ultimately causes inflammation and adversely affects lung function and development. Both AECs I and II are highly important for the maintenance of normal lung tissue function. AECs constitute the majority of the alveolar epithelium in terms of surface area and are responsible for the vital process of gas exchange [ 32 ] . AEC II is present in large numbers in normal lung tissue and is the progenitor cell of AEC I. In the event of damage to the alveolar epithelium, AEC II will differentiate into AEC I to repair damaged tissue [ 33 ] . The findings of this study indicate that, compared with that in normal lung tissue, the AEC II in CPAM samples was lower. Conversely, no such reduction was observed in AEC I. This finding indicates that in the pathogenesis of CPAM, either AEC II is specifically subjected to a negative influence or AEC I is damaged, and AEC II differentiates and replenishes AEC I over time, resulting in the loss of AEC II. Both factors may also be present. Conclusion In conclusion, this study collected clinical tissue samples and conducted experimental explorations, which revealed a significant increase in the cytoskeleton, a significant decrease in AEC II and an abnormal increase in the expression of cilium-related genes in CPAM. These findings provide crucial insights into the etiology of CPAM, which may inform the development of more effective diagnostic and therapeutic strategies. Declarations Ethics approval and consent to participate The Institutional Review Board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology approved the protocol of the study (Permit Number 20240403 Wuhan, China). All study procedures complied with regulations in the Declaration of Helsinki. Consent for publication Informed written consents were obtained from the patient's parents for publication this study. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding No funding was involved in this study. Authors' contributions T.Z., X.M. and Q.H wrote the main manuscript text, K.C. prepared figures 4A&B. Y.Y., D.Z., Y.H., J.W., J. F. and X.C. provided vital support for writing this manuscript. All authors reviewed the manuscript. Acknowledgements Some experiments in this study were conducted at the Experimental Medicine Center of Tongji Hospital, which is affiliated with Tongji Medical School at Huazhong University of Science and Technology. The authors would like to express their gratitude to the staff at the Experimental Medicine Center for their assistance in this study. References PEDERIVA F, ROTHENBERG S S, HALL N et al (2023) Congenital lung malformations [J]. Nat Rev Dis Primers 9(1):60 EL AMRAOUI W, BENTALHA A, HAMRI H et al (2017) Congenital cystic adenomatoid malformation - dangers of misdiagnosis: a case report [J]. J Med Case Rep 11(1):212 STRUMIŁŁO B, JóŹWIAK A, PAŁKA A et al (2018) Congenital cystic adenomatoid malformation - diagnostic and therapeutic procedure: 8-year experience of one medical centre [J]. Kardiochir Torakochirurgia Pol 15(1):10–17 IOACHIMESCU O C, MEHTA AC (2005) From cystic pulmonary airway malformation, to bronchioloalveolar carcinoma and adenocarcinoma of the lung [J]. Eur Respir J 26(6):1181–1187 CHANG W C (2021) Mucinous adenocarcinoma arising in congenital pulmonary airway malformation: clinicopathological analysis of 37 cases [J]. 78(3):434–444ZHANG Y Z MOROTTI R A, CANGIARELLA J, GUTIERREZ M, C et al (1999) Congenital cystic adenomatoid malformation of the lung (CCAM): evaluation of the cellular components [J]. Hum Pathol 30(6):618–625 WANG X, WOLGEMUTH D J, BAXI LV (2011) Overexpression of HOXB5, cyclin D1 and PCNA in congenital cystic adenomatoid malformation [J]. Fetal Diagn Ther 29(4):315–320 LIECHTY K W, CROMBLEHOLME T M, QUINN T M et al (1999) Elevated platelet-derived growth factor-B in congenital cystic adenomatoid malformations requiring fetal resection [J]. J Pediatr Surg, 34(5): 805-9; discussion 9–10 WITHERDEN I R, TETLEY TD (2001) Isolation and Culture of Human Alveolar Type II Pneumocytes [J]. Methods Mol Med 56:137–146 BROOKES O BOLANDS (2021) Co-culture of type I and type II pneumocytes as a model of alveolar epithelium [J]. 16(9):e0248798 ZHANG S, YE C, XIAO J et al (2020) Single-cell transcriptome profiling reveals the mechanism of abnormal proliferation of epithelial cells in congenital cystic adenomatoid malformation [J]. Exp Cell Res 396(2):112299 POLLARD T D, GOLDMAN RD (2018) Overview of the Cytoskeleton from an Evolutionary Perspective [J]. Cold Spring Harb Perspect Biol, 10(7) ANVARIAN Z, MYKYTYN K (2019) Cellular signalling by primary cilia in development, organ function and disease [J]. 15(4):199–219 HUA K, FERLAND RJ (2018) Primary cilia proteins: ciliary and extraciliary sites and functions [J]. Cell Mol Life Sci 75(9):1521–1540 ZHANG G, LOU L (2024) The underlying molecular mechanism of ciliated epithelium dysfunction and TGF-β signaling in children with congenital pulmonary airway malformations [J]. Sci Rep 14(1):4430 ZHANG G, CAI C, LI X et al (2022) Application of second-generation sequencing in congenital pulmonary airway malformations [J]. Sci Rep 12(1):20459 TAN Z, LI F, CHEN Q et al (2023) Integrated bulk and single-cell RNA-sequencing reveals SPOCK2 as a novel biomarker gene in the development of congenital pulmonary airway malformation [J]. Respir Res 24(1):127 PHILLIPS JD (2020) Inhalational Indocyanine Green to Visualize Lung Tumors—Defining the Margin of Error [J]. JAMA Surg 155(8):741 FLODBY P, LI C, LIU Y et al (2017) Cell-specific expression of aquaporin-5 (Aqp5) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation [J]. Sci Rep 7(1):3473 JIANG M, ROTH M G, CHUN-ON P et al (2020) Phenotypic Diversity Caused by Differential Expression of SFTPC-Cre-Transgenic Alleles [J]. 62(6):692–698 POSPICH S, MERINO F (2020) RAUNSER S. Structural Effects and Functional Implications of Phalloidin and Jasplakinolide Binding to Actin Filaments [J]. Structure, 28(4): 437 – 49.e5. ROMANI M (2021) Phalloidin Staining of Actin Filaments for Visualization of Muscle Fibers in Caenorhabditis elegans [J]. Bio Protoc 11(19):e4183 CAO W, XING H, LI Y et al (2022) Claudin18.2 is a novel molecular biomarker for tumor-targeted immunotherapy [J]. Biomark Res 10(1):38 VAN ROY F BERXG (2008) The cell-cell adhesion molecule E-cadherin [J]. Cell Mol Life Sci 65(23):3756–3788 NEYRINCK-LEGLANTIER D, LESAGE J, BLACHER S et al (2021) ZO-1 Intracellular Localization Organizes Immune Response in Non-Small Cell Lung Cancer [J]. Front cell Dev biology 9:749364 DOUGHERTY G W, LOGES N T KLINKENBUSCHJA et al (2016) DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes [J]. Am J Respir Cell Mol Biol 55(2):213–224 KOBAYASHI D, IIJIMA N, HAGIWARA H et al (2010) Characterization of the medaka (Oryzias latipes) primary ciliary dyskinesia mutant, jaodori: Redundant and distinct roles of dynein axonemal intermediate chain 2 (dnai2) in motile cilia [J]. Dev Biol 347(1):62–70 YANG W, CHEN L, GUO J et al (2022) Multiomics Analysis of a DNAH5-Mutated PCD Organoid Model Revealed the Key Role of the TGF-β/BMP and Notch Pathways in Epithelial Differentiation and the Immune Response in DNAH5-Mutated Patients [J]. 