For pan-cancer analysis, the expression data of ETHE1 in clinical samples from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets were downloaded from the University of California, Santa Cruz database (http://xena.ucsc.edu/); the details are shown in Table SI. The difference in ETHE1 expression in tumor and normal tissues was analyzed using a non-parametric test (Wilcoxon rank-sum test). The immunohistochemical results of ETHE1 in GC tissues and normal tissues from healthy controls were analyzed using the Human Protein Atlas (HPA) database (https://www.proteinatlas.org/). Survival analysis was performed based on the expression data of ETHE1 in patients with stomach adenocarcinoma (STAD) from the data obtained from TCGA using Kaplan-Meier analysis. Survival analysis results and survival plots were generated using Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer–pku.cn/) (20), and the high and low cutoff values were set to 50%. The Spearman correlation analysis was performed to determine the correlation between ETHE1 expression in STAD and immunomodulator expression using the TISIDB database (http://cis.hku.hk/TISIDB/index.php) (21). ETHE1 expression in regulatory T cells (Tregs) and M2 macrophages in STAD tumor tissues and paracancerous tissues were analyzed using GEPIA based on the data from TCGA.

A total of 30 pairs of fresh cancerous and paracancerous tissues from 30 patients with GAC who underwent surgical resection at The Second Hospital of Dalian Medical University (Dalian, China) were collected between April 15, 2023 and October 30, 2023. The patients were aged 39–85 (mean age, 65.7 years), 80% were men and 20% were women. These fresh tissues were used to detect ETHE1 expression. In addition, the clinical characteristics of 50 patients with GAC who had not received preoperative chemotherapy or radiotherapy were collected from The Second Hospital of Dalian Medical University between April 15, 2023 and October 30, 2023. These patients were aged 47–85 year (mean age, 65.2 years), 78% were men and 22% were women. The paraffin-embedded cancerous tissue samples of the 50 patients were used to detect ETHE1 expression. The association between ETHE1 expression and the clinical characteristics of patients with GAC was analyzed using a Yates's corrected χ² test or Fisher's exact test. All of the specimens were collected under a protocol approved by the Ethics Committee of The Second Hospital of Dalian Medical University (2023; approval no. 136). Each patient provided written informed consent, and the study was performed in accordance with The Declaration of Helsinki.

All cell lines were purchased from iCell Bioscience Inc. The human GAC AGS cell line was cultured in Ham's F-12K medium (Wuhan Servicebio Technology Co., Ltd.) supplemented with 10% FBS (Zhejiang Tianhang Biotechnology Co., Ltd.), 1% penicillin and 1% streptomycin. GES-1, SNU-1, NCI-N87 and MKN-45 cells were cultured in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 10% FBS. All of the cells were maintained at 37°C in a humidified incubator supplied with 5% CO₂ air.

