Spatial transcriptomic characteristics of gastric cancer in young and the expression and role of TMEM176B in gastric cancer cells

Abstract

Background

Gastric cancer in young (GCY) is increasing in incidence with poor prognosis. Current screening and molecular methods are inadequate, necessitating new approaches to explore its pathogenesis. This study used spatial transcriptomic sequencing (ST-seq) to analyze the cellular composition of gastric cancer (GC) tumors, compare gene expression patterns, explore signaling pathways, and investigate the role of the differentially expressed gene (DEG) TMEM176B in GCY.

Methods

The surgical specimens of six patients with GCY were included to construct a tissue microarray containing the tumor core region (TCR), cancer-adjacent tissue (CAT), and normal gastric tissue (NGT). ST-seq was performed to obtain the transcript expression levels at different spatial locations. After quality control, normalization, standardization, clustering, dimensionality reduction, and cell-type prediction analyses were carried out to identify the DEGs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to clarify the underlying mechanisms of GC. Based on the results, TMEM176B was selected for functional analysis. Western blotting was used to assess TMEM176B expression in normal gastric cells and cancerous cells. shRNA-mediated TMEM176B knockdown in cancer cells was used for phenotypic analysis, proliferation assays, and apoptosis experiments.

Results

This study identified heterogeneous cell populations in GCY tissues. Exactly 18,082 DEGs were found between the TCR and CAT, mainly enriched in the IL-17, AGE-RAGE, and relaxin pathways. Moreover, 17,586 DEGs were identified between the TCR and NGT, primarily related to the HIF-1 and apoptosis pathways. TMEM176B was a key DEG in the TCR vs. CAT and TCR vs. NGT comparisons. It was highly expressed in GCY tissues and GC cell lines. Further analysis using The Cancer Genome Atlas database confirmed its oncogenic effects. TMEM176B knockdown in GC cell lines inhibited cell proliferation (reduced CCK8 and colony formation), increased apoptosis (higher Bax/Bcl2 ratio), and arrested the cell cycle in the G0/G1 phase.

Conclusions

This study used ST-seq to map the transcriptomic profiles of the TCR, CAT, and NGT in patients with GCY, investigating gene spatial expression patterns and tumor heterogeneity. We identified TMEM176B’s role in GC development and progression, offering molecular targets and a foundation for future treatments.

Background

Gastric cancer (GC), a malignant tumor originating from the gastric mucosa epithelium [1], ranks high in global and domestic incidence and mortality rates, posing a serious threat to human health [2]. Some studies defined gastric cancer in young (GCY) as the initial diagnosis age of 30 years or younger [3]. Our previous studies indicated that it is characterized by an increasing incidence rate, low histological grade, advanced clinical stage at initial diagnosis, and poor treatment prognosis [4]. Moreover, it is gradually garnering more attention and focus in ongoing research [5]. Given the insufficient sensitivity of existing screening strategies for young high-risk populations [6] and the difficulty of traditional molecular classification strategies in explaining the heterogeneity of its pathogenesis, new technologies and perspectives are needed to reveal the spatiotemporal features of GCY.

Spatial transcriptomic sequencing (ST-seq), a novel gene expression analysis technique, enables precise gene expression mapping at the cellular level. Unlike traditional transcriptome sequencing, it reveals the correlation between the cellular position within tissues and gene expression patterns, providing new insights into tumor heterogeneity [7]. In this study, we applied ST-seq to GCY tissue samples and identified gene expression variations in the tumor core region (TCR), cancer-adjacent tissue (CAT), and normal gastric tissue (NGT), exploring the molecular mechanisms underlying heterogeneity. By integrating ST-seq data, TMEM176B was identified as a core differentially expressed gene (DEG) in the TCR vs. CAT and TCR vs. NGT comparisons. Combined with bioinformatics analysis, TMEM176B was ultimately selected as a candidate gene. We further examined the expression pattern and cellular function of TMEM176B, selected based on spatial transcriptomic data. We believe that our findings would deepen the understanding of GCY heterogeneity and open new avenues for molecular classification and personalized therapies.