11(24) KUMARI A, CALIZ A D, YOO H J et al (2024) TNF-alpha promotes cilia elongation via mixed lineage kinases signaling in mouse fibroblasts and human RPE-1 cells [J] ZHOU W, YAN K, XI Q (2023) BMP signaling in cancer stemness and differentiation [J]. 12(1):37 TERLIZZI M, COLARUSSO C, SOMMA P et al (2022) S1P-Induced TNF-α and IL-6 Release from PBMCs Exacerbates Lung Cancer-Associated Inflammation [J]. Cells, 11(16) YAMAMOTO K, FERRARI JD, CAO Y et al (2012) Type I alveolar epithelial cells mount innate immune responses during pneumococcal pneumonia [J]. J Immunol 189(5):2450–2459 CASTRANOVA V, RABOVSKY J, TUCKER JH et al (1988) The alveolar type II epithelial cell: a multifunctional pneumocyte [J]. Toxicol Appl Pharmacol 93(3):472–483 LAMBERT MW (2019) Cytoskeletal and nucleoskeletal interacting protein networks play critical roles in cellular function and dysfunction [J]. Exp Biol Med (Maywood) 244(15):1233–1239 BARSCH F, NIEDERMAIR T, MAMILOS A et al (2020) Physiological and Pathophysiological Aspects of Primary Cilia-A Literature Review with View on Functional and Structural Relationships in Cartilage [J]. 21(14) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Pediatric Surgery International → Version 1 posted Editorial decision: Revision requested 20 Jul, 2025 Reviews received at journal 11 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers invited by journal 08 Jul, 2025 Editor assigned by journal 04 Jul, 2025 Submission checks completed at journal 03 Jul, 2025 First submitted to journal 03 Jul, 2025 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-7036277","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482872032,"identity":"30debeff-34f9-4a7a-bcb7-938718f82ef6","order_by":0,"name":"Tianqi Zhu","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Tianqi","middleName":"","lastName":"Zhu","suffix":""},{"id":482872033,"identity":"0dfe5df0-3246-466b-aba9-24431b1d866a","order_by":1,"name":"Xinyao Meng","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinyao","middleName":"","lastName":"Meng","suffix":""},{"id":482872034,"identity":"c7a2f76a-ab0e-40c1-a088-9fb789b00692","order_by":2,"name":"Qingxuan Hu","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Qingxuan","middleName":"","lastName":"Hu","suffix":""},{"id":482872035,"identity":"74293301-dc6a-4f4d-a593-140bcc845fbe","order_by":3,"name":"Ke Chen","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Chen","suffix":""},{"id":482872036,"identity":"99dbd3eb-a1c1-47d2-8d9e-4a48bf445ebb","order_by":4,"name":"Xiaofeng Xiong","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Xiong","suffix":""},{"id":482872037,"identity":"40d8c226-a0e1-45c7-a27b-09689421f5d7","order_by":5,"name":"Ye Yin","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Yin","suffix":""},{"id":482872038,"identity":"ea225d14-4016-4288-8d5f-d21b022273eb","order_by":6,"name":"Didi Zhuansun","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Didi","middleName":"","lastName":"Zhuansun","suffix":""},{"id":482872039,"identity":"4336d109-1c0c-44d6-b295-b7c3f74aed65","order_by":7,"name":"Ying He","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"He","suffix":""},{"id":482872040,"identity":"0d872636-b184-4b3b-bf66-411edd9ca12c","order_by":8,"name":"Jun Wu","email":"","orcid":"","institution":"Kunming Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Wu","suffix":""},{"id":482872041,"identity":"268a2738-7715-424d-806b-4da0c8751a6a","order_by":9,"name":"Xuan Zhang","email":"","orcid":"","institution":"Pingshan District Maternal \u0026 Child Healthcare Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Zhang","suffix":""},{"id":482872043,"identity":"417ac83b-131a-49ba-bf37-7b57bd71e860","order_by":10,"name":"Jiexiong Feng","email":"","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiexiong","middleName":"","lastName":"Feng","suffix":""},{"id":482872045,"identity":"c6b89bab-6e9a-4edb-a2c8-f46711559c6a","order_by":11,"name":"Xuyong Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYDACCTiLsflBAo8NDz9/A9FamNsMHsikyUjOOEC0FvYGyQc2h20MGhLw6+Cf3XzsMW/bYTlz/oUNBgk553kMGA4wfviYg8eSO8fSDWe2HTa2nPGw4UHCmds85swNzJIzt+HWYiCRYybxse1w4oYbBxsMEntu81g2HGBj5sWrJf+bRCJUi0Tiv3M8BgcSCGnJYYPYcr6xQSKB5wBhLRI30swkZ5xLNza4wdhmkMCTzCM542AzXr/wz0h+Js1TZi1ncP7444c/eOzs+fmbD374iEcLFDQD7UuAcRgbCKoHgjqgfQeIUTgKRsEoGAUjEQAAd/ZYRjjCLDIAAAAASUVORK5CYII=","orcid":"","institution":"Huazhong University of Science \u0026 Technology","correspondingAuthor":true,"prefix":"","firstName":"Xuyong","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-07-03 09:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7036277/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7036277/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00383-025-06250-0","type":"published","date":"2025-11-20T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86655743,"identity":"7a6b9afd-7f7c-43bd-854f-5a817a6ab7c6","added_by":"auto","created_at":"2025-07-14 10:15:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":364599,"visible":true,"origin":"","legend":"\u003cp\u003eCT scan, thoracoscopic views and H\u0026amp;E staining image of CPAM lesions\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7036277/v1/4ef187a7e0bc6f9af48796b8.png"},{"id":86655742,"identity":"e48e39de-7662-42ce-bbe3-4775e9a12b71","added_by":"auto","created_at":"2025-07-14 10:15:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96558,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs in CPAM\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7036277/v1/12f3bf192627eb875797e51c.png"},{"id":86655745,"identity":"00a78bdd-ac19-4d0b-9b0e-e8e22d8305c9","added_by":"auto","created_at":"2025-07-14 10:15:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130028,"visible":true,"origin":"","legend":"\u003cp\u003eA GO analysis and GSEA of DEGs in CPAM\u003c/p\u003e\n\u003cp\u003eB PPI network of DEGs in CPAM\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7036277/v1/9317ed4cc0ae24a2ef71d28d.png"},{"id":86655746,"identity":"06563a77-06cc-4f19-82a8-3b89134595e5","added_by":"auto","created_at":"2025-07-14 10:15:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":182066,"visible":true,"origin":"","legend":"\u003cp\u003eA Immunofluorescence staining of AQP5 and SFTPC, the marker of AEC I and AEC II\u003c/p\u003e\n\u003cp\u003eB Immunofluorescence staining of phalloidin, claudin-2, E-cadherin and ZO-1\u003c/p\u003e\n\u003cp\u003eC Comparisons of immunofluorescence staining results between normal lung tissue and CPAM lesion tissue\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7036277/v1/84fa19ef757603738fe8aabc.png"},{"id":96650344,"identity":"8e33cfa4-930b-4e4d-88c3-fbf25f5af5d8","added_by":"auto","created_at":"2025-11-24 16:11:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1556101,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7036277/v1/c62d84f2-0ede-44db-a573-47158f9da2b4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The essential role of cytoskeleton and ciliary system alterations in the development of congenital pulmonary airway malformations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital pulmonary airway malformation (CPAM) of the lung, also known as congenital