Application of SNF2 protein derived from streptococcus angina extracellular vesicles in gastric cancer prognosis

Abstract

The invention discloses an application of SNF2 protein derived from streptococcus angina extracellular vesicles in gastric cancer prognosis. The chromatin remodeling protein SNF2 derived from S.anginosus EVs can be combined with a transcription factor TEAD1, so that the transcription of the palmitoyl transferase ZDHHC11 is promoted together. Then, the stability of the ZDHHC11 is enhanced by catalyzing palmitoylation of PD-L1, and finally immune escape is induced. In addition, SNF2 also can activate AXL, CTGF, CYR61 and other carcinogenic targets at the downstream of TEAD1, thereby further accelerating the malignant progression of gastric cancer. In an in-vivo experiment, the intragastric administration of the S.anginosus EVs not only promotes the tumor growth of mice, but also significantly inhibits the infiltration of CD8 + T cells. Blocking of ZDHHC11 can effectively reverse immune escape, and has a synergistic effect with an anti-PD-1 therapy, so that the treatment effect is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of microbial applications, and in particular to the application of SNF2 protein derived from extracellular vesicles of Streptococcus pharyngitis in the prognosis of gastric cancer. Background Technology

[0002] Streptococcus pharyngitis ( Streptococcus anginosus , S. anginosus Streptococcus pharyngis is a ubiquitous Gram-positive commensal bacterium that colonizes multiple mucosal sites, including the oral cavity, nasopharynx, and gastrointestinal tract. It is one of the most abundant gut microbiota in gastric cancer and can induce purulent abscesses. Previous studies have shown that its surface protein TMPC can activate the MAPK pathway by binding to the ANXA2 receptor on gastric epithelial cells, thereby promoting gastric tumorigenesis. However, the exact mechanism by which Streptococcus pharyngis affects the regulation of the tumor immune microenvironment (TIME) remains unclear.

[0003] Bacteria can communicate with host cells through direct contact or by secreting factors such as metabolites, proteins, toxins, and membrane vesicles. Extracellular vesicles (EVs) are spherical, double-membrane-coated nanoparticles produced by Gram-positive or Gram-negative bacteria, typically ranging in diameter from 10 to 400 nanometers. Studies have shown that *Fusobacterium nucleatum* (…) Fusobacterium nucleatum , F. nucleatum Outer membrane vesicles (OMVs) from Streptococcus pharyngitis can deliver their virulence factor FadA to joints and activate synovial macrophages via the Rab5a-YB-1 signaling pathway, thereby exacerbating rheumatoid arthritis (RA). However, the extracellular vesicle protein components secreted by Streptococcus pharyngitis have not yet been systematically characterized, and their regulatory mechanism in gastric cancer progression remains unclear. Summary of the Invention

[0004] This invention identifies *Streptococcus anginosus* (S. anginosus) as a bacterium enriched in gastric cancer tumors and capable of promoting gastric cancer progression. We further demonstrate that extracellular vesicles (EVs) derived from *Streptococcus anginosus* are internalized by gastric cancer cells primarily through dynein-dependent endocytosis, thereby accelerating gastric cancer progression. Mechanistically, the chromatin remodeling protein SNF2, derived from *S. anginosus* EVs, binds to the transcription factor TEAD1, jointly promoting the transcription of palmitoyltransferase ZDHHC11. Subsequently, ZDHHC11 enhances the stability of PD-L1 by catalyzing palmitoylation, ultimately inducing immune escape. Furthermore, SNF2 can also activate downstream oncogenic targets of TEAD1, such as AXL, CTGF, and CYR61, thereby further accelerating the malignant progression of gastric cancer. In in vivo experiments, gavage administration of *S. anginosus* EVs not only promoted tumor growth in mice but also significantly inhibited CD8+ T cell infiltration. Notably, blocking ZDHHC11 effectively reversed immune escape and synergized with anti-PD-1 therapy, significantly improving treatment efficacy. This study establishes that *Streptococcus pharyngis* extracellular vesicles are key drivers of gastric cancer progression and immunosuppression, suggesting that targeting this microbe-host interaction signaling axis may enhance the efficacy of existing immunotherapy strategies. Attached Figure Description

[0005] Figure 1*Streptococcus pharynx* enriches in gastric cancer tissue and promotes tumorigenesis. (A) Differential bacterial abundance at the genus level between gastric cancer tissue (T) and normal tissue (N), measured by linear discriminant analysis (LDA) scores. Only genera with log10 (LDA score) > 3 are shown (n = 40). (B) Comparison of bacterial abundance in primary gastric cancer lesions from patients with (Lp) and without (Ln) lymph node metastasis by LDA analysis. (C) Detection of *Streptococcus pharynx* in gastric cancer tissue by fluorescence in situ hybridization (FISH). Blue (DAPI) indicates cell nuclei, and green signals represent *Streptococcus pharynx*-specific probes (left panel). The percentage of *Streptococcus pharynx*-positive cells in gastric cancer tissue is quantified and shown in a bar chart (right panel). (D) Schematic diagram of the mouse subcutaneous model design. C57BL / 6 mice were administered *Streptococcus pharynx* orally three times a week (n = 5) or PBS (control group, n = 5). (E) Representative image of subcutaneous YTN16 xenograft tumors in mice. (F and G) Tumor growth curve data and tumor weight. (H) Recording of mouse weight changes. (I) Immunohistochemical staining of tumor tissue sections for proliferation markers (Ki67, PCNA) and apoptosis marker (Cleaved caspase-3) (top). Staining intensity was quantified using ImageJ and presented as a bar chart (bottom). (J) Principal coordinate analysis (PCoA) based on Bray-Curtis distance to show the β diversity of gut microbiota operational taxonomic units (OTUs) in mouse fecal samples after gavage with Streptococcus angiotensinus. (K) α diversity of mouse feces after gavage with Streptococcus angiotensinus, measured by the Chao1 index (left) and Observed Species (right). (L) Genus-level heatmap showing bacterial abundance in mouse fecal samples. (M and N) The proliferation and colony-forming abilities of AGS, MKN1, and YTN16 cells were assessed after treatment with *Streptococcus pharyngis* conditioned medium (Sa-CM). The control group was treated with BHI. (O and P) The migration and invasion abilities of AGS and YTN16 cells after treatment with *Streptococcus pharyngis* conditioned medium (Sa-CM) were evaluated by Transwell assay. The control group was treated with BHI. All data are expressed as mean ± standard deviation and were analyzed using unpaired Student's t test. *P < 0.05; **P < 0.01; ***P < 0.001; ns indicates no statistical significance.