The 2nd lentiviral system was used to infect the NCI-N87 and MKN-45 cells. Short hairpin (sh)RNA constructs were synthesized by General Biotech (Anhui) Co., Ltd. and were cloned into the pLKO.1-EGFP-puro vector (Fenghbio). The sequences of the shRNAs were: ETHE1-shRNA (708–728; sh^708–728), 5′-ccgGATAGACTTTGCTGTTCCAGCttcaagagaGCTGGAACAGCAAAGTCTATCtttttt-3′, ETHE1-shRNA (557–577; sh^557-577), 5′-ccgCAGGAGACTGTCTGATCTACCttcaagagaGGTAGATCAGACAGTCTCCTGtttttt-3′, and negative control (NC)-shRNA 5′-ccgTTCTCCGAACGTGTCACGTttcaagagaACGTGACACGTTCGGAGAAtttttt-3′. The numbers 708–728 and 557–577 represent the target sequence position of the shRNA. The uppercase letters represent ETHE1-specific sequences, and the lowercase letters represent hairpin sequences. The ETHE1-targeted interference vectors (14 µg), packaging plasmids (10.5 µg) and envelope plasmids (3.5 µg) were co-transfected into 293T cells (iCell Bioscience Inc.) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) to produce lentiviral particles that were used to infect cells. The 293T cells were cultured at 37°C with 5% CO₂, and 48 h post-transfection with the plasmid vectors, the supernatant containing lentiviral particles was collected. Subsequently, 48 h after transduction, stably infected cells were screened, and subsequent experiments were performed 5 days later. In detail, the NCI-N87 [multiplicity of infection (MOI)=10] and MKN-45 (MOI=20) cells were seeded in 6-well plates and cultured in media containing lentiviral particles (2×10⁸ TU/ml; NCI-N87 cell, 10 µl/well; MKN-45 cell, 20 µl/well) at 37°C and 5% CO₂ for 24 h, the media were then replaced with fresh media and the cells were cultured for a further 24 h. Control cells were transfected with a lentivirus carrying a shRNA that lacked a specific target sequence (NC-shRNA). The cells were cultured in complete culture medium containing puromycin (NCI-N87, 1.5 µg/ml; MKN-45, 2.5 µg/ml) for 5 days to screen for stably infected cells. The stably infected NCI-N87 and MKN-45 cells were maintained in media containing 0.3 and 0.5 µg/ml puromycin, respectively.

A total of 10 male BALB/c nude mice (age, 6 weeks; body weight, 17–19 g) supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were randomly divided into the following two groups: Control and sh^708-728 (n=5/group). The mice were reared under a 12-h light/dark cycle, at a temperature of 22±1°C and a humidity level of 45–55%. The mice were allowed to eat and drink freely, and their health and behavior were monitored every 4–6 h. After 1 week of adaptive feeding, the mice were subcutaneously injected with the NCI-N87 cell line (2×10⁶ cells; 0.2 ml/mouse). The sh^708-728 group received ETHE1-knockdown NCI-N87 cells, while the control group was injected with cells infected with the NC-shRNA lentivirus. Tumor volume was measured every 3 days following tumor formation. At the end of the experiment (day 25), mice were euthanized using CO₂ with a volume displacement rate of 50%/min, followed by cervical dislocation. Tumor tissues were collected for subsequent experiments.

For animal welfare reasons, the animals were kept in a constant temperature and humidity environment, and the experimental procedures were carried out by skilled experimenters to reduce the temporary tension and pain caused by subcutaneous injection. Based on the National Institutes of Health Guidelines for Endpoints in Animal Study Proposals (22), humane endpoints in the study included tumors growing to >15 mm in diameter, and ulceration or infection of the growth site. A total of 10 animals were used in the study, all of which were euthanized at the end of the experiment. No animals were found dead during the experiment. All the animal experiments were approved by the Animal Care and Welfare Committee of Dalian Medical University (approval no. AEE23074).

Total RNA from clinical samples or cells (NCI-N87 and MKN-45) were extracted using TRIpure lysis buffer (Bioteke Corporation) and cDNA was obtained by RT. The mixture including oligo (dT)₁₅ (Takara Biotechnology Co., Ltd.) and random primers synthesized by General Biotech (Anhui) Co., Ltd. was heated at 70°C for 5 min, and was cooled on ice for 2 min. Subsequently, dNTP (Beijing Solarbio Science & Technology Co., Ltd.), buffer (Beyotime Institute of Biotechnology), RNase inhibitor (Sangon Biotech Co., Ltd.) and reverse transcriptase (Beyotime Institute of Biotechnology) were added to the mixture, which was heated at 25°C for 10 min and 42°C for 50 min, before the reaction was terminated by heating at the mixture at 80°C for 10 min. qPCR was performed using SYBR GREEN (Beijing Solarbio Science & Technology Co., Ltd.) and 2X Taq PCR MasterMix (Beijing Solarbio Science & Technology Co., Ltd.) on a fluorescence quantifier (Exicycler™ 96, Bioneer Corporation). The qPCR thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min(), followed by 40 cycles at 95°C for 10 sec (denaturation), 60°C for 10 sec (annealing) and 72°C for 15 sec (extension). The mRNA levels were quantified using the 2^{−ΔΔCq(Cq test-Cq reference)} formula (23). The sequences of the primers used were: ETHE1 forward, 5′-GTCATCTCCCGCCTTAGTG-3′ and reverse, 5′-CGGATCAACAGGGCATCT-3′; and GAPDH forward, 5′-GACCTGACCTGCCGTCTAG-3′ and reverse, 5′-AGGAGTGGGTGTCGCTGT-3′. GAPDH was used as the internal control gene.