cystic adenomatoid malformation (CCAM), is a rare developmental anomaly characterized by the proliferation of distal bronchi\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. It accounts for 25% of congenital pulmonary malformations, with the majority of cases occurring in neonates and infants \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The initial categorization of CPAM was based on the predominant characteristics of cyst size and morphology, which were grouped into three categories. Type I is characterized by the presence of single or multiple large cysts (\u0026gt;\u0026thinsp;2 cm) with pseudostratified columnar epithelium. Type II is defined by the occurrence of multiple smaller cysts (\u0026lt;\u0026thinsp;1 cm) with cuboidal or columnar epithelia. Type III is associated with a microcystic lesion with a cuboidal epithelium. Two additional categories, Type 0 (solid lesions) and Type IV (large cysts with flattened epithelium), were subsequently incorporated into this classification system. Both Type 0 and Type IV fractures are relatively rare\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. A large lesion may cause a respiratory disorder, resulting in a mediastinal shift, infections, edema and even malignant transformation\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The pathogenesis of CPAM is not fully understood, but it is thought to result from the arrest of lung development at different stages of branching of the bronchopulmonary tree. This can lead to the formation of cysts lined with different types of epithelium, reflecting the stage of development at which arrest occurs\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The proposed mechanism is related to the overgrowth of mesenchymal elements with subsequent suppression of alveolar growth and decreased apoptosis. Potential genes of influence, such as HOXB5, cyclin D1, and PDGF-BB, have been identified; however, the complete mechanism has not yet been elucidated\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Therefore, the etiology of this disease remains unclear and controversial.\u003c/p\u003e\u003cp\u003eThe human lung contains two main types of alveolar epithelial cells: alveolar type I epithelial cells (AECs) and alveolar type II epithelial cells (AEC IIs). These cells are responsible for maintaining the normal function of the alveolar epithelium through their distinctive morphological and functional characteristics \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The results of a recent study revealed an increase in the number of these two cell types in CPAM\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, few studies have been conducted with the objective of either corroborating or refuting the findings of this research.\u003c/p\u003e\u003cp\u003eThe cytoskeleton is a complex network of protein filaments that play pivotal roles in maintaining cellular shape, enabling cellular motion, and facilitating intracellular transport. \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Cilia are cellular organelles that project from the cell surface and are involved in a number of critical functions, including the sensing of environmental signals and the facilitation of cellular movement\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Both the cytoskeleton and cilia are integral structures that facilitate cell movement. A significant body of research has highlighted the interconnection between cilia and CPAM\u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. However, the relationship between CPAM and the cytoskeleton remains relatively understudied. In this study, we collected tissue samples from surgical patients, performed experiments utilizing RNA-seq and immunohistochemistry, and explored alterations in cellular structures, such as the cytoskeleton and cilia, during the pathogenesis of CPAM, as well as the impact and role of two types of alveolar epithelial cells in this progression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eStudy design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis is a prospective study of 34 children patients (aged from 4 months to 13 years) who had received a preoperative diagnosis of CPAM and were surgically treated from 2022 to 2024 at Tongji Hospital. All patients were histopathologically confirmed as CPAM after surgery. During hospitalization, the children\u0026rsquo;s caregivers were informed about the study and they provided written consent.\u003c/p\u003e\u003cp\u003eChest computed tomography (CT) scans were performed before surgery to clarify the classification(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The intraoperative findings in all cases were consistent with the preoperative classification. In all cases,, we used aerosolized indocyanine green to visualize the lesion and show its margin\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We obtained samples of the lesion and adjacent normal tissue for subsequent experimental comparison. All adjacent normal tissues were confirmed by histopathology. The sampling was approved by the ethics committee and the patient consented. Tissue samples were immediately preserved after obtained from surgery. In every case, a portion of the tissue sample was fixed with formalin for HE staining, and the remainder was snap-frozen in liquid nitrogen for other experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong the patients, 7 were selected for RNA sequencing, and the relevant information is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Total RNA was extracted from the frozen lung tissues of CPAM patients via TRIzol Reagent (Invitrogen). Four micrograms of total RNA were used for cDNA sequencing library generation via the NEBNext Ultra RNA Library Prep Kit (NEB, USA). The purified products were evaluated with an Agilent Bioanalyzer (Agilent Technologies). Eligible libraries were sequenced on the Illumina HiSeq 2000 platform via 100-bp paired-end reads. The original fastq data were evaluated, and low-quality reads were filtered by the Trimmomatic tool. The filtered clean reads were aligned to the human reference genome (GRCh38) via HISAT2. Only protein-coding genes were retained for analysis, and genes with no mapped reads in at least half of the samples were excluded. Gene expression levels were quantified with raw counts (FPKM) via StringTie.