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Negative Regulation of Cell Adhesion as a Driver of Brain Metastasis in NSCLC Patients with EGFR Amplification

Biological Procedures Online volume 27, Article number: 15 (2025) Cite this article

Abstract

Brain metastases are strongly associated with a poor prognosis. Experimental animal models have provided valuable insights into the complex biology underlying brain metastasis, and translating these findings could pave the way for innovative management strategies for patients with brain metastases. Between May 2019 and June 2023, twenty-four lung cancer patients and thirty patients with brain metastases from lung cancer were enrolled at Guangdong Sanjiu Brain Hospital. Next-generation targeted panel sequencing (NGS) was performed on lung cancer tissue and surgical specimens from brain tumors for each patient. Brain metastasis mouse models were established through intracardiac injections, and the brain metastasis rate was analyzed. Our results showed that the rate of EGFR amplification was significantly higher in patients with brain metastases compared to lung cancer patients (40% vs. 12%). EGFR-overexpressing PC9 cell lines demonstrated significantly enhanced proliferation and infiltration abilities compared to their parental PC9 counterparts, as evidenced by CCK-8, wound healing, and transwell assays. Moreover, we observed a much higher brain metastasis rate in mice injected with EGFR-overexpressing PC9 cells compared to those injected with parental PC9 cells. RNA sequencing and Gene Ontology (GO) analysis revealed that differentially expressed genes were primarily associated with the “negative regulation of cell adhesion” in biological processes (BP) and “collagen-containing extracellular matrix” in cellular components (CC). This study identifies the negative regulation of cell adhesion as a key driver of brain metastasis in NSCLC patients with EGFR amplification.

Importance of the study

In comparing the sequencing results of brain metastatic tumor tissue and primary lung cancer tumor tissue, this study found that the detection rate of EGFR amplification was significantly higher in brain metastatic tumors than in lung cancer tissues. It is speculated that EGFR amplification may promote the development of brain tumors. Using lentiviral technology to establish cell lines overexpressing the EGFR gene, in vitro experiments showed that the proliferation and migration abilities of these cells were significantly enhanced. Furthermore, in vivo experiments confirmed that overexpression of the EGFR gene could promote the incidence of brain metastasis. These results suggest that targeted interventions against EGFR amplification in the future may reduce the incidence of brain metastasis.

Introduction

Patients with brain metastases are often associated with a poor prognosis, combined with a high incidence rate, which has attracted widespread attention from scholars. Population-based research collected clinical data from patients with primary lung, melanoma, breast, renal, or colorectal cancer from 1973 to 2001 to calculate the incidence rate of brain metastases and draw the conclusion that the total incidence proportions of brain metastases was 9.6% for all primary sites combined, and patients with lung cancer accounting for the highest rate of 19.9% [1]. Evidence from prior research indicated that there was an increasing frequency of brain metastases with increasing tumor grade of patients within non–small cell lung cancer [2], other study showed that for lung cancer patients with metastases disease, the proportion of brain metastases is almost the same between small cell and non-small cell lung cancer presenting 23.5% and 15–26%, respectively [3].

Treatment options for patients with brain metastases typically include surgical removal, radiation therapy, and chemotherapy, and other treatment alternatives include targeted therapy, and immunotherapy [4]. However, patients with brain metastases appear to be refractory to all current treatments, leading to a continuous decline in quality of life. Whole-brain radiation therapy (WBRT) prolongs median survival from 1 to 3–6 months. The one-year survival rate for lung cancer patients with BM treated with whole-brain radiation therapy (WBRT) is approximately 10–20% and surgical resection or radiosurgery (RS) combined with adjuvant WBRT prolongs survival to approximately 8–11 months [5]. The adverse prognosis has presented novel challenges and also addresses increased research efforts to better understand the underlying mechanism of brain metastasis.

In this research, we conducted a comparative analysis of the NGS results between the lung and brain metastases tumor issue and further validated our hypothesis through in vitro experiments and subsequent in vivo animal studies.

Patients and Methods

Patient Population

Between May 2019 and June 2023, twenty-four patients with lung cancer and thirty patients with brain metastases from lung cancer were enrolled at Guangdong Sanjiu Brain Hospital. Next-generation targeted panel sequencing (NGS) was performed on lung cancer tissue and surgical specimens of brain tumors for each patient.