Methods

Patients

This study involved specimens from six patients with GCY who underwent surgery and were diagnosed at Zhengzhou University People’s Hospital between June 2022 and June 2023. The tissue samples included the TCR, CAT (non-cancerous tissues within 1–2 cm from the tumor edge in the same patient), and NGT (non-cancerous tissues more than 5 cm from the tumor edge in the same patient). The exclusion criteria included patients with other malignancies, those with metabolic or immune disorders, or those who declined participation. Clinical data, such as sex, age, and tumor size and location, are presented in Table 1. This study was approved by the Ethics Committee of Zhengzhou University People’s Hospital.

Table 1 Clinical data of the six patients with gastric cancer in young

Experimental procedures

Preparation of the spatial transcriptome samples

Tissues from the three sites were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 24 h. Dehydration was performed using an automated system [8], followed by paraffin embedding for wax block preparation. Sections of 5 μm were cut and processed through dehydration, clearing, and staining. The staining procedure included hematoxylin (10 min), differentiation (10 s), washing (10 min), and eosin (30 s). Hematoxylin and eosin (HE) slides were examined by experienced pathologists to identify cancerous, para-cancerous, and normal tissues. A 2.0 mm tissue microarray punch was used to core the marked sites, with nine cores per recipient block (three patient specimens). Two recipient blocks were created in a 3 × 3 pattern for tissue microarray construction from the six samples.

ST-seq

This study adopted the 10× Genomics Visium platform. Tissue microarray sections were prepared on standard glass slides. Overnight hybridization with the left hybridization sequence (LHS) and right hybridization sequence (RHS) probes was used to generate RTL capture probes. The CytAssist system facilitated probe transfer to visible capture areas, and spatial barcode probes revealed the transcript expression levels.

The gene expression arrays underwent probe extension followed by library construction. Sequencing was performed on an Illumina HiSeq or NovaSeq platform, with quality control using FastQC. Tissue coverage was assessed using the SpaceRanger software.

The Seurat SCTransform method was used to normalize and scale data by identifying highly variable genes using diffusion coefficients. Principal component analysis of the genes enabled linear dimensionality reduction, followed by clustering using the Louvain algorithm. Dimensionality reduction was visualized using t-SNE and UMAP. Moreover, SingleR software was used to predict the cell types.

DEGs were identified using Seurat’s FindAllMarkers. Gene Ontology (GO) functional analysis was performed using TopGO, and pathway enrichment was conducted in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Disease annotation and classification were performed using the DisGeNET software. Protein interaction networks for significant genes in each cluster were constructed using the STRING database.

Experimental cells and preliminary treatment

Three cell lines were investigated: GES-1 (normal gastric mucosa) and the GC cell lines BGC-823 (intestinal type, HER2 positive) and NUGC-3 (diffuse type, highly invasive). The latter represented the two main molecular subtypes of GC, and the complementarity of the two in terms of molecular characteristics and functional phenotypes met the need for mechanism exploration. All lines were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C and 5% CO₂. Cell handling included thawing, sub-culturing, and cryopreservation. For thawing, the cells were rapidly warmed in a 37 °C water bath, centrifuged, resuspended, and plated. Confluent cells were sub-cultured, washed with PBS, trypsinized, centrifuged, and plated in a 1:1 ratio. For cryopreservation, logarithmic-phase cells were trypsinized, centrifuged, resuspended in a serum-free cryopreservation solution, transferred to cryovials, and stored at − 80 °C.

RNA extraction

The culture medium was discarded to isolate RNA, and the cells were washed with PBS before adding TRIzol reagent for lysis. The lysate was mixed, transferred to a centrifuge tube, and 200 μL of chloroform was added. After vortexing for 15 s, the mixture was incubated for 10 min. After centrifugation at 12,000 rpm for 10 min, the upper phase was transferred to an RNase-free tube, mixed with 500 μL isopropanol, and incubated for 20 min at room temperature (20 ℃). The mixture was then centrifuged at 10,000g for 10 min. The supernatant was discarded, and the pellet was washed with 75% ethanol and centrifuged at 7500g for 5 min. The ethanol was removed, and the tube was air-dried for 5 min before adding RNase-free water. The RNA concentration was determined using a UV spectrophotometer.

Real-time fluorescence quantitative polymerase chain reaction (qPCR)

For cDNA synthesis, 1 μg of RNA, 4 μL of ALL Mix, and 1 μL of cDNA Eraser were combined in a 20 μL reaction with RNase-free DEPC-treated water, following the manufacturer’s protocol. The mixture was incubated at 42 °C for 15 min in a water bath and heated to 85 °C for 5 s on a heat block. Then, 80 μL of RNase-free DEPC-treated water was added to dilute the cDNA to a final concentration of 100 ng/μL. The qPCR program was set up, and the results were analyzed using the 2^−ΔΔCT method.