[0006] Figure 2*Streptococcus pharyngis* accumulates in tumor tissues and acts as a microbial driver for gastric cancer development. (A) LEfSe analysis of fecal microbiota in mice after gavage administration of *Streptococcus pharyngis* (LDA score [log10]>2). (B) Analysis of microbial community composition by linear discriminant analysis (LDA score [log10]>2). (C and D) Detection of proliferation and colony-forming ability of AGS, MKN1, and YTN16 cells after co-culturing with *Streptococcus pharyngis*. (E and F) Assessment of migration and invasion ability of AGS, MKN1, and YTN16 cells after co-culturing with *Streptococcus pharyngis* using Transwell assay. (G and H) Assessment of migration and invasion ability of MKN1 cells after treatment with *Streptococcus pharyngis* conditioned medium (Sa-CM) using Transwell assay. The control group was treated with BHI. Data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0007] Figure 3Extracellular vesicles of *Streptococcus angiosum* are internalized by gastric cancer cells via dynein-dependent endocytosis, driving tumor progression. (A) Proliferation capacity of MKN1, YTN16, and AGS cells was assessed after treatment with *Streptococcus angiosum* conditioned medium (Sa-CM), nuclease-treated Sa-CM, or heat-treated Sa-CM, respectively. The control group was treated with BHI. (B and C) *S. angiosum*-derived EVs were characterized by scanning electron microscopy (SEM) (B) and nanoparticle tracking analysis (NTA) (C). Scale bar: 100 nm. (D and E) Proliferation and colony-forming capacity of MKN1, YTN16, AGS, and MKN45 cells were assessed after treatment with *S. angiosum* EVs. The control group was treated with PBS. (F and G) Migration and invasion capacity of MKN1, YTN16, AGS, NUGC3, and YTN16 cells after treatment with *S. angiosum* EVs were evaluated by Transwell assay. The control group was treated with PBS. (H) Confocal microscopy images showing the endocytosis of EVs by AGS cells (20 μg / mL DiO-labeled EVs, 2 hours) (left). The number of DiO-labeled EVs-positive cells was quantified and shown in a bar chart (right). Scale bar: 100 μm. (I) Confocal images of MKN1 cells pretreated with a specific endocytosis inhibitor (1 hour) and then exposed to DiO-labeled S. anginosus EVs (20 μg / mL) for 2 hours (left). The number of DiO-EVs-positive cells was quantified and shown in a bar chart (right). Scale bar: 100 μm. (J) Schematic diagram of the mouse subcutaneous model design. C57BL / 6 mice were administered S. eosinophilic EVs (n=5) or PBS (n=5) orally three times a week. (K) Representative images of YTN16 xenograft tumors in mouse subcutaneous tissue. (L and M) Tumor growth curve data and tumor weight. (N) Record changes in mouse body weight. (O) Perform immunohistochemical staining for proliferation markers (Ki67, PCNA) and apoptosis markers (Cleaved caspase-3) on tumor tissue sections (top figure). Staining intensity was quantified using ImageJ and presented as a bar chart (bottom figure). All data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0008] Figure 4Extracellular vesicles of *Streptococcus pharyngitis* drive gastric cancer immune escape by stabilizing PD-L1 via ZDHHC11. (A) Gene ontology (GO) enrichment analysis of RNA sequencing data from Sa-EV-treated AGS cells. (B) Volcano plot of differentially expressed genes in pathway (A) after Sa-EV treatment. (C) qRT-PCR analysis showing upregulated ZDHHC11 expression in Sa-EV-treated gastric cancer cells. (D) Peak diagram of PD-L1 in immunoprecipitation mass spectrometry. HA-tagged ZDHHC11 plasmid was transiently transfected into AGS cells, and ZDHHC11 interacting proteins were pulled down using HA antibody and Protein A / G magnetic beads. (E) MKN1 cells transfected with HA-tagged ZDHHC11 were immunoprecipitated using HA antibody and Protein A / G magnetic beads, and Western blotted with a specified antibody. (F) Three-dimensional structural diagram of the potential binding site of ZDHHC11 and PD-L1, with hydrogen bonds marked (top). The binding free energy is -25.0 kcal / mol (bottom figure). (G) Immunoblotting analysis of PD-L1 upregulation in AGS cells after ZDHHC11 overexpression. (H) Immunofluorescence staining shows PD-L1 upregulation in MKN1 cells after ZDHHC11 overexpression (left figure). PD-L1 fluorescence intensity was quantified using ImageJ (right figure). (I) After ZDHHC11 overexpression, MKN1 cells were treated with actinomycin (CHX; 100 μg / mL) for a specified time. PD-L1 protein stability was assessed by immunoblotting and quantified using ImageJ. (J) MKN1 cells transfected with the specified plasmid were collected after 6 hours of treatment with 25 μM MG132. Cell lysates were subjected to nickel bead pull-down assays and immunoblotting was performed using the specified antibody. (K) Immunofluorescence staining shows PD-L1 upregulation in mouse xenograft tissue after oral administration of Sa-EVs (left figure). PD-L1 fluorescence intensity was quantified using ImageJ (right figure). (L) Immunofluorescence staining showed upregulation of PD-L1 in MKN1 cells after Sa-EV treatment (left panel). PD-L1 fluorescence intensity was quantified using ImageJ (right panel). (M) Immunoblot analysis showed upregulation of PD-L1 protein after Sa-EV treatment (left panel). qRT-PCR analysis showed no change in PD-L1 mRNA levels in Sa-EV treated MKN1 cells (right panel). (N) After Sa-EV treatment, MKN1 cells were treated with actinomycin (CHX; 100 μg / mL) for a specified time. PD-L1 protein stability was assessed by immunoblotting and quantified using ImageJ. (O) Sa-EV treated MKN1 cells were collected after treatment with 25 μM MG132 for 6 hours. Cell lysates were subjected to nickel bead pull-down assays and immunoblotting was performed using the specified antibody. (P) Schematic diagram of the co-culture process of YTN16-OVA cells (stable expression of ovalbumin) and CD8+ T cells.(Q to S) YTN16-OVA cells were pretreated with Sa-EVs and then co-cultured with activated spleen CD8+ T cells from OT-1 mice for 24 hours. The cytotoxic activity of CD8+ T cells against YTN16-OVA cells was detected (Q). The concentrations of IL-2 and IFN-γ in the supernatant were determined by ELISA (R). The expression levels of IL-2 and IFN-γ mRNA in CD8+ T cells were analyzed by qRT-PCR (S). All data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0009] Figure 5 Extracellular vesicles of *Streptococcus pharyngitis* drive gastric cancer immune escape by stabilizing PD-L1 via ZDHHC11. (A) qRT-PCR analysis showed no change in the expression levels of ZDHHC3 and ZDHHC9 in gastric cancer cells treated with Sa-EV. (B) Immunoblotting analysis of PD-L1 upregulation in MKN1 and MKN45 cells after ZDHHC11 overexpression. (C) MKN45 cells were treated with actinomycin (CHX; 100 μg / mL) for a specified time after ZDHHC11 overexpression. PD-L1 protein stability was assessed by immunoblotting and quantified using ImageJ. (D) After treatment with 25 μM MG132 for 6 hours, doxycycline-induced ZDHHC11 knockdown AGS and MKN45 cells were collected. Cell lysates were subjected to nickel bead pull-down assays and immunoblotting with specified antibodies. (E) Immunofluorescence staining showed upregulation of PD-L1 and ZDHHC11 in mouse xenograft tissue after gavage administration of Sa-EVs (top panel). The fluorescence intensity of PD-L1 and ZDHHC11 was quantified using ImageJ (bottom panel). (F) Immunoblot analysis showed upregulation of PD-L1 protein after Sa-EV treatment (top panel). qRT-PCR analysis showed no change in PD-L1 mRNA levels in Sa-EV treated MKN45 and YTN16 cells (bottom panel). (G) After Sa-EV treatment, MKN45 cells were treated with actinomycin (CHX; 100 μg / mL) for a specified time. PD-L1 protein stability was assessed by immunoblotting and quantified using ImageJ. (H) Sa-EV treated MKN45 and YTN16 cells were collected after treatment with 25 μM MG132 for 6 hours. Cell lysates were subjected to nickel bead pull-down assays and immunoblotting was performed using the specified antibody. Data are presented as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0010] Figure 6Extracellular vesicles of *Streptococcus pharyngitis* promote protein stability through ZDHHC11-mediated PD-L1 palmitoylation. (A) Schematic diagram of the LC-MS analysis of PD-L1 modification status in MKN1 cells treated with Sa-EVs or PBS (control group). MKN1 cells transiently transfected with the Flag-tagged PD-L1 plasmid were treated with Sa-EVs or PBS, and PD-L1 was immunoprecipitated using anti-Flag M2 beads. The palmitoylation status was analyzed by LC-MS. (B) PD-L1 peaks detected by LC-MS after Sa-EVs or PBS (control group) treatment. (C) Schematic diagram of the acyl-biotin exchange (ABE) method for palmitoylation detection (top figure). The IP-ABE assay was used to detect the level of palmitoylated PD-L1 (Palm-PD-L1) in AGS cells expressing Flag-PD-L1 after treatment with Sa-EVs (50 μg / mL, 24 h) or 2-BP (50 μM, 24 h) (Figure below). (D) Immunofluorescence staining shows the upregulation of PD-L1 in MKN1 cells after treatment with Sa-EVs (50 μg / mL, 24 h) and Palmostatin B (5 μM, 24 h). (E) Immunofluorescence staining shows that PD-L1 was upregulated in MKN1 cells after treatment with Sa-EVs (50 μg / mL, 24 h), and this effect was reversed by treatment with the palmitoylation inhibitor 2-BP (50 μM, 24 h) (Figure above). The fluorescence intensity of PD-L1 was quantified using ImageJ (Figure below). (F) Immunofluorescence staining showed that PD-L1 was upregulated in AGS and MKN45 cells after treatment with Sa-EVs (50 μg / mL, 24 h), and this effect was reversed by ZDHHC11 knockdown (induced by doxycycline Dox) (top panel). PD-L1 fluorescence intensity was quantified using ImageJ (bottom panel). (G) After treatment with 25 μM MG132 for 6 hours, AGS and MKN45 cells transfected with the specified plasmid were collected. Cell lysates were subjected to nickel bead pull-down assays and immunoblotting with the specified antibody. (H to J) YTN16-OVA cells were pretreated with Sa-EVs and 2-BP and co-cultured with activated spleen CD8+ T cells from OT-1 mice for 24 hours. The cytotoxic activity of CD8+ T cells against YTN16-OVA cells was detected (H). The concentrations of IL-2 and IFN-γ in the supernatant were determined by