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. 2025 Mar-Apr;30(2):e70022.
doi: 10.1111/hel.70022.

Proteomic Profiling of Extracellular Vesicles Reveals Potential Biomarkers for Helicobacter pylori Infection and Gastric Cancer

Phawinee Subsomwong  1   2 , Krisana Asano  1 , Junko Akada  2 , Takashi Matsumoto  2   3 , Akio Nakane  1   4 , Yoshio Yamaoka  2   3   5
Affiliations

Proteomic Profiling of Extracellular Vesicles Reveals Potential Biomarkers for Helicobacter pylori Infection and Gastric Cancer

Phawinee Subsomwong et al. Helicobacter. 2025 Mar-Apr.

Abstract

Background: Helicobacter pylori (H. pylori) has been identified as a type I carcinogen and contributes to a high rate of gastric cancer (GC), especially in Eastern Asia. Extracellular vesicles (EVs) have the potential to be used to detect various cancer types and diseases. However, the protein markers in EVs for the prognosis of H. pylori infection and GC are unknown. We aim to identify the proteins within EVs derived from a gastric epithelial cell line (AGS) infected with H. pylori by using LC-MS/MS.

Materials and methods: EVs were isolated from AGS cells infected with high- and low-virulence H. pylori (strains TN2wt and Tx30a) by ultracentrifugation. Proteins within these EVs were identified and analyzed for potential marker candidates through bioinformatics. Proteins in H. pylori-derived EVs (HpEVs) from bacterial culture supernatant and HpEVs derived from H. pylori-infected AGS cells were elucidated.

Results: Differentially expressed proteins by proteomic analysis in AGSEVs-Tx30a vs. AGSEVs-noninfected (NI) and AGSEVs-TN2wt vs. AGSEVs-NI were 107 and 55 proteins, respectively. Bioinformatics of these proteomes revealed that essential proteins for H. pylori survival and pathogenicity including outer membrane proteins, metabolism-related, host cell infection-related, and virulence-related proteins were observed in HpEVs. Interestingly, EVs derived from AGS cells infected with H. pylori TN2wt significantly contained multiple proteins related to GC (ATP6V0A1, GAPDH, HINT1, LYZ, and RBX1).

Conclusion: This study provides a comprehensive protein profile of EVs from H. pylori-infected AGS cells and HpEVs, which could serve as liquid-based biomarkers in the future for screening H. pylori infection, especially GC-related.

Keywords: Helicobacter pylori; biomarker; extracellular vesicles; gastric cancer; proteomics.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Overview of the experiment flow and proteomic analysis by bioinformatic methods. NI, Non‐infection with H. pylori .
FIGURE 2
FIGURE 2
The detection of AGS cell‐derived EVs (AGSEVs). (A) Immunoblotting analysis using TSG101 antibody to dot blots of AGSEVs from non‐infected cells (AGSEVs‐NI), cells infected with H. pylori Tx30a (AGSEVs‐Tx30a), and cells infected with H. pylori TN2wt (AGSEVs‐TN2wt). (B) Negative staining of AGSEVs under electron microscopy. Scale bars: 500 nm; magnifications: 25,000× and 50,000×.
FIGURE 3
FIGURE 3
Enrichment by Gene Ontology (GO) and pathway terms were visualized using the ClueGO/CluePedia plugin from Cytoscape. (A) The biological process (BP), molecular function (MF), and cellular component (CC) of DEGs PPI network of AGSEVs‐Tx30a and AGSEVs‐NI are shown. (B) The REACTOME pathway. The enrichment shows only significant GO terms and pathways (p < 0.05). The node color indicates the specific functional class that they are involved in. The colors represent various BP, MF, CC, and molecular pathways involved in the enrichment analysis of identified DEGs. The bold fonts indicate the most important functional GO terms and name of signaling pathway of each group. The names of the DEGs involved in each group are displayed in red font. The DEGs related to gastric cancer were shown in the black block.
FIGURE 4
FIGURE 4
Enrichment by Gene Ontology (GO) and pathway terms were visualized using the ClueGO/CluePedia plugin from Cytoscape. (A) The biological process (BP), molecular function (MF), and cellular component (CC) of DEGs PPI network of AGSEVs‐TN2wt and AGSEVs‐NI are shown. (B) The REACTOME pathway. The enrichment shows only significant GO terms and pathways (p < 0.05). The node color indicates the specific functional class that they are involved in. The colors represent various BP, MF, CC, and molecular pathways involved in the enrichment analysis of identified DEGs. The bold fonts indicate the most important functional GO terms and name of signaling pathway of each group. The names of the DEGs involved in each group are displayed in red font. The DEGs related to gastric cancer were shown in the black block.
FIGURE 5
FIGURE 5
Volcano plot of differential protein expression (DEPs) and protein–protein interaction (PPI) network of DEPs analyzed by STRING. (A) Volcano plot of DEPs between AGSEVs‐Tx30a and AGSEVs‐NI. (B) PPI network of DEPs between AGSEVs‐Tx30a and AGSEVs‐NI. (C) Volcano plot of DEPs between AGSEVs‐TN2wt and AGSEVs‐NI. (D) PPI network of DEPs between AGSEVs‐TN2wt and AGSEVs‐NI. Volcano plot: Black dots represented proteins that did not show significantly differential expression, while the red dots indicated the proteins with significantly differential expression. Cut‐off: Log2FC > 1.3 or log2FC < −1.3 and FDR < 0.05.
FIGURE 6
FIGURE 6
Venn diagram of proteomic analysis of HpEVs. The Venn diagram displays the proteomic analysis of HpEVs derived from bacteria culture supernatant (HpEVs) and from H. pylori ‐infected AGS cell culture supernatant (AHpEVs). (A) HpEV‐Tx30a and AHpEVs‐Tx30a. (B) HpEVs‐TN2wt and AHpEVs‐TN2wt. (C) AHpEVs‐Tx30a and AHpEVs‐TN2wt. Identified proteins from AHpEVs‐TN2wt were listed in Table S5.
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