Cell Culture

The parental PC-9 cells (EGFR exon 19 E746–A750 deletion) were obtained from the Cell Bank/Stem Cell Bank, Chinese Academy of Sciences. These cells were then transfected to express green fluorescence and luciferase, enabling fluorescence quantification and bioluminescence tracking. The signal intensity was measured using the Bright-Glo Luciferase Assay System (Promega). Cells were grown in DMEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 10 µL/mL of corresponding antibiotics to ensure selection of transduced cells. Cells were kept at 37 ℃ and 5% CO2. All cells used for in vivo and in vitro experiments were between passages 1–10.

Lentivirus Transduction

Luciferase lentivirus (pcSLenti-EF1-Luc2-P2A-BSR-CMV-MCS-WPRE) and EGFR-overexpressing lentivirus (pcSLenti-EF1-EGFP-P2A-Puro-CMV-EGFR-3xFLAG-WPRE) were purchased from OBiO Technology (Shanghai, China).

The procedure was as follows: (1) On Day 0, cells were plated in 10 cm dishes and allowed to adhere to the plate overnight. (2) On Day 1, the media was aspirated from the cell plates and replaced with fresh DMEM. The concentrated lentivirus was quickly thawed in a 37 °C water bath and immediately placed on ice. The virus was added to the cells dropwise, with the volume determined based on viral titration, and the plates were incubated overnight at 37 °C. (3) On Day 2, the lentiviral media was aspirated from the plates and replaced with fresh DMEM supplemented with 10% FBS. Cell selection started on Day 3. (4) To isolate clones stably expressing EGFR, the media was aspirated again and replaced with fresh DMEM supplemented with puromycin for selection.

Western Blot

The experimental procedure was performed as follows: (1) Cells were lysed on ice using RIPA buffer (YAMI, Shanghai, China) containing protease inhibitors. The lysates were then mixed with loading buffer and denatured at 100 °C for 10 min. (2) Protein concentration was determined using the Bicinchoninic Acid (BCA) assay, after which the samples were subjected to SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis, SDS-PAGE). 3)The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Prior to transfer, the PVDF membranes were activated by soaking them in methanol. 4) The membranes were incubated overnight at 4 °C with primary antibodies: EGFR (Abcam, 1:3000) and GAPDH (Affinity, 1:1000). 5) The following day, the membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (HRP; anti-rabbit IgG, CST, 1:5000) at room temperature for 2 h. 6) After incubation, the membranes were washed three times with TBST (Tris-Borate-Sodium Tween-20 buffer), with each wash lasting 15 min. 7) Finally, the immunoreactive bands were visualized using Image Lab software after the addition of a luminol-based chemiluminescent substrate (ECL; Millipore).

CCK-8 Assay

The CCK-8 kit (Dojindo, Shanghai, China) was used to measure the proliferation of PC9 cells, which overexpress the EGFR gene, and parental PC9 cells. The procedure was as follows: (1) A total of 1000 cells in 100 µL of medium per well were cultured in five replicate wells of a 96-well plate containing medium with 10% FBS. A concentration gradient was established using serial dilution, and different groups were set up (control group and EGFR-overexpressing group). (2) The 96-well plate was incubated in a cell culture incubator at 37 °C with 5% CO2 for 24 h, allowing the cells to reach the logarithmic growth phase. (3) CCK-8 reagent (10 µL) was added to 90 µL of DMEM to prepare a working solution, and 100 µL of this solution was added to each well. The plate was then incubated for 30 min. (4) After incubation, the optical density (OD) of each well was measured at a wavelength of 450 nm using a microplate reader. (5) The blank wells (without cells or CCK-8 reagent) were used as a control, with their OD values considered as background signal and subtracted from the experimental values.

Wound Healing Assay

Cells in the logarithmic growth phase were plated in a six-well plate at a density of 5 × 10⁵ cells per well. Two experimental groups were established: one group consisting of PC9 cells that overexpress the EGFR gene, and the other group consisting of parental PC9 cells. The procedure was as follows: (1) A uniform scratch was made on the cell monolayer using a scratcher, with a scratch width of 300–500 μm, ensuring the scratch was straight and covered the center of the well. (2) After scratching, the wells were washed with PBS and then cultured in an incubator at 37 °C with 5% CO₂. (3) Photographs were taken at 0 h and 24 h under a microscope, and the experiment was repeated three times.