ELISA (I). qRT-PCR was used to analyze the expression levels of IL2 and IFNG mRNA in CD8+ T cells (J). All data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0011] Figure 7SNF2 family proteins derived from *Streptococcus pharyngitis* extracellular vesicles synergistically enhance ZDHHC11 transcription with the transcription factor TEAD1. (A) ZDHHC11 transcription factor predicted based on the JASPAR database. (B) qRT-PCR analysis showed that ZDHHC11 expression was upregulated in MKN1 cells after TEAD1 overexpression. (C) Five potential TEAD1 binding sites (S1–S5) were predicted within the ZDHHC11 promoter region (-2,000 to +100 bp). (D) Chromatin immunoprecipitation (ChIP) experiments in MKN1 cells validated the binding of TEAD1 at multiple specific sites in the ZDHHC11 promoter region. (E) Protein components in Sa-EVs identified by 4D label-free quantitative proteomics. (F) The top four transcriptional regulatory proteins identified in Sa-EVs. (G) 293T cells transfected with Flag-tagged TEAD1 and HA-tagged Sa-SNF2 were subjected to immunoprecipitation using M2 beads and Western blotting with a specified antibody. (H) Neighbor-linkage (PLA) assay was performed in 293T cells co-transfected with Flag-tagged TEAD1 and HA-tagged Sa-SNF2. Detection based on HA / Flag antibody showed neighbor-to-neighbor interactions (red signal), and the cell nuclei were counterstained with DAPI (blue). (I) Three-dimensional structural diagram of the potential binding site of Sa-SNF2 and TEAD1, with hydrogen bonds marked. The binding free energy was -10.0 kcal / mol. (J) Molecular dynamics simulations showed the root mean square fluctuation (RMSF) values ​​of Sa-SNF2 (orange) and TEAD1 (blue). (K) qRT-PCR analysis showed that ZDHHC11 expression was upregulated in MKN1 cells after Sa-SNF2 overexpression. (L) ChIP assays showed that Sa-SNF2 overexpression enhanced TEAD1 recruitment to the ZDHHC11 promoter. (M and N) Immunoblotting analysis of ZDHHC11 and PD-L1 expression in AGS and MKN45 cells after Sa-SNF2 overexpression and ZDHHC11 knockdown (induced by doxycycline). (O) IP-ABE assay was used to detect the level of palmitoylated PD-L1 (Palm-PD-L1) in AGS cells expressing Flag-PD-L1 under different treatment conditions. (P) MKN45 cells transfected with the specified plasmid were collected after treatment with 25 μM MG132 for 6 hours. Cell lysates were subjected to nickel bead pull-down assays and immunoblotting was performed with the specified antibody. (Q to S) YTN16-OVA cells transfected with the Sa-SNF2 overexpression plasmid were pretreated with 2-BP and then co-cultured with activated spleen CD8+ T cells from OT-1 mice for 24 hours. The cytotoxic activity of CD8+ T cells against YTN16-OVA cells was detected (Q). The concentrations of IL-2 and IFN-γ in the supernatant were determined by ELISA (R).qRT-PCR was used to analyze the expression levels of IL2 and IFNG mRNA in CD8+ T cells (S). All data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0012] Figure 8 SNF2 family proteins derived from extracellular vesicles of *Streptococcus pharyngitis* synergistically enhance ZDHHC11 transcription with the transcription factor TEAD1. (A and B) qRT-PCR analysis showed the expression levels of ZDHHC11 in MKN1 and AGS cells after overexpression of MEF2A, IRF1, or TEAD1. (C) Pan-cancer analysis using GEPIA showed increased TEAD1 expression in STAD tumor tissues compared to normal tissues, and differential expression patterns in other malignancies. (D and E) Kaplan-Meier survival curves from the TCGA database were used to assess the prognostic significance of TEAD1 and ZDHHC11 in gastrointestinal cancers (STAD / COAD / READ), and statistical significance was determined by log-rank test (interquartile stratification). (F) GEPIA analysis of TCGA data revealed a positive correlation between TEAD1 and ZDHHC11 expression in gastrointestinal cancers (STAD, COAD, READ, ESCA, LIHC). (G) Immunohistochemical staining of tumor tissue sections for ZDHHC11 and TEAD1 (left panel). Staining intensity was quantified using ImageJ and presented as a bar chart (right panel). (H) Comparison of the ATP-binding domains of SNF2 (Streptococcus pharyngis) and BRG1 (human) shows highly conserved ATP-binding sites. (I) Molecular dynamics simulations (100 ns) using root mean square deviation (RMSD) analysis demonstrate the structural stability of the Sa-SNF2-TEAD1 complex. (J) qRT-PCR analysis shows upregulation of ZDHHC11 expression in AGS and YTN16 cells after Sa-SNF2 overexpression. (K and L) Immunoblot analysis of ZDHHC11 and PD-L1 expression in MKN1, YTN16, or AGS cells after Sa-SNF2 overexpression and ZDHHC11 knockdown (induced by doxycycline). (M) AGS cells transfected with the specified plasmid were collected after treatment with 25 μM MG132 for 6 hours. Cell lysates were subjected to nickel bead pull-down assays and immunoblotting with specified antibodies. Data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0013] Figure 9SNF2 derived from extracellular vesicles of *Streptococcus pharyngitis* regulates TEAD1-mediated transcription and BAF complex assembly through its conserved ATP-binding site. (A) Comparative analysis of SNF2 protein sequence similarity among different species. (B) qRT-PCR analysis showing the expression level of ZDHHC11 in AGS and YTN16 cells after overexpression of wild-type or mutant Sa-SNF2. (C) GEPIA analysis of TCGA data revealed a positive correlation between TEAD1 and its target gene expression in gastric adenocarcinoma (STAD). (D and E) Proliferation and colony-forming ability of MKN1 cells were detected after overexpression of Sa-SNF2 and knockdown of AXL (siRNA). (F and G) Transwell assay was used to evaluate the migration and invasion abilities of MKN1 cells after overexpression of Sa-SNF2 and knockdown of AXL (siRNA). Data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0014] Figure 10SNF2, derived from extracellular vesicles of *Streptococcus pharyngitis*, regulates TEAD1-mediated transcription and BAF complex assembly through its conserved ATP-binding site. (A) Comparison of ATP-binding sites and their mutants in different species. (B) Schematic diagram of the secondary structure of Sa-SNF2 (three domains) and its ATP-binding site (wild type: DDMGLGKT; mutant: AAAAAAAA) visualized using PyMOL. (C) qRT-PCR analysis showing the expression level of ZDHHC11 in MKN1 cells after overexpression of wild-type or mutant Sa-SNF2. (D) ChIP experiment showing the binding of TEAD1 to the ZDHHC11 promoter region in MKN1 cells after overexpression of wild-type or mutant Sa-SNF2. (E) qRT-PCR analysis showing the expression level of TEAD1 target genes in AGS cells after overexpression of TEAD1. (F) qRT-PCR analysis showing the expression level of TEAD1 target genes in AGS cells after overexpression of wild-type or mutant Sa-SNF2. (G and H) Proliferation and colony-forming abilities of AGS cells after overexpression of Sa-SNF2 and knockdown of AXL (siRNA). (I and J) Migration and invasion abilities of AGS cells after overexpression of Sa-SNF2 and knockdown of AXL (siRNA) were assessed by Transwell assay. (K) Schematic diagram of the structure of the human BAF (BRG1 / BRM-related factor) chromatin remodeling complex. (L) Gel filtration chromatography analysis of AGS cell lysates expressing empty vector (control group), wild-type Sa-SNF2 (SNF2-WT), or mutant Sa-SNF2 (SNF2-mut). Molecular weights of eluted fractions are labeled above. Data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0015] Figure 11Streptococcus pharyngitis SNF2 can serve as a tumor marker. Targeting the Sa-SNF2-ZDHHC11 axis can inhibit tumor growth and synergize with anti-PD-1 therapy. (A) Schematic diagram of mouse subcutaneous model design. YTN16 cells stably overexpressing Sa-SNF2 were subcutaneously injected into C57BL / 6 mice. (B) Representative images of mouse tumor xenografts. (C and D) Tumor growth curve data and tumor weight. (E) Immunohistochemical staining of tumor tissue sections for HA, Sa-SNF2, Ki-67, Cleaved caspase-3, ZDHHC11, and PD-L1 expression (top). Staining intensity was quantified using ImageJ and presented as a bar chart (bottom). (F) Immunohistochemical staining of Sa-SNF2 expression in clinical specimens (left). Staining intensity in the cytoplasm and nucleus was quantified based on H-score and presented as a violin chart (n = 141) (right). (G) Kaplan-Meier analysis was used to assess the prognostic significance of Sa-SNF2 for overall survival and disease-free survival (n=141). (H) ZDHHC11 expression in different cell types of gastric cancer tissue was analyzed by single-cell RNA sequencing (scRNA-seq) from the TISCH database (http: / / tisch.comp-genomics.org / ). (I) scRNA-seq data from the TIMER2.0 database (http: / / timer.cistrome.org / ) were used to analyze the correlation between ZDHHC11 expression and CD8+ T cell infiltration in gastric cancer. (J) Immunohistochemical staining of CD8 expression in tumor tissue sections (top). ImageJ was used to quantify the staining intensity and the results are presented in a bar chart (bottom). (K) Schematic diagram of the mouse subcutaneous model design. C57BL / 6 mice were orally administered Streptococcus pharyngitis EVs three times a week. Mice were intraperitoneally injected with anti-PD-1 antibody (3 mg / kg, twice a week) and 2-BP (40 mg / kg, every other day). (L) Representative images of mouse tumor xenografts. (M and N) Tumor growth curve data and tumor weight. (O) Immunohistochemical staining of tumor tissue sections for Ki-67, Cleaved caspase-3, ZDHHC11, PD-L1, and CD8 expression (top). Staining intensity was quantified using ImageJ and presented as a bar chart (bottom). Data are expressed as mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0016] Figure 12Preparation of monoclonal antibodies (anti-SNF2). (A) Full-length amino acid sequence of Streptococcus pharyngitis SNF2. Blue text indicates the immunogen sequence used for antibody production. Red text indicates the ATP binding site. (B) SNF2-specific monoclonal antibody preparation process. (C) Balb / c mouse immunization program and serum titer assessment. SDS-PAGE purity analysis of the final antibody under non-reducing (left) and reducing (right) conditions. Detailed Implementation