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of patients\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAge(Years old)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLesion location\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale:16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;1:21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLeft lung:12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale:18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;1:13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRight lung:20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBoth lungs:2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferential expression analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDifferentially expressed genes (DEGs) between cysts and simple controls were identified via the DESeq2 package in R. DEGs were selected on the basis of the thresholds of both a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and a fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;2. DEGs were visualized via volcano plots. Principal component analysis (PCA) was performed to detect the overall differences between the individual samples via the normalized counts of all the genes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional enrichment and protein‒protein interaction network analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the functions of the differentially expressed genes (DEGs) identified in CPAM, Gene Ontology (GO) enrichment analysis was conducted via the clusterProfiler package with the upregulated genes and downregulated genes. Only those GO terms with an FDR value of less than 0.05 were considered to represent a significant event. The PPI data were downloaded from the STRING database to investigate protein interactions among the DEG sets that were enriched in the aforementioned significant GO terms. Only those interactions with a PPI score greater than 500 were retained for further analysis, and the PPI network was visualized via Cytoscape software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe paraffin-embedded sections underwent deparaffinization in xylene and subsequent rehydration through a descending ethanol concentration series. The sections were subsequently subjected to microwave treatment for 16 minutes in Tris-EDTA buffer for antigen retrieval. To quench endogenous peroxidase activity, a solution of 3% hydrogen peroxide was applied at room temperature for 5\u0026ndash;10 minutes.\u003c/p\u003e\u003cp\u003eThe slides were then treated to block nonspecific binding by immersion in a mixture of 5% bovine serum albumin (BSA) and 0.1% Triton X-100 in phosphate-buffered saline (PBS) for a period of 1 hour at 37\u0026deg;C. Next, the primary antibody was added to the slides. The slides were incubated with the primary antibody overnight at 4\u0026deg;C, after which they were treated with a secondary horseradish peroxidase-conjugated anti-mouse IgG antibody for an additional hour.\u003c/p\u003e\u003cp\u003eThe chromogenic reaction was developed by applying a 3,3'-diaminobenzidine (DAB) substrate kit from Servicebio to the slides, allowing visualization of color. Examination under a high-powered microscope involved scrutinizing five randomly selected fields from each affected tissue segment.\u003c/p\u003e\u003cp\u003eConsistent imaging conditions were maintained by capturing all images with the same microscope and camera setup. The optical density and mean optical density of positively stained cell structures were quantified via ImageJ software (version 1.43u).\u003c/p\u003e\u003cp\u003eThe following dyes and primary antibodies were used for staining: phalloidin (dilution 1:100, Abbkine, Wuhan, P.R.C.), DAPI (Servicebio, Wuhan, P.R.C.), AQP5 (dilution 1:100, Rabbit, ABclonal, Wuhan, P.R.C.), SFTPC (dilution 1:100, Rabbit, ABclonal, Wuhan, P.R.C.), Claudin-2 (dilution 1:500, Rabbit, Abcam, Cambridge, UK), E-cadherin (dilution 1:300, Rabbit, Proteintech, Rosemont, USA) and ZO1 (dilution 1:1000, Rabbit, Proteintech, Rosemont, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistopathology\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe tissue samples were fixed in 10% neutral buffered formalin, decalcified, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H\u0026amp;E) via standard histological methods. Microscopy images were taken via a Zeiss Axioscope A1 microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll the data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs from three or more independent experiments. Two-tailed Student's unpaired t tests were used to assess the statistical significance of the data. All the statistical analyses were performed via SPSS 16.0. Statistical significance was set at probability values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eClinical features and pathological changes in CPAM patients\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 34 patients with CPAM were recruited for the study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All 34 patients underwent successful thoracoscopic wedge resection, lobectomy, or segmentectomy, with no cases requiring conversion to thoracotomy. The mean operation time was 100.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.6 minutes, with a mean intraoperative blood loss of 30.5\u0026thinsp;\u0026plusmn;\u0026thinsp;25.6 millilitres. The mean postoperative chest closed drainage time was 48.5\u0026thinsp;\u0026plusmn;\u0026thinsp;24.3 hours, with a mean postoperative hospital stay of 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 days. All patients were successfully treated and discharged, with no deaths. No adverse reactions, including allergic reactions, respiratory depression, or respiratory failure, were observed. Postoperative complications included one case of postoperative pneumothorax and three cases of subcutaneous emphysema. The pneumothorax spontaneously healed with prolonged indwelling chest closed drainage tube, while the subcutaneous emphysema did not receive special treatment. All patients were followed up in an outpatient setting for a period of three to six months following surgery, with a 100% follow-up rate. The follow-up content included a chest CT scan and a pulmonary ventilation function test (tidal breathing pulmonary function test). A reexamination of the chest CT scan revealed the absence of residual lesions or recurrence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome sequencing revealed significant abnormalities in the cytoskeleton\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe selected 7 patients out of 34 for RNA sequencing analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), including 5 males and 2 females, with surgical ages ranging from 7 months to 3 years. Their lesions were located in the right lower lobe in 4 cases, the left lower lobe in 1 case, the right middle lobe in 1 case and the left upper lobe in 1 case.