Transwell Migration Assay

The procedure was as follows: (1) The PC9 cells that overexpress the EGFR gene and the parental PC9 cells were diluted to a concentration of 10 × 10⁴ cells/mL in serum-free DMEM medium. (2) A 200 µL aliquot of the cell suspension was added to the upper chamber (top well) with serum-free DMEM medium, and 500 µL of the cell suspension was added to the lower chamber (bottom well) with DMEM medium containing 20% fetal bovine serum. 3)The upper chamber was carefully immersed into the lower chamber liquid using sterile forceps. The 24-well plate containing the transwell inserts was then placed in the incubator (typically at 37 °C with 5% CO₂) for 24 h. 4) After incubation, the cell suspension was removed from the upper chamber, and the chamber was washed three times with PBS. 5) Fixative (4% paraformaldehyde) was added to the lower chamber to fix the cells, typically for 15 min. After fixation, the chamber was washed three times with PBS. 6) The cells were stained with crystal violet for 15 min. After staining, the upper chamber was observed under a microscope and photographed. Each experiment was repeated three times.

Animals and Brain Tumor Model Development

The tumor cells were subcutaneously inoculated into experimental mice, and fluorescence intensity as well as mouse body weight were measured weekly to assess tumor growth and plot growth curves. Mice were anesthetized using 2% isoflurane. After placement into a stereotactic device, approximately 150,000 PC-9 cells in 100 µL of PBS were injected into the left cardiac ventricle, with a similar process in previous research [6, 7]. Bioluminescence signals and body weight were measured weekly to evaluate the growth of the tumor and verify the presence of metastases in the brain. In vivo metastasis experiments were conducted to examine the impact of selected PC9 cells overexpressing EGFR. Luciferase reporters were employed to track and determine the existence of brain metastases.

RNA Sequencing Was Performed on PC9 Cells that Overexpress the EGFR Gene and Parental PC9 Cells

The sequencing process involves the following steps: (1) total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer’s instructions. (2) RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions (Illumina, San Diego, CA). (3) the raw paired end reads were trimmed and quality controlled by fastp with default parameters. Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 software. (4) differential expression analysis and functional enrichment. To identify DEGs (differential expression genes) between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. In addition, functional-enrichment analysis including GO was performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at Bonferroni-corrected P<0.05 compared with the whole-transcriptome background.

Statistical Analysis

Student’s t-test and one-way ANOVA followed by a Fisher’s LSD test were applied to determine the difference in the results of cell proliferation and viabilities. On survival endpoints, mice were sacrificed and the date of death was recorded. Kaplan-Meier curves were generated and compared using log-rank statistics. Significance for all tests was defined as p < 0.05. Statistical analysis was performed on GraphPad Prism software, SPSS and R statistical software (version 4.2.1).

Results

The Flowchart of this Study and Also the Basic Characteristic between Lung Cancer Patients and Patients with BM

Our main purpose was to identify the differences between lung cancer tissue specimens and brain metastasis tumor specimens and try to explore the mechanisms underlying the occurrence of brain metastasis (Fig. 1). This research enrolled 30 lung cancer patients with brain metastases. The median age was 55.5 (ranging from 37 to 76 years), and 53% (16/30) of the patients were female in this study. EGFR mutation accounts for the majority of patients in this study presenting 43% (13/30), including EGFR L858R, EGFR 19del, EGFR L861Q, EGFR G719A, EGFR G719S accounting for 13% (4/30), 17% (5/30), 7% (2/30), 3% (1/30), and 3% (1/30), respectively. In addition, all patients enrolled in this research had surgically removed brain tissue and were further verify by pathology (Table 1).

Fig. 1
figure 1

Flowchart of this study design. Abbreviation: NSCLC, non-small cell lung cancer; NSCLC-BM, NSCLC patients with brain metastases

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Table 1 The basic character of the patients in this study cohort
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In Lung Adenocarcinoma, the Presence of EGFR Amplification Indicates a Poorer Prognosis

Survival data were downloaded from the public UCSC database (https://xenabrowser.net/datapages) and subjected to Kaplan-Meier survival analysis.

The results indicate that patients with EGFR amplification tend to have a poorer prognosis than those without EGFR amplification, with a statistically significant difference (Fig. 2; HR: 0.61, 95% CI: 0.350–1.064, P = 0.031).