Protein electrophoresis (Western blot, WB)

The cells were washed with pre-cooled PBS, lysed by scraping, and centrifuged. Protein concentration was measured using a BCA working solution. An SDS-PAGE gel was prepared and left to solidify for 10 min. Next, 30 μg of the protein sample was loaded into each well, and electrophoresis was conducted at 80 V for 30 min. Once the samples entered the separating gel, the voltage was increased to 100 V, and electrophoresis continued for 100 min. The PVDF membrane was immersed in methanol for 10 s, transferred to an electrophoresis buffer for 100 min, and then blocked with 5% non-fat milk at room temperature for 2 h. It was incubated with the primary antibody overnight at 4 °C. After washing, the membrane was incubated with a secondary antibody for 2 h at room temperature. Protein imaging was conducted using a protein imaging system, and the images were analyzed using ImageJ software.

Immunofluorescence (IF)

The cells were washed twice with PBS for 5 min each, then fixed with 100 μL of 3.7% formaldehyde at room temperature for 20 min. After fixation, they were washed thrice with PBS containing 0.1% Triton X-100 for 5 min each and then incubated with 200 μL of Actin-Tracker Green and 2% BSA at room temperature in the dark for 60 min. After incubation, the cells were washed thrice with PBS containing 0.1% Triton X-100 for 5 min each and observed under a confocal microscope.

Extraction of the TMEM176B shRNA plasmid and virus packaging

TMEM176B shRNAs (shRNA1: GCUGAAAGCUGGAGGUUCUTT; shRNA2: CAAACUUGCUGGCUAUAUATT; shRNA3: AGGAAGUUGUUCACAGCAATT) were selected from the GIPZ lentiviral shRNA library (Cambridge, USA). The bacterial culture was incubated in a shaking incubator at 37 °C for 12 h or until turbidity was observed. The shRNA plasmid was extracted using a plasmid extraction kit. 293 T cells in the logarithmic growth phase were trypsinized and seeded in 10 cm culture dishes at approximately 40% confluence. The cells were cultured in antibiotic-free DMEM for 24 h for viral packaging. The three plasmids (packaging plasmid pCMV-dR8.9, envelope plasmid pVSV-G, and target plasmid pCDH-shTMEM176B) and Lipofectamine 2000 were mixed and incubated for 15 min. The mixture was then slowly added to the supernatant of the 293 T cells and cultured in an incubator. After 48 h, the virus-containing supernatant was collected, centrifuged, and stored at -80 °C for future use.

Construction of the TMEM176B stably knocked down GC cells

GC cells in the logarithmic growth phase were trypsinized and centrifuged, seeded into a six-well plate at 10,000 cells/well density, and cultured overnight. The virus stored at − 80 °C was thawed on ice and diluted at a virus:polybrene ratio of 1000:1. The culture medium was discarded, and the virus solution was added to the cells cultured in an incubator for 48 h. Total cell protein was extracted, and WB was performed to assess the knockdown efficiency of the virus on TMEM176B.

Cell viability assay (CCK8 method)

The cells were trypsinized, centrifuged to prepare a suspension, and seeded into a 96-well plate at a density of 2,000 cells per well, with three parallel wells for each condition. After cell attachment, shNC, sh-TMEM176B-1, and sh-TMEM176B-3 were added. Following 48 h of incubation, the medium in each well was replaced with DMEM containing 10 μL of CCK8 solution, and the plate was incubated for an additional 0.5–2 h. Each well’s absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay microplate reader.

Cell proliferation assay (EdU method)

Logarithmic-phase cells were trypsinized, centrifuged, and seeded in a 24-well plate (three parallel wells per condition). After adherence, shNC, sh-TMEM176B-1, and sh-TMEM176B-3 were added, and the cells were cultured for 48 h. EdU working solution was added for 2 h. The medium was discarded, and 200 μL of 4% paraformaldehyde was added to fix the cells. The cells were permeabilized, treated with the click reaction solution, and incubated at room temperature in the dark for 30 min. They were washed thrice for 3 min each. Then, Hoechst dye was added for nuclear staining, and fluorescence microscopy images were captured.