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of patients selected for RNA sequencing\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLesion\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSurgery\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1y3m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRight lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRight lower lung wedge resection\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRight lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType III\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRight lower lobectomy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7 m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRight lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRight lower lobectomy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLeft lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLeft lower lobectomy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7 m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRight lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRight lower lung segment resection\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRight middle lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType III\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRight middle lobectomy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9 m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLeft upper lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eType I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLeft upper lung segment resection\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA total of 380\u0026nbsp;million reads were generated from the 14 transcriptome sequencing datasets. The mean proportion of reads mapped to the human reference genome was 97%. Principal component analysis(PCA) of the gene expression profile revealed that CPAM samples exhibited a clear separation from the control group. A consolidated analysis of all CPAM samples was conducted to facilitate comparison with the control group. This revealed the identification of 1252 differentially expressed genes (DEGs) between CPAM areas and the paired control areas. Of these, 1041 were found to be upregulated, while 211 were downregulated. Interestingly, a large number of DEGs are related to cell cilia, and these genes are marked in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenetic abnormalities leading to ciliary dysfunction play an important role in CPAM\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGO analysis revealed that the upregulated genes were significantly enriched in a number of biological processes, including cilium organization (FDR\u0026thinsp;=\u0026thinsp;1.80\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;49\u003c/sup\u003e) and cilium movement (FDR\u0026thinsp;=\u0026thinsp;8.49\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;45\u003c/sup\u003e). The GO analysis revealed that the upregulated genes were significantly enriched in several biological processes, including epithelial cilium movement involved in extracellular fluid movement (FDR\u0026thinsp;=\u0026thinsp;4.94\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;23\u003c/sup\u003e), phagocytosis and recognition (FDR\u0026thinsp;=\u0026thinsp;5.14\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;21\u003c/sup\u003e), and the humoral immune response mediated by circulating immunoglobulin (FDR\u0026thinsp;=\u0026thinsp;5.85\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;21\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The downregulated genes were found to be significantly enriched in a number of biological processes, including positive regulation of response to external stimulus (FDR\u0026thinsp;=\u0026thinsp;8.38\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), positive regulation of defense response or cytokine production (FDR\u0026thinsp;=\u0026thinsp;1.28\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e, FDR\u0026thinsp;=\u0026thinsp;1.64\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), leukocyte migration (FDR\u0026thinsp;=\u0026thinsp;6.61\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), and regulation of the inflammatory response (FDR\u0026thinsp;=\u0026thinsp;8.99\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e) (FDR\u0026thinsp;=\u0026thinsp;1.12\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). GO enrichment, which is based on molecular function, also revealed that the differentially expressed genes (DEGs) were predominantly involved in microtubule motor and immune factor activities. The genes were found to be significantly associated with the components of the cilium and granule epithelial cells. The GSEA yielded analogous results, with gene sets enriched in ciliary movement, ciliary development, ciliopathies, and axoneme assembly.\u003c/p\u003e\u003cp\u003eA total of 747 differentially expressed genes (DEGs) were filtered into the protein‒protein interaction (PPI) network, with a PPI score ranging from 500\u0026ndash;999. The network consisted of 492 nodes and 765 edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among the identified genes, BMP2, TNF, DNAI1, DNAI2, and DNAH5 presented relatively high degrees and betweenness centrality, indicating strong interactions with other genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence staining revealed the presence of cytoskeletal abnormalities and a reduction in the number of AEC IIs in the CPAM\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe immunofluorescence staining procedure involved the use of DAPI, phalloidin, and antibodies, as illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B. The subsequent quantification of the fluorescence intensity was conducted using Image J. To ensure the accuracy of the normalization process, the fluorescence intensity of the target protein was divided by the fluorescence intensity of the nucleus (stained with DAPI), thereby yielding the relative mean fluorescence intensity. A two-sample t-test was employed to compare the relative mean fluorescence intensity of CPAM lesion tissue with that of normal lung tissue, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA comparison of CPAM and normal lung tissue revealed a significantly greater mean fluorescence intensity in CPAM lesions when they were stained with phalloidin and a lower ratio when they were stained with the SFTPC antibody. No significant differences were detected when the AQP5, claudin-2, E-cadherin, or ZO-1 antibodies were used. The present study found that AQP5 is generally considered to be a marker for AEC I, while SFTPC is a marker for AEC II\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Phalloidin has been demonstrated to stain the cytoskeleton, while claudin-2, E-cadherin, and ZO-1 have been shown to play important roles in cell junctions\u003csup\u003e[\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Immunofluorescence staining revealed the presence of cytoskeletal abnormalities and a reduction in the number of AEC IIs in the CPAM.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCPAM is a congenital pulmonary malformation that poses a significant risk to the health and well-being of children\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The pathological features of CPAM are characterized by the presence of adenomatous hyperplasia and cyst formation in bronchial-like structures. The cysts are lined by a variety of epithelial cells, including pseudostratified ciliated columnar epithelium and low cuboidal epithelium\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The pathogenesis of CPAM remains incompletely understood. However, it is hypothesized that this condition results from the arrest of lung development at various stages of branching of the bronchopulmonary tree. This can result in the formation of cysts lined with different types of epithelium, which is reflective of the stage of development at which arrest occurs\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, the underlying cellular and molecular mechanisms remain to be discovered.