Fig. 2
figure 2

Kaplan-Meier curves for overall survival of lung adenocarcinoma patients with or without EGFR amplification

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The Distribution of the Mutation Genes in the Lung and Brain Metastases Tissues

TP53 mutation was the most frequent mutation in brain metastases tissues presenting 60%. The rate of EGFR amplification was closely followed by and was much higher in patients with brain metastases compared to lung cancer patients (40% vs. 12%). Gene signatures, such as LRP1B, RNF43, and MYC were also commonly detected with the same percentage of 17%. Other mutation genes, including EPAH3, CCND1, FGF4, CSMD3, CDKN2A, and PMS2 were also frequently seen, showing the same percentage of 13% (Fig. 3A).

The driver of EGFR account for the majority (67%, 16/24) of the mutation in lung cancer tissue, with EGFR L8585R, EGFR 19del and EGFR L861Q presenting 25%, 38%, and 4%, respectively. Without the driver genes, TP53 was the most mutation gene in the lung cancer tissue with a mutation rate of 75%, and APC also has the higher mutation rate in the lung cancer tissue with a mutation rate of 21%, gene signature, such as PMS2 and CDK4 has the same rate of 17% in lung cancer tissues (Fig. 3B).

Fig. 3
figure 3

Shows the oncoprint displaying the distribution of mutated genes in brain metastases tissue (A) and lung cancer tissue (B)

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EGFR Overexpression Was Confirmed by Western Blot

The parental PC-9 cells (EGFR exon19 E746–A750 deletion) was transfected by EGFR-overexpressing lentivirus (pcSLenti-EF1-EGFP-P2A-Puro-CMV-EGFR-3xFLAG-WPRE) to display green color and overexpression of EGFR protein was further confirmed by western blot (Fig. 4).

Fig. 4
figure 4

Images from the light microscope showed that the PC9 cell line (A) was successfully transfected by EGFR-overexpressing lentivirus. Western blot showed that the expression of EGFR proteins was successfully increased in PC9 cell lines (B) due to Lentivirus transduction

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Overexpression of EGFR Protein Enhanced the Proliferation and Migration Ability of NSCLC Cell Lines in Vitro

We assessed the proliferation and migration capabilities of NSCLC cell lines PC9 in vitro CCK8 assay, wound healing, and transwell assays. Our findings indicate that EGFR overexpression led to increased proliferation in CCK8 (Fig. 5A) and this trend was also evident in wound healing (Fig. 5B) and transwell assays (Fig. 5C).

Fig. 5
figure 5

Proliferation and migration ability of NSCLC cell lines in vitro. (A) CCK8 test was performed to assess the proliferation ability of PC9 cell line, Data are represented as means ± SD. (B) wound healing was performed to assess the migration ability of PC9 cell line. (C) Transwell assays was performed to assess the infiltration ability of PC9 cell line

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Lentivirus Transduction of PC9 Cells Can Successfully Grow Tumor Subcutaneously

Tumor cells were injected subcutaneously into mice for the experiment, and fluorescence intensity was measured weekly, along with monitoring the mice’s body weight, to evaluate tumor growth and create growth curves (Supplementary Data Fig. 1). Mice were sacrificed and the date of death was recorded, the subcutaneous tumor was removed and subjected to hematoxylin and eosin (HE) staining for further identification of tumor cells (Supplementary Data Figs. 2 and 3).

Overexpression of EGFR Protein Enhanced the Brain Metastases Rate Compared To the Control Group

The mice were randomly assigned to control and experimental groups. In the control group, approximately 150,000 parental PC9 cells suspended in 100 µL of PBS were injected into the left cardiac ventricle. In the EGFR amplification group, approximately 150,000 PC9 cells overexpressing the EGFR gene in 100 µL of PBS were injected into the same site. After injection, the mice were marked with ear tags for accurate group identification. Luciferase reporters were used to monitor brain metastases, with bioluminescence signals measured weekly. The operator performing IVIS (In Vivo Imaging System) imaging was blinded to group assignments, ensuring the study was conducted in a blinded manner to minimize bias and maintain result integrity.

The mice were anesthetized with 2% isoflurane and positioned in a stereotactic device before intracardiac injection. Weekly assessments of bioluminescence signals and body weight were conducted to evaluate tumor growth and confirm brain metastases. Our results demonstrated that mice injected with EGFR-overexpressing PC9 cells exhibited a significantly higher rate of brain metastases compared to those injected with parental PC9 cells (P < 0.05) (Fig. 6).