Cell colony formation assay

The cells were seeded at 1,000 cells/well in a 6-well plate and cultured for 24 h before adding shNC, sh-TMEM176B-1, or sh-TMEM176B-3. After a 72-h incubation, the medium was replaced with fresh sh-TMEM176B-containing medium, which was changed every 3 days until clones were visible. The culture process was terminated, and the medium was discarded. The cells were fixed with 4% paraformaldehyde for 15 min, and then 2 mL of 0.1% crystal violet solution was added to each well. The plate was incubated at room temperature in the dark for 15 min. After washing thrice with PBS, the plates were dried in a ventilated area, photographed using a smartphone, and the cell colony formation rate was determined.

Flow cytometry for cell cycle analysis

The cells were seeded in culture dishes and, once attached, treated with shNCs, sh-TMEM176B-1, or sh-TMEM176B-3 for 48 h. After treatment, the cells were trypsinized and centrifuged, and the supernatant was discarded. The cell pellet was washed twice with pre-cooled PBS, resuspended in 250 μL of pre-cooled PBS, and slowly mixed with 750 μL of pre-cooled absolute ethanol for fixation at 4 °C for 12 h. After centrifugation at 1200 rpm for 5 min, the cells were washed twice with PBS and incubated with 500 μL of PI/RNase staining solution at room temperature in the dark for 15 min. The cell cycle was then analyzed using flow cytometry.

Mitochondrial membrane potential detection

The cells were seeded in a 6-well plate and cultured for 24 h before adding shNC, sh-TMEM176B-1, or sh-TMEM176B-3. They were then cultured for an additional 24 h. Next, 1 mL of the JC-1 staining solution was added to each well, and the plate was returned to the incubator. After removing the supernatant, the cells were washed twice with JC-1 staining buffer and replenished with a culture medium. The cells were then observed and imaged using a confocal microscope.

Statistical analysis

All data were analyzed using R software (http://www.r-project.org) and visualized using R Studio. Regarding the spatial transcriptomic data, genes with a fold change > 2 and an adjusted P value < 0.05 were considered significantly differentially expressed. Cytological data are expressed as mean ± standard deviation (x ± s). One-way analysis of variance was used for multiple group comparisons, and independent-sample t-tests were used for pairwise comparisons. The level of statistical significance was set at P < 0.05.

Results

Overview of the GCY ST-seq results

ST-seq was performed on tissue microarrays from six pathologically confirmed patients with GCY (S1-S6). The tissues, including the TCR, CAT, and NGT, were selected after HE staining and processed into microarrays for sequencing (Fig. 1). After normalization, Seurat was used to integrate and analyze data from both chips. In chip 1, 92.5% of the spots with tissue coverage yielded 2030 specific spots, with an average of 165,061 reads per spot, 2636 genes, and a gene expression level of 18,061. In chip 2, 90.1% of the spots with tissue coverage had 2060 specific spots, with an average of 154,081 reads per spot, 2770 genes, and a gene expression level of 18,064. The spatial distribution and proportion of each cluster were visualized in the HE-stained scans. Cell types in each spot were annotated using automated software. In the TCR region, the majority of cells were epithelial cells. In contrast, the CAT region primarily consisted of epithelial cells, B cells, adipocytes, mesangial cells, and fibroblasts. The TCR was mainly composed of epithelial cells, with a small number of mesangial cells and fibroblasts.

Fig. 1

Hematoxylin and eosin (HE)-stained images of the six specimens. a HE-stained specimens from cases 1, 2, and 3; b HE-stained specimens from cases 4, 5, and 6. Scale bar 150 μm

DEGs and functional analysis regarding TCR vs. CAT

Differential gene analysis of the TCR and CAT in the six samples revealed 18,082 genes with differential expression between the two groups. Compared to the CAT, 3,632 genes were upregulated in the TCR (top 10 genes: HSPB1, S100A16, SFN, EMP1, ANXA1, CSTB, TMEM176B, S100A14, KRT13, and S100A8), whereas 14,450 genes were downregulated (top 10 genes: FCGBP, KRT8, AGR2, S100P, TFF1, S100A6, TSPAN8, MMP7, KCTD1, and RNASE1) (Fig. 2a, b). GO functional analysis revealed that the DEGs were primarily associated with pancreatic neuroendocrine tumors, GC, and adenocarcinoma (Fig. 2c). KEGG pathway analysis indicated that these genes were mainly involved in the IL-17, AGE-RAGE, and relaxin signaling pathways (Fig. 2d).