[0017] Abbreviations: STAD: Stomach adenocarcinoma; EV: Extracellular vesicle; CHX: Cycloheximide; PD: Pulldown; BHI: Brain heart Infusion Broth; TMA: Tissue microarray; ip: Intraperitoneal injection; Sc: Subcutaneous injection; ig: Oral gavage; αPD-1: Anti-PD-1 antibody; BAF: BRG1 / BRM-associated factor; ChIP: Chromatin immunoprecipitation; PLA: Proximity ligation assay; RMSF: Root-mean-square fluctuation; RMSD: Root-mean-square deviation; ABE: Acyl-biotin exchange: acyl-biotin exchange; 2-BP: 2-Bromohexadecanoic acid; IB: immunoblot; LC-MS: Liquid chromatography-mass spectrometry; OVA: ovalbumin; CM: conditioned medium; COAD: Colon adenocarcinoma; READ: Rectumadenocarcinoma; ESCA: Esophageal carcinoma; LIHC: Liverhepatocellular carcinoma; ABX: Antibiotics; OTU: Operational Taxonomic Units; Ctrl: control.

[0018] Materials and Methods Tissue specimens from patients Gastric cancer tumor tissue and matched adjacent non-tumor tissue were collected from the Department of Gastric Surgery, The Sixth Affiliated Hospital of Sun Yat-sen University. In addition, a human gastric cancer tissue microarray containing 141 paired tumors and adjacent tissues, along with corresponding clinical and survival data, was used.