Fig. 6
figure 6

Figure A-B presents the distribution of brain metastases in the control group. C-D presents the distribution of brain metastases in overexpression of the EGFR protein group (EGFR-amp). E shows the brain metastasis rate between EGFR-amp and the control group. F shows the distribution of brain metastases in the loss of function of EGFR protein group (EGFR-sh)

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RNA Sequencing Results of EGFR Overexpression in The PC-9 Cell Line Vs. The Parental PC-9 Cell Line

The heatmap of gene expression revealed that the samples were classified into two groups: control and EGFR amplification. The results showed that genes with significantly higher expression included H2AX, TERT, HSD17B10, CITED4, COL13A1, NOMO3, NCEH1, SOCS2, LGALS1, and ANKRD29. Genes with significantly lower expression included TINCR, CYP4F12, PGLYRP3, CYP4F3, KRT16, BCAS1, KDM7A, TLE2, CD82, and ACKR3. To explore the potential biological functions of the differentially expressed genes (DEGs), we performed Gene Ontology (GO) enrichment analyses, focusing on biological processes (BP), cellular components (CC), and molecular functions (MF). The results showed that DEGs in BP were significantly enriched in epidermis development, skin development, and the negative regulation of cell adhesion. For cellular components, DEGs were mainly enriched in collagen-containing extracellular matrix, apical plasma membrane, and endoplasmic reticulum lumen (Fig. 7).

Fig. 7
figure 7

Heatmap of differentially expressed genes between EGFR-amp versus control group (Left). Bubble chart plotted against the results of GO analysis for the differential expression (Right)

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Discussion

Upon diagnosis, cancer patients with metastases to the brain continue to experience a poor prognosis, characterized by increased morbidity, mortality, and a substantial rise in the cost during their treatment [8, 9]. The incidence of brain metastases continues to increase over time. The cumulative incidence after 5 years was estimated at 16.3% in patients with lung carcinoma [10]. The poor prognosis of lung cancer patients with brain metastases has led to increased focus in this area.

To figure out the different mutation genes between lung cancer and brain metastases tissue, our research compares the NGS results between lung cancer tissues and brain metastases samples trying to figure out the difference. Results demonstrated that the mutation rate of EGFR amplification was much higher in brain metastases tissue compared to the lung cancer samples (40% vs. 12%). In vitro experiments, we observed that compared to the control group, the EGFR gene overexpression group exhibited significantly enhanced cell proliferation in CCK-8 assays and greater migration ability in wound healing and transwell experiments. Based on the promising results from our in vitro experiments, we proceeded to conduct in vivo experiments. Firstly, subcutaneous tumor formation experiments were performed by subcutaneously implanting PC9 cells which were overexpressing the EGFR gene into experimental mice. Weighed and tumor volume of the experiment mice were measured every 7 days, and results indicated that the tumors were well-established in the subcutaneous (Supplementary Data Fig. 1). Evidence from previous research suggests that EGFR gene overexpression is often associated with poor prognosis [11,12,13,14]. Further studies have identified TP53 mutation and EGFR amplification as independent risk factors for recurrence-free survival (RFS) in resected EGFR-mutant lung adenocarcinoma [15]. To further confirm the function of EGFR overexpression in lung cancer patients with brain metastases, an animal model of brain metastases was built. And results from our study found that the brain metastases rate was much higher in the EGFR amplification group compared to the control group (P<0.05).

RNA sequencing analysis comparing EGFR-overexpressing PC-9 cells with parental PC-9 cells revealed that DEGs in biological processes were significantly enriched in pathways related to the negative regulation of cell adhesion. Through a series of literature reviews, we identified that LGALS1 is associated with poor prognosis across various tumor types. For instance, a previous study on relapsed and refractory multiple myeloma (RRMM) analyzed differentially expressed genes in CD138⁺ plasma cells (PCs) at various disease stages and found a significant upregulation of LGALS1 in proliferating stem-like plasma cells (PSPCs). Furthermore, survival analysis indicated that LGALS1 is linked to unfavorable prognostic outcomes in multiple myeloma [16]. A similar study utilized RNA sequencing and identified galectin-1, encoded by LGALS1, as a critical factor contributing to resistance in the treatment of esophageal squamous cell carcinoma (ESCC) [17]. Additionally, another study demonstrated that Lgals1 was overexpressed in the interstitial kidney of chronic kidney disease (CKD) mice. Treatment with Nobiletin (NOB) significantly reduced Lgals1 expression while inhibiting PI3K and AKT phosphorylation [18].