Fig. 2

Functional analyses of the differentially expressed genes in the tumor core region (TCR) vs. cancer-adjacent tissue (CAT) groups. a Heatmap of the differentially expressed genes between TCR and CAT; b Volcano plot showing the upregulated (red) and downregulated (blue) genes; c Gene Ontology (GO) functional analysis; d Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

DEGs and functional analysis regarding TCR vs. NGT

Differential gene expression analysis between the TCR and NGT groups in the six samples identified 17,586 DEGs. Compared to the NGT, 4,860 genes were upregulated in the TCR (top 10 genes: STARD3, MIEN1, GSDMB, ERBB2, PGAP3, TMEM176B, ORMDL3, TCAP, ZPBP2, and IKZF3), whereas 12,726 genes were downregulated (top 10 genes: FDFT1, PSCA, CTSB, URI1, KRAS, CD44, MMP7, VEGFA, ENO1, and KCTD1) (Fig. 3a, b). GO analysis revealed that the DEGs were primarily associated with malignant lymph node metastasis, GC, and adenocarcinoma (Fig. 3c). KEGG pathway analysis highlighted their involvement in relaxin-related, HIF-related, and apoptosis-related signaling pathways (Fig. 3d).

Fig. 3

Functional analyses of the differentially expressed genes in the tumor core region (TCR) vs. normal gastric tissue (NGT) groups. a Heatmap of the differentially expressed genes; b Upregulated and downregulated differentially expressed genes; c Gene Ontology (GO) functional analysis; d Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

TMEM176B was highly expressed in various cancer diseases

Analysis of the top 10 upregulated DEGs in TCR–CAT and TCR–NGT comparisons revealed TMEM176B as the only gene with significantly elevated expression in both groups (Fig. 4a). Further analysis of the cancer genome atlas (TCGA) database revealed high TMEM176B expression in various cancers (Fig. 4b, c) and an association with shorter disease-free survival (DFS) in patients with GC with high TMEM176B expression compared to those with low expression (Fig. 4d).

Fig. 4

The Cancer Genome Atlas database revealed high TMEM176B expression in various cancers. a TMEM176B was highly expressed in the tumor core region (TCR)-cancer-adjacent tissue (CAT) and TCR–normal gastric tissue (NGT) comparisons. b The expression of TMEM176B was analyzed across various cancers. c In stomach adenocarcinoma (STAD), the expression pattern of TMEM176B was investigated. d The relationship between the TMEM176B expression and disease-free survival in patients with gastric cancer was assessed

qPCR analysis of the normal human gastric mucosa cells (GES-1) and human GC cell lines (BGC-823 and NUGC-3) revealed significantly higher TMEM176B mRNA levels in BGC-823 and NUGC-3 cells than in GES-1 cells (P < 0.05) (Fig. 5a). WB analysis revealed significantly upregulated TMEM176B protein levels in BGC-823 and NUGC-3 cells than in GES-1 cells (P < 0.05) (Fig. 5b, c), indicating elevated TMEM176B expression in GC.

Fig. 5

TMEM176B was highly expressed in gastric cancer (GC). a Analysis of normal human gastric mucosa cells (GES-1) and human GC cell lines (BGC-823 and NUGC-3) revealed significantly higher TMEM176B mRNA levels in BGC-823 and NUGC-3 cells than in GES-1 cells; b, c Western blot analysis revealed significantly upregulated TMEM176B protein levels in the BGC-823 and NUGC-3 cells than in the GES-1 cells, indicating elevated TMEM176B expression in GC. ***P < 0.001

Knockdown of TMEM176B inhibited the growth rate and proliferative capacity of GC cells

Given the high expression of TMEM176B in the GC cells, we constructed three TMEM176B-specific shRNAs using lentivirus. WB analysis demonstrated that all three shRNAs exhibited significantly reduced TMEM176B protein levels in BGC-823 (Fig. 6a, b) and NUGC-3 (Fig. 6c, d) cells compared to the control group, confirming successful shRNA construction.