[0019] Cell culture and transfection Human gastric cancer cell line AGS was purchased from Meisen Chinese Tissue Culture Collections (MeisenCTC), while MKN1 and MKN45 were purchased from Servicebio. These cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. Mouse gastric cancer cell line YTN16 was kindly provided by Professor Sachiyo Nomura (University of Tokyo, Japan) and cultured according to the previously described conditions. HEK293T cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium containing 10% fetal bovine serum. All cells were cultured in a humidified environment at 37°C with 5% CO2. For transient transfection experiments, plasmids were introduced into cells using linearized polyethyleneimine (PEI) (Polysciences Inc., no. 24765-1) according to the manufacturer's protocol.

[0020] Bacterial strains co-culture with cells Streptococcus pharyngitis (ATCC 33397) strain was purchased from ATCC. Single clones were obtained by culturing bacteria on Brain Heart Infusion (BHI) agar plates and then incubated overnight in BHI broth at 37°C under aerobic conditions. For co-culture experiments, tumor cells were seeded into 60 mm culture dishes and allowed to adhere until a density of 70-90% was reached. Streptococcus pharyngitis in the logarithmic growth phase was collected, and the bacterial-to-cell ratio was determined using the multiplicity of infection (MOI). For in vitro studies, an MOI of 10-30 is routinely used.

[0021] Extraction and purification of bacterial extracellular vesicles Extracellular vesicles (EVs) were isolated from *Streptococcus pharyngitis* cultures using the previously described optimized ultracentrifugation protocol. Briefly, the bacteria were cultured aerobically at 37°C to the logarithmic phase in 180 mL of BHI. After culturing, bacterial cells were removed by centrifugation (10,000 × g, 20 min, 4°C) and filtration through a 0.22 μm pore size (Millipore) membrane. The supernatant was ultracentrifuged (Beckman Coulter, USA) at 170,000 × g, 4°C for 3 h. The resulting EV pellet was resuspended in 300 μL of PBS for subsequent experiments.

[0022] Extracellular vesicle labeling and cellular uptake analysis Extracellular vesicles of *Streptococcus pharyngitis* were fluorescently labeled with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) (20 μg / mL; MCE) at 37°C for 40 min. Unbound dye was removed by centrifugation at 4,000 × g for 45 min using a 30 kDa Amicon Ultra centrifuge filter. Gastric cancer cells cultured on slides were co-cultured with DiO-labeled EVs. After washing with PBS, cells were fixed with 4% paraformaldehyde (20 min), counterstained with DAPI (15 min), and imaged using confocal microscopy.

[0023] reagents and inhibitors The endocytosis inhibitors were purchased from Aladdin and used at the following working concentrations: chlorpromazine (5 μM, clathrin-mediated endocytosis inhibitor), cytochalasin D (2.5 μM, actin-mediated endocytosis inhibitor), and Dynasore (40 μM, dynein-mediated endocytosis inhibitor). Protein palmitoylation regulators, including 2-bromopalmitate (2-BP; 50 μM for in vitro cell experiments; 40 mg / kg for in vivo mouse administration) and palmostatin B (5 μM), were purchased from Sigma-Aldrich.

[0024] Mouse subcutaneous xenograft tumor model Animal studies were conducted in specific-pathogen-free (SPF) laminar flow ventilated cages with a 12 / 12-hour light / dark cycle. A xenograft tumor model was established using 6-week-old male C57BL / 6 mice. After 2 weeks of antibiotic treatment (administered in drinking water containing vancomycin [0.5 mg / mL; Aladdin], colistin [0.3 mg / mL; Aladdin], and ampicillin [1 mg / mL; Aladdin]), mice were subsequently provided with normal drinking water. YTN16 cells suspended in 50% Matrigel were subcutaneously implanted into the mice. For bacterial administration, mice in the experimental group were administered Streptococcus pharyngitis (1 × 10⁻⁶) by gavage every two days. 8 CFU), and the control group was administered PBS by gavage. For treatment with *Streptococcus pharyngitis* extracellular vesicles, the experimental group mice were administered CFU by gavage every two days throughout the experiment. S. anginosus EVs (100 ug / mouse) were administered, while the control group received PBS via gavage. For anti-PD-1 treatment, mice received intraperitoneal injections of anti-PD-1 antibody (3 mg / kg; BioXcell) twice weekly. Tumor size was monitored periodically, and volume was calculated using the formula: volume = (length × width²) / 2. At the experimental endpoint, all xenograft tumors were surgically removed and weighed.

[0025] 16S rRNA Sequencing and Data Analysis The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primer pairs 338F and 806R, and a unique 7-base barcode was added to enable multiplex analysis of samples. Each PCR reaction system contained: 5 μl 5× reaction buffer, 0.25 μl Fast pfu DNA polymerase, 2 μl dNTPs, 1 μl forward and reverse primers (10 μM), 1 μl DNA template, and 14.75 μl sterile distilled water. The thermal cycling program consisted of: initial denaturation at 98°C for 5 min; 25 cycles of 98°C for 30 s, 53°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. PCR products were purified using Vazyme VAHTS™ DNA Clean Beads and quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Equimolar amounts of purified amplicones were pooled and paired-end sequencing was performed at Shanghai Paisenuo Biotechnology Co., Ltd. using the Illumina NovaSeq platform and the NovaSeq 6000 SP Reagent Kit.

[0026] Fluorescence in situ hybridization Bacterial colonization in human gastric cancer tissue was assessed using a FITC-labeled Streptococcus pharyngitis-specific probe via fluorescence in situ hybridization (FISH). FISH was performed according to the manufacturer's protocol (EXONBIO, D-0016). Bacterial localization was observed using confocal microscopy.

[0027] Cell proliferation and colony formation experiments For cell proliferation analysis, 15,000 cells per well were seeded into 12-well plates. A live-cell imaging system (Incucyte S3, Sartorius, Germany) was used for continuous monitoring. Acquired images were processed, and data were analyzed using the manufacturer's software. Quantitative data were extracted using analysis software (Incucyte 2019B Rev2). Simultaneously, cells were seeded into 6-well plates at a density of 1,000 cells per well and cultured for 7–14 days. Cells were then fixed with 4% paraformaldehyde for 15 minutes, stained with 0.5% crystal violet for 30 minutes, and the resulting colonies were counted.