\u003c/p\u003e\u003cp\u003eRNA-seq analysis revealed a significant number of differentially expressed genes (DEGs) between CPAM and normal lung tissues. For these genes, we employed both Gene Ontology (GO) and gene set enrichment analysis (GSEA) for functional enrichment analyses. Notably, the enrichment of pathways related to the ciliated epithelium suggests its potential roles in the pathogenesis of CPAM. This finding is somewhat consistent with existing research results\u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our PPI network, BMP2, TNF, DNAI1, DNAI2, and DNAH5 exhibited relatively high degrees and betweenness centrality, which were confirmed to play important roles in the regulation and driving of cilia movement\u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Notably, some of these proteins also significantly influence the development and progression of lung cancer\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, which may help explain the link between CPAM and lung cancer\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, further clinical research is needed to substantiate this perspective.\u003c/p\u003e\u003cp\u003ePhalloidin is a fungal toxin that has become an essential tool in cell biology because of its high affinity for filamentous actin (F-actin)\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In immunohistochemistry, phalloidin and its fluorescently labeled derivatives are widely used for the visualization of F-actin in cells and tissues, providing valuable insights into cytoskeletal organization\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Aquaporin-5 (AQP5) is a water channel protein predominantly found in the lung, salivary gland and lacrimal gland. In the lung, it is specifically expressed in alveolar type I cells (AEC I)\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Surfactant protein C (SFTPC) is expressed specifically in type II alveolar epithelial cells (AEC II), making it a useful marker for these cells in immunohistochemical studies\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Claudin-2 is a protein that belongs to the claudin family of integral membrane proteins found in tight junctions\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. E-cadherin, a transmembrane glycoprotein, is essential for the structural and functional maintenance of adherens junctions in epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Zonula occludens-1 (ZO-1) is a tight junction protein that plays a key role in maintaining the structural integrity and barrier function of epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe results of immunohistochemistry demonstrated that CPAM tissue samples presented greater levels of cytoskeleton fluorescent markers than normal lung tissue samples did. This finding indicates an increase in cytoskeleton structure during the occurrence and development of CPAM, which may represent a crucial biological process in the onset and progression of CPAM. In addition, AEC II decreased in CPAM lesion tissue, whereas the amount of AEC I did not change significantly. Claudin-2 and ZO-1, which are associated with cell tight junctions, did not change significantly, and E-cadherin, which is associated with cell adherens junctions, did not change significantly.\u003c/p\u003e\u003cp\u003eIt is widely acknowledged that alveoli are responsible for gas exchange in the respiratory system. They are composed of two main types of alveolar epithelial cells: alveolar type I epithelial cells (AECs), which constitute the majority (approximately 96%) of the alveolar surface area, and alveolar type II epithelial cells (AEC IIs), which constitute the majority (approximately 60%) of the alveolar cells\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. AECs are notably thin, which allows for efficient gas exchange between the alveolar air space and the pulmonary capillaries\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Their distinctive flattened morphology enables them to cover a vast surface area with minimal thickness, thereby reducing the diffusion distance for gases\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. AEC IIs are cuboidal in shape and perform a vital function in the production and secretion of pulmonary surfactant, which is indispensable for reducing surface tension within the alveoli and preventing their collapse\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Additionally, AEC II cells serve as progenitor cells for AEC I and are involved in regeneration of the alveolar epithelium following injury\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Both cell types are vital for maintaining the integrity and function of the alveolar epithelium. The cytoskeleton is a complex network of protein filaments that play pivotal roles in maintaining cellular shape, enabling cellular motion, and facilitating intracellular transport. It comprises three principal types of protein filaments: actin filaments, microtubules, and intermediate filaments\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The cytoskeleton plays a pivotal role in maintaining cellular structure, intracellular transport, signal transduction and mechanotransduction\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Cilia are cellular organelles that project from the cell surface and are involved in a number of critical functions, including the sensing of environmental signals and the facilitation of cellular movement\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. They can be broadly classified into two categories: motile cilia and primary (nonmotile) cilia\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Both the cytoskeleton and cilia are integral structures that facilitate cell movement.\u003c/p\u003e\u003cp\u003eThese results suggest that the cells in CPAM lesions have undergone significant alterations in comparison with normal lung tissue cells. On the one hand, an increase in the cytoskeleton has been observed, and on the other hand, the expression of multiple genes related to cilia has been found to be elevated. Lesion tissue has lost the physiological function of normal lung tissue cilia, which move debris and mucus out of the airways by beating in a coordinated and continuous manner\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Therefore, we tend to assume that these changes in the expression of cilium-related genes lead to changes in ciliary ultrastructure and motility, thus resulting in functional failure of ciliary movement and clearance. Severe mucus accumulation in the airway ultimately causes inflammation and adversely affects lung function and development.