Studies on the Role of S100A9-RAGE/TLR4 Signaling Axis in Regulating Brain Metastasis and Endothelial Adhesion of Non-Small Cell Lung Cancer

Yiduo XU; Yanqi ZHOU; Jian WANG; Jiang LONG

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Yiduo XU, Yanqi ZHOU, Jian WANG, Jiang LONG. Studies on the Role of S100A9-RAGE/TLR4 Signaling Axis in Regulating Brain Metastasis and Endothelial Adhesion of Non-Small Cell Lung Cancer[J]. Journal of Kunming Medical University.

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Studies on the Role of S100A9-RAGE/TLR4 Signaling Axis in Regulating Brain Metastasis and Endothelial Adhesion of Non-Small Cell Lung Cancer

Department of Neurosurgery，The First Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032，China

Received Date: 2025-05-19
Available Online: 2025-08-13

Abstract

Abstract

Objective To explore the mechanism of S100A9 derived from non-small cell lung cancer （NSCLC） in regulating invasion, metastasis and activating the brain microvascular endothelium of the metastatic niche. Methods R language was used to extract RNAseq data from the TCGA database and a paired-sample T-test was employed to analyze the expression of S100A9 in NSCLC tissues and normal lung tissues. Visualization was conducted using the ggplot2 package; the proportional hazards assumption test and survival regression were performed using the survival package to compare the prognosis between the high/low expression groups of S100A9, and visualization was carried out using the survminer package and ggplot2 package. RT-qPCR and Western Blot were used to detect the expression differences of S100A9 in NSCLC cell lines （A549, NCI-H1299） and normal lung epithelial cells （BEAS-2B）. An in vitro co-culture of A549 cells and human brain microvascular endothelial cells （HCMEC/D3） was established to construct a blood-tumor barrier （BTB） model. Additionally, siS100A9 knockdown A549 cell strains were constructed. Scratch healing and Transwell assays were performed to assess the changes in the migration and invasion abilities of A549 cells in different treatment groups. CCK-8 and flow cytometry were used to examine the proliferative activity and cell cycle effects of HCMEC/D3 cells treated with varying concentrations of S100A9. RT-qPCR and Western Blot were employed to investigate the expression changes of receptors for advanced glycation endproducts （RAGE）, Toll-like receptor 4 （TLR4）, and tumor transendothelial migration-related adhesion molecules （ICAM-1, VCAM-1, ALCAM） in hCMEC/D3 cells treated with different concentrations of S100A9. Furthermore, CCK-8, RT-qPCR, and Western Blot were utilized to assess the recovery of proliferative activity and adhesion molecule expression in hCMEC/D3 cells stimulated with different concentrations of S100A9 after pretreatment with FPS-ZM1 and TAK242 to block RAGE and TLR4 pathways, respectively. Results The RNAseq data mining and analysis from the TCGA database revealed that the expression of S100A9 in lung cancer tissue samples was significantly higher than that in normal lung tissue samples （P = 0.03）. The Kaplan-Meier survival curve graph showed that the survival probability of the S100A9 high-expression group was lower than that of the S100A9 low-expression group, suggesting that the high expression of S100A9 was significantly associated with a poorer overall survival period for patients （HR = 1.46 （1.10 - 1.95）, P = 0.01）. In the cell experiments, S100A9 was highly expressed in NSCLC （P < 0.05）. Knockdown of S100A9 inhibited the migration and invasion of A549 cells （P < 0.05）. The average migration inhibition rate of the knockdown group at 6, 12, 24, 36, and 48 hours was 80.61%, 75.70%, 73.78%, 69.54%, and 56.96% respectively, and the average invasion inhibition rate was 57.38% （at 48 hours）. Meanwhile, the proliferative activity and cell cycle of hCMEC/D3 cells in the BTB model were regulated positively （P < 0.05）. Mechanistically, S100A9 promoted the crosstalk between A549 and hCMEC/D3 cells through RAGE and TLR4, upregulating the expression of ICAM-1, VCAM-1, and ALCAM in hCMEC/D3 cells （P < 0.05）. Recovery experiments confirmed that the S100A9-RAGE/TLR4 regulatory axis could affect the endothelial adhesion process during lung cancer brain metastasis （P < 0.05）. Conclusion The S100A9-RAGE/TLR4 axis is associated with the progression of lung cancer brain metastasis. Knockdown of S100A9 can inhibit the invasion and metastasis of lung cancer cells. Blocking downstream RAGE and TLR4 receptors can attenuate the proliferative growth of brain microvascular endothelium and inhibit the formation of a pre-metastatic adhesive microenvironment between lung cancer cells and brain microvascular endothelium. This has potential implications for exploring early diagnosis and therapeutic targets for lung cancer brain metastasis.
- S100A9,
- Non-small cell lung cancer,
- Brain metastasis,
- Brain microvascular endothelial cells （BMECs）

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