[0028] Cell migration and invasion experiments Cell migration and invasion assays were performed using Transwell chambers (Corning, NY, 353097) with 8 μm pores. For invasion assays, the membranes were pre-coated with 100 µL of Matrix-Gel™ basement membrane matrix and incubated at 37°C for 1 hour. Cells were suspended at a specific density in serum-free medium and seeded in the upper chamber, while the lower chamber contained medium with 10% FBS. After approximately 22 hours of incubation, cells were fixed and stained. Unmigrated / invaded cells inside the chambers were wiped away with cotton swabs. Cell counts were performed in ≥3 random fields under an optical microscope, with each assay repeated three times.

[0029] Immunofluorescence staining For tissue immunofluorescence staining, frozen tissue sections were equilibrated to room temperature for 15 minutes, fixed with 4% paraformaldehyde for 30 minutes, blocked with 5% BSA / PBS for 1 hour, and then incubated with primary antibody overnight at 4°C. Secondary antibody (Invitrogen) labeled with Alexa Fluor was incubated at room temperature for 1 hour (protected from light), followed by DAPI staining of cell nuclei. Imaging was performed using confocal microscopy. For cell immunofluorescence staining, cells grown on slides were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 5% BSA / PBS containing 0.5% Triton X-100 for 1 hour, and then incubated with primary antibody overnight at 4°C. After washing, secondary antibody (Invitrogen) labeled with Alexa Fluor was added and incubated at room temperature for 1 hour (protected from light), followed by DAPI staining. Images were acquired using confocal microscopy.

[0030] RNA extraction and real-time quantitative PCR Total RNA was extracted from cells using a Total RNA Extractor (Servicebio) and reverse transcribed into cDNA using the GoScript™ Reverse Transcription Kit (Promega) according to the manufacturer's instructions. Real-time quantitative PCR was performed on a LightCycler® 480II system (Roche) using the SYBR Green qPCR Master Mix (TransGen Biotech). All reactions were performed in triplicate, with Actin as an internal control.

[0031] Immunoblot analysis and immunoprecipitation Cells were collected and lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 0.1% NP-40, 1 mM EDTA). Protein concentration was determined by BCA assay, and equal volumes of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with primary antibody, and then incubated for 1 hour at room temperature with HRP-labeled secondary antibody (ThermoFisher; 1:10,000). Protein bands were detected by enhanced chemiluminescence and visualized on X-ray film. For cell immunoprecipitation experiments, cells pretreated with MG132 (MedChemExpress) for 6 hours were lysed in ice-cold lysis buffer. Cell lysates were immunoprecipitated overnight at 4°C using anti-Flag M2 agarose beads (Sigma-Aldrich). Wash the beads four times with lysis buffer and then boil them in 2× SDS loading buffer for 10 min. Perform immunoblotting using the specified antibody.

[0032] Mass spectrometry analysis and data processing MKN1 cells transiently transfected with the Flag-tagged PD-L1 plasmid were used Sa -EVs or PBS were used for treatment. PD-L1 was immunoprecipitated using anti-Flag M2 agarose beads. After washing, the bound protein was eluted and separated by SDS-PAGE. Protein bands corresponding to PD-L1 were excised from the gel, destained, reduced, alkylated, washed, digested with trypsin in the gel, extracted peptides, and vacuum dried. Peptides were separated by nanoLC and analyzed using a Q Exactive HF-X mass spectrometer. Data were processed using Proteome Discoverer 2.4.

[0033] Acyl-Biotin Exchange Palmitylation Assay AGS cell lysates transiently transfected with the Flag-tagged PD-L1 plasmid were immunoprecipitated using anti-Flag M2 agarose beads. Protein palmitoylation levels were then assessed according to the manufacturer's protocol using an acyl-biotin exchange assay kit (AM10314; AIMSMASS, Shanghai Huber Biotechnology Co., Ltd.).

[0034] Protein turnover rate determination Cells were seeded in six-well plates and treated with actinomycin (CHX; Sigma). Cells were collected at specified time points for immunoblotting analysis. Protein levels were quantified using ImageJ software.

[0035] ubiquitination assay Gastric cancer cells were co-transfected with a His-labeled ubiquitous plasmid and a designated expression plasmid. After 48 hours of incubation, cells were treated with the proteasome inhibitor MG132 (25 μM; MedChemExpress) for 6 hours before collection. Cells were then collected and lysed in denaturing buffer A (6 M guanidine-HCl, 0.1 M Na2HPO4 / NaH2PO4, 10 mM imidazole, pH 8.0), followed by sonication. The lysates were gently incubated overnight at 4°C with Ni-NTA agarose beads by rotation. The beads were washed sequentially, and the eluted proteins were analyzed by SDS-PAGE and Western blotting.

[0036] Proximity Ligation Assay (PLA) PLA experiments were conducted using the Duolink® PLA kit (Sigma-Aldrich, DUO92101) according to the manufacturer's protocol. In short, the HA-labeled [product / product] was co-transfected... Sa 293T cells containing the SNF2 and Flag-tagged TEAD1 expression plasmids were fixed, permeabilized, and blocked, then incubated overnight at 4°C with HA and Flag primary antibodies. Subsequently, ligation and amplification were performed using PLA secondary antibody probes. After washing, the cells were mounted with DAPI-containing mounting medium and imaged using confocal microscopy.

[0037] Immunohistochemical staining Paraffin-embedded tissue sections were dewaxed with xylene and hydrated with graded ethanol. Antigen retrieval was performed by microwave heating in sodium citrate buffer. After cooling, endogenous peroxidase activity was quenched with 3% H₂O₂, followed by blocking with goat serum for 1 hour. Primary antibody was incubated overnight at 4°C, followed by incubation with secondary antibody at room temperature for 20 minutes. After diaminobenzidine (DAB) staining, hematoxylin counterstaining, dehydration, and mounting were performed. Staining scores were determined by calculating percentage scores or intensity scores. Primary antibodies included ki67 (27309-1-AP, Proteintech, 1:2000), PCNA (ab29, abcam, 1:1000), Cleaved caspase-3 (9664P, CST, 1:1000), ZDHHC11 (GTX106800, GeneTex, 1:1000), PD-L1 (66248-1-g, Proteintech, 1:1000), CD8 (98941s, CST, 1:200), HA (3724T, CST, 1:1500), and SNF2 mouse monoclonal antibody (prepared and manufactured by Beijing Yiqiao Shenzhou Technology Co., Ltd., 1:1000). Secondary antibody was horseradish peroxidase-labeled goat anti-mouse / rabbit IgG polymer (PV-6000, Zhongshan Jinqiao).