In our study, we found that EGFR overexpression enhances cell proliferation and migration. In vivo experiments further demonstrated that, compared to the control group, animals with high EGFR expression exhibited a significantly higher incidence of brain metastases. Sequencing data suggested that the negative regulation of cell adhesion might be a key factor promoting brain metastasis. However, our study has several limitations. First, the biological validation of LGALS1, the gene we identified through differential analysis, is still in its early stages and requires further investigation. Second, the mechanistic link between EGFR and the negative regulation of cell adhesion in contributing to brain metastasis remains unclear, as does the interplay between EGFR and LGALS1 and the downstream pathways involved. These aspects warrant further exploration in future studies. Current therapies are mainly palliative and do not improve survival for most patients and advances in several therapeutic modalities have effectively challenged the lethal nature of brain metastasis for specific subsets of patients [19]. Our results suggest that overexpression of the EGFR gene may play an important role in the process of brain metastasis in lung cancer patients. Interventions targeting this gene could potentially offer new treatment options for lung cancer patients with metastasis to the brain.

Data Availability

No datasets were generated or analysed during the current study.

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Acknowledgements

Not applicable.

Funding

This study is funded by The Scientific Research Program of Shanghai Pudong New Area Health Commission (the General Program, Grant No.PW2024A-58), National Natural Science Foundation of China (82174189, 82304920), Discipline Development Program of the Shanghai Pudong New Area Health Commission (PWxq2022-09).

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Author notes

  1. Hainan Yang, Congli Hu and Yu Zhou contributed equally to this work.

Authors and Affiliations

  1. Department of Critical Care Medicine, Seventh People’s Hospital of Shanghai University of Traditional Chinese Medicine, 358 Datong Road, Pudong New District, Shanghai, 200137, China

    Hainan Yang, Luyao Qi, Weifang Yuan & Ming Lei

  2. Department of Oncology, School of Medicine, Shanghai Pulmonary Hospital & Thoracic Cancer Institute, Tongji University, Shanghai, 200092, China

    Hainan Yang, Congli Hu & Caicun Zhou

  3. Department of Medical Genetics, School of Medicine, Tongji University, Shanghai, 200092, China

    Yu Zhou

  4. School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China

    Taoyuan Tong

  5. Department of Oncology, Guangdong Sanjiu Brain Hospital, Guangzhou, China

    Changguo Shan & Weiping Hong

  6. Department of Radiation Oncology, Zhujiang Hospital, Southern Medical University, 253 Gongye Dadao, Guangzhou, Guangdong, 510280, China

    Lei Wen

  7. Department of Medical Oncology, School of Medicine, Shanghai Pulmonary Hospital & Thoracic Cancer Institute, Tongji University, No. 507, Zheng Min Road, Shanghai, 200433, China

    Caicun Zhou

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Contributions

Hainan Yang, Congli Hu, Yu Zhou, designed the experiments and wrote the manuscript. Taoyuan Tong, luyao Qi, Yuan Weifang, helped in reviewing, acquisition, analysis and interpretation of clinical data for the work. Changguo Shan, Weiping Hong did the statistical analysis. Wen Lei, Caicun Zhou, Ming Lei revised critically the manuscript for important intellectual content. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Lei Wen, Caicun Zhou or Ming Lei.

Ethics declarations

Ethics Approval and Consent to Participate

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Guangdong Sanjiu Brain Hospital. We informed the participants about the study and obtained informed consent from each patient.

Consent for Publication

Written informed consent was obtained from all patients for the study.

Competing Interests

The authors declare no competing interests.

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12575_2025_277_MOESM1_ESM.docx

Supplementary Material 1: Supplementary Data Fig. 1 Tumor cells were injected subcutaneously in the experiment mice and growth curves was plotted. Supplementary Data Fig. 2-3 H&E stained images of resected mouse subcutaneous tumors.

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Yang, H., Hu, C., Zhou, Y. et al. Negative Regulation of Cell Adhesion as a Driver of Brain Metastasis in NSCLC Patients with EGFR Amplification. Biol Proced Online 27, 15 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12575-025-00277-2

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Biological Procedures Online

ISSN: 1480-9222

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