Fig. 6

Western blot analysis demonstrated that all three shRNAs exhibited significantly reduced TMEM176B protein levels in the BGC-823 (a, b) and NUGC-3 (c, d) cells. **P < 0.01, ***P < 0.001

Using sh-TMEM176B-1 and sh-TMEM176B-3 for subsequent experiments, CCK8 assays showed no significant change in the OD values between the shNC group and the sh-TMEM176B-1 or sh-TMEM176B-3 groups after 1 and 2 days of culture in the BGC-823 cells (P > 0.05). However, after 3 and 4 days, the OD values of the sh-TMEM176B-1 and sh-TMEM176B-3 groups were significantly lower (Fig. 7a). In the NUGC-3 cells, TMEM176B knockdown significantly slowed cell growth compared to the control group as the culture time increased (Fig. 7b). This suggested that TMEM176B knockdown reduced the proliferation rate of the GC cells. EdU assays showed that TMEM176B knockdown significantly inhibited the proliferation of BGC-823 cells compared to the shNC group (P < 0.05) (Fig. 7c). A similar effect was observed in NUGC-3 cells (Fig. 7d), consistent with the CCK8 assay results, indicating that TMEM176B knockdown suppresses GC cell proliferation.

Fig. 7

Knockdown of TMEM176B significantly reduced the growth rate of BGC-823 (a) and NUGC-3 cells (b). EdU assays showed that TMEM176B knockdown significantly inhibited the proliferation of BGC-823 cells compared to the shNC group (P < 0.05) (c). A similar inhibitory effect on proliferation was observed in NUGC-3 cells (d). **P < 0.01, ***P < 0.001

Knockdown of TMEM176B inhibited the colony formation ability of GC cells

The clonogenic assay demonstrated that TMEM176B knockdown in the sh-TMEM176B-1 and sh-TMEM176B-3 groups significantly reduced the number and size of tumor cells of BGC-823 cells compared to the shNC group (P < 0.05) (Fig. 8a, b). Similarly, TMEM176B knockdown significantly inhibited the clonogenic ability of NUGC-3 cells (Fig. 8a, c).

Fig. 8

Knockdown of TMEM176B inhibited the colony formation ability of gastric cancer (GC) cells. a Clonogenic formation of GC cells in the shNC, sh-TMEM176B-1, and sh-TMEM176B-3 groups in the NUGC-3 and BGC-823 cells; b Number of tumor cells in each group of BGC-823 cells; c Number of tumor cells in each group of NUGC-3 cells. Compared to the NC group, **P < 0.01, ***P < 0.001

TMEM176B was localized in the mitochondria of the GC cells

Fluorescent probe detection using Actin-Tracker, which labels cell microfilaments, revealed the localization of TMEM176B in the mitochondria of GES-1, BGC-823, and NUGC-3 cells, as observed under confocal microscopy (Fig. 9).

Fig. 9

TMEM176B was localized in the mitochondria of gastric cancer (GC) cells. The subcellular localization of TMEM176B in three GC cell lines (GES-1, BGC-823, and NUGC-3) was observed using confocal microscopy. DAPI (blue): Nucleus. Actin-Tracker (green): Cytoskeleton. Mito-Tracker (red): Mitochondria. TMEM176B (gray): Target protein. Merge: TMEM176B and Mito-Tracker colocalization (pale red). Scale bar 40 μm

Knockdown of TMEM176B promoted GC cell apoptosis

To investigate the effect of TMEM176B knockdown on GC cell apoptosis, WB analysis was performed to examine the expression levels of PARP1, Bax, Bcl2, and Caspase3 in GC cells. In BGC-823 cells (Fig. 10a–c), TMEM176B knockdown significantly increased Bax expression and decreased PARP1 and Bcl2 expressions compared to the control group. A similar trend was observed in NUGC-3 cells (Fig. 10d–f), although Caspase3 expression remained unchanged in both cell lines. These findings suggest that TMEM176B knockdown promoted apoptosis in GC cells, potentially through a Caspase3-independent pathway. The JC-1 assay was used to further confirm the impact of this knockdown on GC cell apoptosis. The results showed that in BGC-823 (Fig. 11a) and NUGC-3 cells (Fig. 11b), the JC-1 expression was significantly reduced in the shNC-TMEM176B-1 and sh-TMEM176B-3 groups, aligning with the WB results and indicating that TMEM176B knockdown promoted apoptosis in the GC cells.