[0038] OVA-specific CD8⁺ T cell isolation and co-culture experiment CD8⁺ T cells were isolated from the spleen of OT-1TCR transgenic mice using the MojoSort™ Mouse CD8 T Cell Isolation Kit (BioLegend, 480007). The isolated cells were then incubated with 10% fetal bovine serum, 1% penicillin / streptomycin, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 10 ng / ml recombinant mouse IL-2 (BioLegend, 575402), and 100 nMOVA. 257-264Primary stimulation was performed on the peptide (MCE, HY-P1489) in RPMI 1640 medium. After activation and expansion for 48 hours, cells were washed and cultured again for 48 hours in OVA peptide-free medium. For functional assays, activated CD8⁺ T cells were co-cultured with YTN16-OVA cells for 24 hours. The culture supernatant was collected for quantifying IL-2 and IFN-γ secretion using a commercial ELISA kit, while CD8⁺ T cells were collected for RNA extraction and subsequent qPCR analysis of IL2 and IFN-γ transcription levels. YTN16-OVA cell viability was monitored by measuring the cell index, which was normalized to baseline values ​​at the start of co-culture. As a control, YTN16-OVA cells were cultured alone under the same conditions to assess cell death.

[0039] Chromatin immunoprecipitation (ChIP) Cells were cultured in 150 mm culture dishes and then cross-linked with 1% paraformaldehyde at room temperature for 20 minutes. The reaction was terminated with 0.125 M glycine. After lysis in ChIP lysis buffer, the samples were centrifuged, and the precipitate was resuspended in nuclear lysis buffer and then sonicated. 10 μl of sheared chromatin was reserved as an input control, and the remainder was incubated overnight at 4 °C with ChIP beads, anti-Flag antibody (Sigma), or control IgG. The immunoprecipitated chromatin was washed sequentially with low-salt, high-salt, LiCl, and Tris-EDTA buffers. After reversing protein-DNA cross-linking by proteinase K treatment, DNA was isolated using a PCR purification kit (Omega). The enrichment of TEAD1 binding sites was quantified by qRT-PCR.

[0040] Protein structure preparation and molecular docking The target protein sequence and structure were retrieved from the UniProt database (https: / / www.uniprot.org / ). The obtained structure was imported into Discovery Studio 2019 for structure optimization. The optimized protein structure was used for molecular docking. Protein-protein docking was performed using GRAMM (https: / / gramm.compbio.ku.edu / ). The docking results were visualized and analyzed using PyMOL 3.1.

[0041] Molecular dynamics simulation Molecular dynamics simulations were performed using GROMACS 2022 software. Protein-ligand systems were treated with a CHARMM36 force field for proteins and GAFF2 for ligands, and solvated in a TIP3P water box. Long-range electrostatic interactions were handled using the particle mesh Ewald method, while short-range interactions were handled using the Verlet algorithm. The system was first equilibrated by 100,000 steps each in the NVT and NPT ensembles. Subsequently, a productive molecular dynamics run with a total simulation time of 100 ns was performed using Gromacs 2022 under constant temperature and pressure conditions.

[0042] Gel filtration chromatography AGS cell lysates transiently transfected with HA-tagged wild-type SNF2 (HA-SNF2-WT) or its mutant (HA-SNF2-mut) expression plasmids were loaded onto a Superose 6 column. Chromatographic analysis was performed using a GE AKTA avant150 system at 4°C and a flow rate of 0.4 mL / min. All fractions were collected and subsequently analyzed by SDS-PAGE and Western blotting with specified antibodies.

[0043] Monoclonal antibodies (anti-) Sa Hybridoma technology for producing -SNF2) A specific antibody against Streptococcus pharyngitis SNF2 was developed by Beijing Yiqiao Shenzhou Technology Co., Ltd. To generate this antibody, Balb / c mice were immunized with a VLP-conjugated peptide. Immunogenicity was confirmed by serum ELISA after four protein immunizations and three peptide immunizations. Then, spleen cells from high-titer mice were fused with SP2 / 0 myeloma cells, and antigen-specific clones were obtained through hybridoma screening. These clones were amplified and purified by Protein A affinity chromatography, achieving a purity >90%. Binding specificity was verified by ELISA.

[0044] Statistical analysis All experiments were repeated at least three times, and data are presented as mean ± standard deviation. Statistical comparisons between two groups were performed using a two-tailed Student's score. t For comparisons of multiple groups, one-way ANOVA is used. For paired samples, paired ANOVA is used. t The growth curves were analyzed using a two-way ANOVA. The Kaplan-Meier survival curves were evaluated using the log-rank test. P <0.05 indicates statistical significance (*) P <0.05,** P <0.01, *** P <0.001). See the corresponding figure captions for further details on the statistical methods. Data analysis was performed using SPSS and GraphPad Prism software.

[0045] Research Results 1. Accumulated in tumor tissue S. anginosus It is a microbial driver of gastric cancer. The role of the gut microbiota in carcinogenesis, particularly in gastric cancer, has become a key area of ​​cancer research. Recent studies have shown that, compared to normal tissues, Streptococcus spp. (…) Streptococcus Significant enrichment of ) in gastric cancer tumor tissue (linear discriminant analysis, LDA) Figure 1 A). Notably, this microbial imbalance was more pronounced in cases with lymph node (LN) metastasis, where the abundance of Streptococcus was significantly higher than in non-metastatic cases. Figure 1 B). Based on previous findings of Streptococcus pharyngitis S. anginosus It was enriched in gastric cancer mucosa and feces, and we further validated it using fluorescence in situ hybridization (FISH) technology, confirming its presence. S. anginosus It mainly colonizes gastric cancer tumor tissue, rather than adjacent normal tissue. Figure 1 C). To investigate S. anginosus Functional effects in gastric cancer progression: We administered YTN16 cells subcutaneously implanted into C57BL / 6 mice via gavage. S. anginosus or PBS ( Figure 1 D). The results showed that, compared with the control group, S. anginosus Treatment significantly promoted tumor growth, as evidenced by increased tumor volume ( Figure 1 E, F) and weight ( Figure 1 An increase in G) did not cause a significant change in the body weight of mice. Figure 1 H). Furthermore, the expression of proliferation markers (Ki-67 and PCNA) was upregulated in tumor tissues of the treatment group, while the expression of the apoptosis marker cleaved caspase-3 was decreased. Figure 1 I). Principal coordinate analysis (PCoA) was performed by sequencing 16S rRNA from mouse feces. S. anginosus There were significant differences in the gut microbiota composition between the gavage group and the control group. Figure 1 J). Alpha diversity analysis showed that the microbial richness (Chao1 index and observed species) of the treatment group was significantly increased (J). Figure 1 K). Heatmap analysis further reveals that, S. anginosus Gavage promotes the growth of various potential pathogens (such as Bacteroides). Bacteroides Prevotella Prevotella Klebsiella spp. Klebsiella Enterococcus Enterococcus and Streptococcus Streptococcus The colonization of Clostridium species was reduced. Clostridium Lactobacillus Lactobacillus Abundance of beneficial probiotics ( Figure 1 L). Linear discriminant analysis (LEfSe) (LDA>2, p (<0.05) further confirms that S. anginosus Administration induced a significant remodeling of the gut microbiota, characterized by the enrichment of pro-tumor bacteria and the simultaneous depletion of beneficial bacteria. Figure 2 A, B). These results collectively indicate that, S. anginosus The intervention profoundly reshaped the gut microbiota structure.