\u003c/p\u003e\u003cp\u003eBoth AECs I and II are highly important for the maintenance of normal lung tissue function. AECs constitute the majority of the alveolar epithelium in terms of surface area and are responsible for the vital process of gas exchange \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. AEC II is present in large numbers in normal lung tissue and is the progenitor cell of AEC I. In the event of damage to the alveolar epithelium, AEC II will differentiate into AEC I to repair damaged tissue \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe findings of this study indicate that, compared with that in normal lung tissue, the AEC II in CPAM samples was lower. Conversely, no such reduction was observed in AEC I. This finding indicates that in the pathogenesis of CPAM, either AEC II is specifically subjected to a negative influence or AEC I is damaged, and AEC II differentiates and replenishes AEC I over time, resulting in the loss of AEC II. Both factors may also be present.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study collected clinical tissue samples and conducted experimental explorations, which revealed a significant increase in the cytoskeleton, a significant decrease in AEC II and an abnormal increase in the expression of cilium-related genes in CPAM. These findings provide crucial insights into the etiology of CPAM, which may inform the development of more effective diagnostic and therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Institutional Review Board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology approved the protocol of the study (Permit Number 20240403 Wuhan, China). All study procedures complied with regulations in the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed written consents were obtained from the patient\u0026apos;s parents for publication this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was involved in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.Z., X.M. and Q.H wrote the main manuscript text, K.C. prepared figures 4A\u0026amp;B. Y.Y., D.Z., Y.H., J.W., J. F. and X.C. provided vital support for writing this manuscript. \u0026nbsp;All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSome experiments in this study were conducted at the Experimental Medicine Center of Tongji Hospital, which is affiliated with Tongji Medical School at Huazhong University of Science and Technology. The authors would like to express their gratitude to the staff at the Experimental Medicine Center for their assistance in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePEDERIVA F, ROTHENBERG S S, HALL N et al (2023) Congenital lung malformations [J]. Nat Rev Dis Primers 9(1):60\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEL AMRAOUI W, BENTALHA A, HAMRI H et al (2017) Congenital cystic adenomatoid malformation - dangers of misdiagnosis: a case report [J]. J Med Case Rep 11(1):212\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSTRUMIŁŁO B, J\u0026oacute;ŹWIAK A, PAŁKA A et al (2018) Congenital cystic adenomatoid malformation - diagnostic and therapeutic procedure: 8-year experience of one medical centre [J]. Kardiochir Torakochirurgia Pol 15(1):10\u0026ndash;17\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIOACHIMESCU O C, MEHTA AC (2005) From cystic pulmonary airway malformation, to bronchioloalveolar carcinoma and adenocarcinoma of the lung [J]. Eur Respir J 26(6):1181\u0026ndash;1187\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCHANG W C (2021) Mucinous adenocarcinoma arising in congenital pulmonary airway malformation: clinicopathological analysis of 37 cases [J]. 78(3):434\u0026ndash;444ZHANG Y Z\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMOROTTI R A, CANGIARELLA J, GUTIERREZ M, C et al (1999) Congenital cystic adenomatoid malformation of the lung (CCAM): evaluation of the cellular components [J]. Hum Pathol 30(6):618\u0026ndash;625\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWANG X, WOLGEMUTH D J, BAXI LV (2011) Overexpression of HOXB5, cyclin D1 and PCNA in congenital cystic adenomatoid malformation [J]. Fetal Diagn Ther 29(4):315\u0026ndash;320\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLIECHTY K W, CROMBLEHOLME T M, QUINN T M et al (1999) Elevated platelet-derived growth factor-B in congenital cystic adenomatoid malformations requiring fetal resection [J]. J Pediatr Surg, 34(5): 805-9; discussion 9\u0026ndash;10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWITHERDEN I R, TETLEY TD (2001) Isolation and Culture of Human Alveolar Type II Pneumocytes [J]. Methods Mol Med 56:137\u0026ndash;146\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBROOKES O BOLANDS (2021) Co-culture of type I and type II pneumocytes as a model of alveolar epithelium [J]. 16(9):e0248798\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG S, YE C, XIAO J et al (2020) Single-cell transcriptome profiling reveals the mechanism of abnormal proliferation of epithelial cells in congenital cystic adenomatoid malformation [J]. Exp Cell Res 396(2):112299\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePOLLARD T D, GOLDMAN RD (2018) Overview of the Cytoskeleton from an Evolutionary Perspective [J]. Cold Spring Harb Perspect Biol, 10(7)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eANVARIAN Z, MYKYTYN K (2019) Cellular signalling by primary cilia in development, organ function and disease [J]. 15(4):199\u0026ndash;219\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHUA K, FERLAND RJ (2018) Primary cilia proteins: ciliary and extraciliary sites and functions [J]. Cell Mol Life Sci 75(9):1521\u0026ndash;1540\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG G, LOU L (2024) The underlying molecular mechanism of ciliated epithelium dysfunction and TGF-β signaling in children with congenital pulmonary airway malformations [J]. Sci Rep 14(1):4430\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG G, CAI C, LI X et al (2022) Application of second-generation sequencing in congenital pulmonary airway malformations [J]. Sci Rep 12(1):20459\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTAN Z, LI F, CHEN Q et al (2023) Integrated bulk and single-cell RNA-sequencing reveals SPOCK2 as a novel biomarker gene in the development of congenital pulmonary airway malformation [J]. Respir Res 24(1):127\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePHILLIPS JD (2020) Inhalational Indocyanine Green to Visualize Lung Tumors\u0026mdash;Defining the Margin of Error [J]. JAMA Surg 155(8):741\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFLODBY P, LI C, LIU Y et al (2017) Cell-specific expression of aquaporin-5 (Aqp5) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation [J]. Sci Rep 7(1):3473\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJIANG M, ROTH M G, CHUN-ON P et al (2020) Phenotypic Diversity Caused by Differential Expression of SFTPC-Cre-Transgenic Alleles [J]. 