Fig. 10

Knockdown of TMEM176B promoted gastric cancer (GC) cell apoptosis. Western blot analysis was performed to examine the expression levels of PARP1, Bax, Bcl2, and Caspase3 in the GC cells. The results are presented for BGC-823 (a–c) and NUGC-3 cells (d–f). Compared to the NC group, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 11

Evaluation of the effect of TMEM176B knockdown on gastric cancer (GC) cell apoptosis. The JC-1 expression was significantly reduced in the sh-TMEM176B-1 and sh-TMEM176B-3 groups both in the BGC-823 (a) and NUGC-3 cells (b). Scale bar 100 μm

Knockdown of TMEM176B blocked the cell cycle

Flow cytometry was used to assess the cell cycle distribution in BGC-823 and NUGC-3 cells after TMEM176B knockdown. In BGC-823 cells, the sh-TMEM176B-1 and sh-TMEM176B-3 groups had significantly more cells in the G0/G1 phase than the shNC group (Fig. 12a, b), which was statistically significant (P < 0.05). Similarly, in NUGC-3 cells, the sh-TMEM176B-1 and sh-TMEM176B-3 groups showed an increased distribution in the G0/G1 phase compared to the shNC group (Fig. 12c, d). This indicates that TMEM176B knockdown arrested GC cells in the G0/G1 phase, preventing entry into the S phase and inhibiting proliferation.

Fig. 12

Knockdown of TMEM176B blocked the cell cycle. A, B In BGC-823 cells, the sh-TMEM176B-1 and sh-TMEM176B-3 groups had significantly more cells in the G0/G1 phase than the shNC group; C, D In NUGC-3 cells, the sh-TMEM176B-1 and sh-TMEM176B-3 groups showed an increased distribution of cells in the G0/G1 phase compared to the shNC group. *P < 0.05

Discussion

In this study, ST-seq was performed on 18 sections from six GCY tissue samples to analyze gene expression in the TCR, CAT, and NGT, illustrating the spatial heterogeneity of GCY. The results comprised the following: (1) Clustering analysis and differential gene expression identified 22 subpopulations and their spatial relationships; (2) DEGs between TCR and CAT and between TCR and NGT were analyzed using GO and KEGG pathway enrichment analyses to reveal potential biological processes and mechanisms in GC development. The risk of GC in the young population has gradually become a new challenge in the public health field [9]. Our previous study revealed that 61.4% of patients with GCY had already progressed to stages III–IV at the initial diagnosis, which was characterized by its distinctive clinical features [4]. However, traditional classification strategies struggle to comprehensively analyze its heterogeneity and invasive mechanisms, highlighting the need for a new perspective to systematically reveal the molecular regulatory network of GCY. Spatial transcriptomic technology precisely determines cell locations within tissues and maps all mRNA in single tissue slices, enabling the localization and differentiation of functional gene expression in specific tissue regions [10, 11].

This study revealed that TCR and CAT were mainly composed of epithelial cells and B cells, suggesting a possible epithelial-immune interaction characteristic in GCY [12]. The enrichment of B cells was likely associated with the formation of tumor-related tertiary lymphoid structures (TLSs) [13]. These TLSs activated anti-tumor immune responses through antigen presentation and antibody secretion [14]. Therefore, they may be associated with chemotherapeutic drug sensitivity [15] and the effectiveness of immunotherapy [16]. However, some subsets of B cells (such as regulatory B cells) may also promote immune tolerance by secreting inhibitory factors, such as IL-10 and TGF-β [17]. Therefore, comprehensive evaluation and further exploration are necessary to understand their role in the development of GCY and the effectiveness of immunotherapy. Epithelial cells constitute the primary cellular component of the gastric mucosa. In physiological conditions, they maintain tissue integrity, prevent the involvement of pathological cells, and ensure continuous cellular renewal through the mechanism of apical extrusion [18]. The disruption of the apical defense mechanism is implicated in the carcinogenesis of epithelial cells, endowing them with invasive characteristics [19].

This study revealed the gene expression differences in the TCR, CAT, and NGT of GCY through ST-seq. There were 18,082 DEGs in the TCR compared to CAT and 17,586 DEGs compared to NGT. These spatial transcriptomic characteristics provided molecular references for multi-dimensional combined treatment research, such as targeting oxidative stress regulation [20], enhancing immunogenic death, and intervening in cholesterol metabolism. The DEGs between TCR and CAT were enriched in the IL-17, AGE-RAGE, and relaxin signaling pathways. Compared to NGT, the DEGs were significantly enriched in the HIF-1 and apoptosis pathways. In this study, spatial transcriptomics was used to analyze the spatial heterogeneity of GCY in tissue microenvironments, providing potential molecular markers and therapeutic targets for understanding the invasive progression of GCY.