Total protein was extracted from clinical samples or cells (NCI-N87 and MKN-45) using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), and the protein concentration was determined using a BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Protein samples (20 µg) were loaded onto SDS-gels (5% stacking gel, 8–12% separation gel), resolved using SDS-PAGE and transferred to PVDF membranes (MilliporeSigma). The membranes were then blocked using a blocking solution (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h and washed. Subsequently, the membranes were incubated with primary antibodies (1:1,000) overnight at 4°C and with the secondary antibody (1:3,000) for 1 h at room temperature. After washing, enhanced chemiluminescence solution (Beijing Solarbio Science & Technology Co., Ltd.) was added to the membranes, which were exposed in a dark room and imaged using a gel imaging system (WD-9413B; Beijing Liuyi Biotechnology Co., Ltd.). The antibodies used in the present study are listed in Table I.

Tumor tissue was fixed in 4% paraformaldehyde overnight at room temperature, and dehydrated in an increasing series of ethanol concentrations (70, 80, 90 and 100%). Dehydrated tissues were permeabilized in xylene and embedded in paraffin. Paraffin-embedded tissues were cut into 5-µm sections, which were immersed in xylene, rehydrated in a descending series of ethanol concentrations (95, 85 and 75%), boiled in antigen retrieval solution (Beyotime Institute of Biotechnology) for 10 min, washed in PBS buffer, and incubated with 3% H₂O₂ (Sinopharm Chemical Reagent Co., Ltd.) for 15 min at room temperature to inactivate endogenous peroxidase activity. Subsequently, the sections were blocked with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at room temperature. After washing, the sections were incubated with primary antibodies (Ki67 antibody, 1:1,000; ETHE1 antibody, 1:200) at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:500) for 1 h at room temperature. The protein expression was visualized using DAB (Fuzhou Maixin Biotechnology Development Co., Ltd.). Finally, sections were counterstained with hematoxylin (Beijing Solarbio Science & Technology Co., Ltd.) and images were captured using a light micrograph system (DP73; Olympus Corporation). Antibody details are provided in Table I.

Cells (NCI-N87 and MKN-45) were seeded in 96-well plates (5×10³ cells/well) and cell viability was assessed using a Cell Counting Kit-8 cell proliferation assay kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Cells were incubated with CCK-8 reagent for 2 h at 37°C. The absorbance was measured at 450 nm using a microplate reader (800TS; BioTek; Agilent Technologies, Inc.).

Cells (NCI-N87 and MKN-45) were stained using a kFluor488 Click-iT EdU imaging assay kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Briefly, virally infected cells were incubated with EdU for 2 h at 37°C, fixed with 4% paraformaldehyde for 15 min at room temperature, and treated with the Click-iT reaction solution in the dark for 30 min at room temperature. Nuclei were counterstained with DAPI for 5 min at room temperature. Images were captured using a fluorescence microscope (IX53; Olympus Corporation).

Cell cycle distribution and apoptosis were analyzed using a flow cytometer (NovoCyte-D2060R; Agilent Technologies, Inc.), and a Cell Cycle and Apoptosis Analysis Kit (BioSharp Life Sciences) or an Annexin V-APC/PI double staining apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.), respectively, according to the manufacturers' protocols. Briefly, cells (NCI-N87 and MKN-45) were incubated in PI/RNase A solution in the dark for 30 min at room temperature, and then the cell cycle distribution was measured. In addition, the cells were incubated with 5-µl Annexin V-APC and 5 µl PI dye in the dark for 10 min at room temperature, and apoptosis was detected. Analysis was conducted using NovoExpress software (version 1.5.6; Agilent Technologies, Inc.).