62(6):692\u0026ndash;698\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePOSPICH S, MERINO F (2020) RAUNSER S. Structural Effects and Functional Implications of Phalloidin and Jasplakinolide Binding to Actin Filaments [J]. Structure, 28(4): 437\u0026thinsp;\u0026ndash;\u0026thinsp;49.e5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eROMANI M (2021) Phalloidin Staining of Actin Filaments for Visualization of Muscle Fibers in Caenorhabditis elegans [J]. Bio Protoc 11(19):e4183\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCAO W, XING H, LI Y et al (2022) Claudin18.2 is a novel molecular biomarker for tumor-targeted immunotherapy [J]. Biomark Res 10(1):38\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVAN ROY F BERXG (2008) The cell-cell adhesion molecule E-cadherin [J]. Cell Mol Life Sci 65(23):3756\u0026ndash;3788\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNEYRINCK-LEGLANTIER D, LESAGE J, BLACHER S et al (2021) ZO-1 Intracellular Localization Organizes Immune Response in Non-Small Cell Lung Cancer [J]. Front cell Dev biology 9:749364\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDOUGHERTY G W, LOGES N T KLINKENBUSCHJA et al (2016) DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes [J]. Am J Respir Cell Mol Biol 55(2):213\u0026ndash;224\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKOBAYASHI D, IIJIMA N, HAGIWARA H et al (2010) Characterization of the medaka (Oryzias latipes) primary ciliary dyskinesia mutant, jaodori: Redundant and distinct roles of dynein axonemal intermediate chain 2 (dnai2) in motile cilia [J]. Dev Biol 347(1):62\u0026ndash;70\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYANG W, CHEN L, GUO J et al (2022) Multiomics Analysis of a DNAH5-Mutated PCD Organoid Model Revealed the Key Role of the TGF-β/BMP and Notch Pathways in Epithelial Differentiation and the Immune Response in DNAH5-Mutated Patients [J]. 11(24)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKUMARI A, CALIZ A D, YOO H J et al (2024) TNF-alpha promotes cilia elongation via mixed lineage kinases signaling in mouse fibroblasts and human RPE-1 cells [J]\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHOU W, YAN K, XI Q (2023) BMP signaling in cancer stemness and differentiation [J]. 12(1):37\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTERLIZZI M, COLARUSSO C, SOMMA P et al (2022) S1P-Induced TNF-α and IL-6 Release from PBMCs Exacerbates Lung Cancer-Associated Inflammation [J]. Cells, 11(16)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYAMAMOTO K, FERRARI JD, CAO Y et al (2012) Type I alveolar epithelial cells mount innate immune responses during pneumococcal pneumonia [J]. J Immunol 189(5):2450\u0026ndash;2459\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCASTRANOVA V, RABOVSKY J, TUCKER JH et al (1988) The alveolar type II epithelial cell: a multifunctional pneumocyte [J]. Toxicol Appl Pharmacol 93(3):472\u0026ndash;483\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLAMBERT MW (2019) Cytoskeletal and nucleoskeletal interacting protein networks play critical roles in cellular function and dysfunction [J]. Exp Biol Med (Maywood) 244(15):1233\u0026ndash;1239\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBARSCH F, NIEDERMAIR T, MAMILOS A et al (2020) Physiological and Pathophysiological Aspects of Primary Cilia-A Literature Review with View on Functional and Structural Relationships in Cartilage [J]. 21(14)\u003c/span\u003e\u003c/li\u003e\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":"pediatric-surgery-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pesi","sideBox":"Learn more about [Pediatric Surgery International](http://link.springer.com/journal/383)","snPcode":"383","submissionUrl":"https://submission.nature.com/new-submission/383/3","title":"Pediatric Surgery International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Congenital pulmonary airway malformation, cytoskeleton, cilia, alveolar epithelial cells","lastPublishedDoi":"10.21203/rs.3.rs-7036277/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7036277/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e Congenital pulmonary airway malformation (CPAM) is a developmental lung malformation that seriously endangers children's health. The objective of this study was to investigate the etiology of CPAM by observing changes at the molecular and cellular levels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e Patient clinical data were collected and analyzed. Tissue samples were collected from CPAM lesions and marginal normal lung tissue during CPAM surgery. The samples were subjected to H\u0026amp;E staining for pathological analysis. Tissue RNA was isolated for RNA sequencing, and the differentially expressed genes (DEGs) were enriched for Gene Ontology (GO) analysis. The cytoskeletal and cell subtypes were subjected to immunofluorescence staining.\u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003eRNA sequencing of 7 CPAM patients revealed 1252 DEGs, with 1041 upregulated and 211 downregulated. GO analysis revealed that biological processes related to cilium organization and movement were strongly enriched. Protein‒protein interaction (PPI) network analysis highlighted genes such as BMP2, TNF, DNAI1, DNAI2, and DNAH5 as potentially important in CPAM. Immunofluorescence staining revealed abnormalities in the cytoskeleton and a reduction in the number of alveolar epithelial type II (AEC II) cells in CPAM lesions compared with normal lung tissue.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e Our study revealed that CPAM is associated with a significant increase in cytoskeletal elements and a decrease in AEC IIs, along with an abnormal increase in the expression of cilium-related genes. These alterations provide critical insights into the etiology of CPAM and may guide the development of improved diagnostic and therapeutic strategies for this condition.\u003c/p\u003e","manuscriptTitle":"The essential role of cytoskeleton and ciliary system alterations in the development of congenital pulmonary airway malformations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 10:15:05","doi":"10.21203/rs.3.rs-7036277/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-20T21:16:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-11T21:04:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225261571218328726992010608634837501733","date":"2025-07-08T15:39:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-08T12:45:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T13:55:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-04T01:41:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pediatric Surgery International","date":"2025-07-03T09:17:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pediatric-surgery-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pesi","sideBox":"Learn more about [Pediatric Surgery International](http://link.springer.com/journal/383)","snPcode":"383","submissionUrl":"https://submission.nature.com/new-submission/383/3","title":"Pediatric Surgery International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4bd9e066-8a35-4973-b0b2-fb699786c2ab","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:06:12+00:00","versionOfRecord":{"articleIdentity":"rs-7036277","link":"https://doi.org/10.1007/s00383-025-06250-0","journal":{"identity":"pediatric-surgery-international","isVorOnly":false,"title":"Pediatric Surgery International"},"publishedOn":"2025-11-20 15:57:15","publishedOnDateReadable":"November 20th, 2025"},"versionCreatedAt":"2025-07-14 10:15:05","video":"","vorDoi":"10.1007/s00383-025-06250-0","vorDoiUrl":"https://doi.org/10.1007/s00383-025-06250-0","workflowStages":[]},"version":"v1","identity":"rs-7036277","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7036277","identity":"rs-7036277","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}