This study analyzed the DEGs in TCR–CAT and TCR–NGT tissues and found that TMEM176B was a key DEG in both groups. Its expression pattern was correlated with that of the S100 family members (S100A8/A14/A16) and the oncogene ERBB2, suggesting that TMEM176B may be involved in GC progression via inflammatory-metabolic crosstalk networks such as the IL-17/HIF-1 axis. Further analysis using the TCGA database confirmed that TMEM176B was overexpressed in multiple malignancies, including GC, with the high expression linked to a shorter DFS. Functional experiments were then conducted to explore the underlying molecular mechanisms.

We conducted cellular experiments to investigate TMEM176B expression and its effects on GC cells. qPCR and WB analyses confirmed significantly higher TMEM176B expression in BGC-823 and NUGC-3 cells, suggesting its potential role in GC progression. RNA viral transfection to knock down TMEM176B expression in BGC-823 and NUGC-3 cells slowed growth rates over time in the CCK8 assays. The EdU and clonogenic assays confirmed that TMEM176B knockdown inhibited the sustained proliferation of BGC-823 and NUGC-3 GC cells.

During tumor development, tumor cells demonstrate continuous proliferation and significantly reduced apoptotic capacity [21,22,23]. The apoptotic process is closely linked to mitochondrial function [24,25,26], with fluorescence probe experiments showing TMEM176B localized in cell mitochondria. This study used WB experiments to detect the expression levels of PARP, Bax, Bcl2, and Caspase3. The results showed that, in the knockdown group, Bax expression was significantly increased, whereas PARP1 and Bcl2 expressions were significantly decreased. This suggests that TMEM176B knockdown can promote apoptosis in GC cells. The apoptotic regulatory function of TMEM176B is implicated in the progression of various non-tumor diseases. In autoimmune diseases, TMEM176B, expressed on immune cells, regulates their activity and contributes to disease progression [27]. In infectious diseases, inhibiting TMEM176B can modulate macrophage apoptosis, enhancing pathogen clearance and reducing infection-related tissue damage [28]. In pulmonary fibrosis, TMEM176B slows disease progression by inhibiting the TGF-β1/Smad signaling pathway [29]. This study confirmed that TMEM176B maintains cell survival in patients with GCY by suppressing the mitochondrial apoptotic pathway, as evidenced by changes in the Bax/Bcl2 protein ratio, highlighting its potential as a therapeutic target for GC.

Our study revealed that TMEM176B is highly expressed in GCY tissues and the GC cell lines (NUGC-3 and BGC-823). Experimental evidence shows that it may promote tumor cell proliferation [30]. This finding offers novel insights into the molecular mechanisms and potential clinical translation for GCY. As a transmembrane protein, its high expression may be associated with the regulation of the immune microenvironment [31], suggesting a similar carcinogenic mechanism in GC. Patients with GCY often exhibit strong invasiveness and poor prognosis, which may be partially explained by the overexpression of TMEM176B. In addition, TMEM176B inhibitors, such as BayK8644, have demonstrated potential for enhancing immunotherapy effects in other cancers [32], providing a reference for the development of TMEM176B inhibitors or antibody-based drugs. However, research on TMEM176B is still in the preclinical stage, and its specific mechanisms of action and interactions with the tumor microenvironment require further exploration.

This study had some limitations. The small sample size may have impacted the generalizability of the findings, requiring further replication and validation. Additionally, while phenotypic experiments for TMEM176B were conducted at the cellular level, no in vivo validation was performed, and the underlying mechanisms of the gene’s effects were not comprehensively investigated. In future studies, we plan to increase the sample size, observe the functional phenotype of TMEM176B in vivo, and investigate related mechanisms to elucidate the relationship between TMEM176B and GC development, potentially identifying new molecular targets for GC diagnosis and treatment.

Conclusions

This study employed ST-seq to map the transcriptomic profiles of TCR, CAT, and NGT in patients with GCY and revealed spatial expression patterns, tumor heterogeneity, and disease-related signaling pathways. We identified the role of TMEM176B in GC development, offering molecular targets and a foundation for future GC treatments.