The TUNEL assay was used to detect apoptosis. Cells (NCI-N87 and MKN-45) grown on glass coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature and were then permeabilized using 0.1% Triton X-100 for 15 min at room temperature. Fluorescent labeling was performed using an In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer's protocol. The cells were incubated with TUNEL reaction liquid for 1 h at 37°C. For tissues, they were embedded in paraffin and cut into 5-µm sections. The sections were dewaxed, permeabilized and then labeled. The nuclei were counterstained with DAPI (Shanghai Aladdin Biochemical Technology Co., Ltd.) for 5 min at room temperature. The sections were sealed using antifading mounting medium (Beijing Solarbio Science & Technology Co., Ltd.) and the images of the sections were captured using a fluorescence micrograph system (three fields/sample).

Caspase-3 and caspase-9 activities in the NCI-N87 and MKN-45 cell lines were assessed using a caspase-3 activity assay kit (cat. no. C1116; Beyotime Institute of Biotechnology) and caspase-9 activity assay kit (cat. no. C1158; Beyotime Institute of Biotechnology), respectively, according to the manufacturer's protocol. Total protein was extracted using the lysis buffer provided by the kits. A Bradford protein concentration assay kit (Beyotime Institute of Biotechnology) was used to determine the total protein concentration, which was used to normalize the data. Caspase-3 and caspase-9 activity were assessed based on the concentration of the product of pNA, which is catalyzed by both caspase-3 and caspase-9. The absorbance of catalytic products was measured at 405 nm using a microplate reader (800TS).

A glucose assay kit (cat. no. F006; Nanjing Jiancheng Bioengineering Institute), lactic acid assay kit (cat. no. A019; Nanjing Jiancheng Bioengineering Institute) and ATP assay kit (cat. no. S0026; Beyotime Institute of Biotechnology) were used to measure glucose consumption, lactate production and ATP content, respectively. The cells (NCI-N87 and MKN-45 cell lines) were lysed using ultrasonic processing (300 W, 3 sec, interval time 25 sec, 5 cycles) and a BCA protein concentration assay kit (Beyotime Institute of Biotechnology) was used to detect total protein concentration, which was used to normalize the data.

The in vitro experiments were repeated three times. Data are presented as the mean ± SD. Statistical analyses were performed using GraphPad Prism version 9.5.0 (Dotmatics). A two-tailed unpaired Student's t-test or one-way ANOVA followed by a Tukey's post hoc test was used to analyze the differences between independent groups. P<0.05 was considered to indicate a statistically significant difference.

Pan-cancer analysis showed that ETHE1 was differentially expressed in multiple types of cancer, such as STAD. Notably, the expression of ETHE1 in STAD samples was significantly higher than that in healthy control samples (Fig. 1A). Furthermore, the immunohistochemical results of ETHE1 in GC and normal tissues were analyzed using the HPA database, and ETHE1 expression was upregulated in tumor tissues (Fig. 1B). The present study detected the expression levels of ETHE1 in 30 pairs of GC tissues and paracancerous tissues. Analysis showed that ETHE1 expression was generally higher in cancerous tissues than that in paracancerous tissues (Fig. 1C and D). Additionally, upregulated expression of ETHE1 was associated with a worse survival time (Fig. 1E). The association between ETHE1 expression levels and clinicopathological characteristics are shown in Table II. A higher tumor stage, lymph node metastasis or higher Tumor-Node-Metastasis (TNM) stage was associated with higher expression of ETHE1, indicating a potential link between ETHE1 and GAC. Representative immunohistochemical images of high and low ETHE1 expression in GAC tissues are shown in Fig. 2A. Furthermore, the expression levels of ETHE1 in GAC cell lines were higher than those in the normal gastric mucosa cell line GES-1, and the expression levels were relatively higher in NCI-N87 and MKN-45 cells than in AGS and SNU-1 cell lines (Fig. 2B and C). Therefore, these two cell lines were selected for further study.

As shown in Fig. 3A, compared with in the control group, intracellular ETHE1 expression was successfully knocked down ~4-fold. Cell proliferation was inhibited following lentiviral infection for 48 h (Fig. 3B). Furthermore, the number of EdU-positive cells was reduced, indicating that GAC cell proliferation was reduced following ETHE1 knockdown (Fig. 3C). Cell cycle distribution was measured using flow cytometry. As shown in Fig. 3D, the knockdown of ETHE1 resulted in a decrease in the number of cells in the S phase, an increase in the number of cells in the G₁ phase, and no significant effect on the number of cells in the G₂ phase. In addition, the proportion of cells in the sub-G₁ phases suggested the occurrence of apoptosis. Western blot analysis showed that ETHE1 knockdown reduced the expression levels of cyclin D1 and CDK4 (Fig. 3E).

Apoptosis was further measured using Annexin V-PI staining combined with flow cytometry. As shown in Fig. 4A, compared with in the control group, knockdown of ETHE1 increased the proportion of apoptotic GAC cells. TUNEL staining also showed that ETHE1 knockdown promoted apoptosis (Fig. 4B). Further analysis of apoptosis-related enzyme activity revealed that knockdown of ETHE1 resulted in an increase in caspase-3 and caspase-9 activity (Fig. 4C and D).

Compared with in the control groups, knockdown of ETHE1 reduced glucose consumption, lactic acid production and ATP levels in GAC cells, as measured by the markers of glycolysis in GAC cells (Fig. 5A-C). In addition, knockdown of ETHE1 reduced the expression levels of glycolysis-related molecules, including glucose transporter type 1 (GLUT1), lactate dehydrogenase A (LDHA), hexokinase 2 (HK2) and pyruvate kinase isozyme type M2 (PKM2) (Fig. 5D).

The growth rate of tumors in the sh^708-728 group was lower than that in the control group in vivo (Fig. 6A). Starting from day 16, the tumor volume in the sh^708-728 group was smaller than that in the control group. The tumor weight of the sh^708-728 group was also lower than that of the control group. Compared with in the control group, Ki67 and ETHE1 expression in the tumor tissues of the sh^708-728 group was reduced (Fig. 6B), whereas apoptosis was increased (Fig. 6C).

To further explore the value of ETHE1 in immune regulation, the correlation between its expression and immunomodulator expression was analyzed in STAD. The expression of ETHE1 in STAD clinical samples was significantly positively correlated with immunosuppressive factors, such as galectin 9 (LGALS9), nectin cell adhesion molecule 2 (PVRL2), IL-10 receptor subunit β, CD274 and indoleamine 2,3-dioxygenase 1 (IDO1) (Fig. 7A). A negative correlation was identified between ETHE1 expression and immune-promoting factors, such as IL-6 receptor (IL6R), CD160, UL16 binding protein 1 (ULBP1), CD276 and CD40 (Fig. 7B). ETHE1 expression was also positively correlated with chemokines, such as C-X-C motif chemokine ligand (CXCL)5, CXCL3, CXCL16, CXCL1 and CXCL2 (Fig. 7C). These findings suggested that ETHE1 may contribute to the development of STAD. Although some correlations were not very strong (r value <0.3), they were still statistically significant, thus indicating that ETHE1 may be involved in the regulation of the expression of immune regulatory factors. In addition, compared with in the normal samples (paracancerous tissues, n=36), ETHE1 expression was upregulated in Tregs and M2 macrophages in STAD tumor tissues (n=414), suggesting that ETHE1 expression may mediate the malignant behavior of STAD (Fig. 7D).