[0046] For research S. anginosus To investigate the functional effects on gastric cancer cells, we established co-culture systems of three gastric cancer cell lines (AGS, MKN1, and YTN16) with live bacteria. Compared with the control group, S. anginosus Co-culture significantly enhanced various malignant phenotypes of cancer cells, including proliferation ( Figure 2 C) Settlement formation ( Figure 2 D) Migration Figure 2 E) and invasiveness ( Figure 2 F). Considering the role of bacterial secretory factors, we then used S. anginosus Gastric cancer cells were treated with conditioned medium (CM). Results showed that, compared to the BHI medium control group, S. anginosus Conditioned culture medium ( Sa-CM Treatment also significantly enhanced the proliferation of cancer cells. Figure 1 M), settlement formation ( Figure 1 N), migration ( Figure 1 O and Figure 2 G) and invasiveness ( Figure 1 P and Figure 2 H). In summary, S. anginosus As a tumor-promoting bacterium, it plays an important role in the pathogenesis of gastric cancer by altering the gut microenvironment and directly enhancing the malignant behavior of cancer cells.

[0047] 2. S. anginosus Extracellular vesicles are internalized by gastric cancer cells through dynein-dependent endocytosis and drive tumor progression. For identification S. anginosus Conditioned culture medium ( Sa-CM We identified key components in [a specific compound] that promote gastric cancer progression, and separately degraded their nucleic acids using nucleases or their proteins using heat treatment. The results showed that nuclease treatment... Sa-CM It can still enhance cell proliferation, while heat treatment Sa-CM The loss of this ability indicates that its protein components are crucial. Figure 3 A). Given that bacterial extracellular vesicles (EVs) can package a variety of biomolecules, including nucleic acids and proteins, and are key mediators of host-microbe interactions, we isolated them using ultracentrifugation. S. anginosus EVs. Scanning electron microscopy (SEM) shows that they exhibit a typical cup-shaped morphology. Figure 3 B), nanoparticle tracking analysis (NTA) confirmed that its size distribution was 50-300 nm, with a peak at 127.6 nm. Figure 3 C), consistent with EVs characteristics. Compared to the PBS control group, S. anginosus EV treatment significantly enhanced the proliferation of gastric cancer cells. Figure 3 D) Settlement formation ( Figure 3 E), Migration ( Figure 3 F) and invasiveness ( Figure 3 G and). To confirm the internalization of EVs, we labeled them with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) dye, and confocal microscopy showed that AGS cells could effectively take up EVs within 2 hours. Figure 3 H). Mechanistically, pretreatment with dynasore (a dyskinin inhibitor) can block EV entry, while chlorpromazine (CPZ, a clathrin inhibitor) or cytochalasin D (an actin inhibitor) has no such effect. Figure 3 (I) demonstrates that its uptake mainly depends on the dynein-mediated endocytosis pathway.

[0048] Having clarified its internalization mechanism, we further evaluated the tumor-promoting function of EVs through in vivo experiments. In a YTN16 cell subcutaneous xenograft model, oral administration... S. anginosus EVs significantly increased tumor volume ( Figure 3 J and Figure 3 K) and weight ( Figure 3 L and Figure 3 M), and does not affect mouse weight (M), Figure 3N). Immunohistochemistry (IHC) analysis further showed that Ki-67 and PCNA (proliferation markers) expression was increased in tumors of the EV-treated group, while cleaved caspase-3 (apoptosis marker) expression was decreased. Figure 3 O). In summary, this study shows that S. anginosus EVs are internalized by gastric cancer cells via a dynein-dependent endocytosis pathway, thereby promoting their malignant phenotype and accelerating tumor growth in vivo.

[0049] 3. S. anginosus Extracellular vesicles drive gastric cancer immune escape through ZDHHC11-mediated PD-L1 stability. To elucidate the molecular mechanism by which *Streptococcus pharyngis* extracellular vesicles (Sa-EVs) promote gastric cancer progression, we performed RNA sequencing on AGS cells treated with Sa-EVs or PBS. Gene Ontology (GO) analysis showed that Sa-EV treatment significantly enriched pathways related to protein palmitoylation, palmitoyltransferase activity, and peptidyl-cysteine ​​modification. Figure 4 A). Volcano plot analysis screened out several candidate genes, among which palmitoyltransferase ZDHHC11 showed significantly increased expression after Sa-EVs treatment ( Figure 4 B). Subsequent qRT-PCR validated the upregulation of ZDHHC11 in multiple gastric cancer cell lines (AGS, MKN1, MKN45, YTN16). Figure 4 C). Given that palmitoylation is a key post-translational modification, we further identified potential downstream targets of ZDHHC11. Immunoprecipitation-mass spectrometry analysis suggested a potential interaction between ZDHHC11 and PD-L1. Figure 4 D), this result was further confirmed by immunoprecipitation (IP) assay. Figure 4 E). Molecular docking analysis predicts that the two have a high binding affinity, with a binding energy of -25 kcal / mol (E). Figure 4 F). Furthermore, previous studies have confirmed that ZDHHC3 and ZDHHC9 are responsible for PD-L1 palmitoylation in colorectal cancer and lung adenocarcinoma, respectively. Therefore, we investigated the effects of Sa-EVs on these two enzymes, but qRT-PCR results showed no change in their expression (F). Figure 5 A). Based on the above findings, we hypothesize that Sa-EVs specifically regulate PD-L1 through ZDHHC11. Functional experiments showed that overexpression of ZDHHC11 increased PD-L1 protein levels, as confirmed by Western blotting. Figure 4 G and Figure 5 B) and immunofluorescence ( Figure 4H) confirmed this. Furthermore, ZDHHC11 overexpression prolonged the half-life of PD-L1 in MKN1 and MKN45 cells ( Figure 4 I and Figure 5 C), and reduces its ubiquitination in a dose-dependent manner (C) Figure 4 J). Conversely, doxycycline (Dox)-induced knockdown of ZDHHC11 enhanced PD-L1 ubiquitination in AGS and MKN45 cells. Figure 5 D). These results suggest that ZDHHC11 stabilizes PD-L1 by inhibiting ubiquitination degradation.

[0050] To investigate whether Sa-EVs regulate PD-L1, we first examined mouse tumor tissues. Immunofluorescence showed that PD-L1 expression was significantly upregulated after administration of Sa-EVs. Figure 4 K and Figure 5 E). Similarly, Sa-EVs treatment also increased PD-L1 protein levels in MKN1 cells (E). Figure 4 L). Notably, the upregulation of PD-L1 protein levels was not accompanied by changes in its mRNA expression (L). Figure 4 M and Figure 5 F), suggesting that its regulation occurs at the post-transcriptional level. Consistent with this, cycloheximide (CHX) tracking experiments showed that Sa-EVs prolonged the half-life of PD-L1 (F). Figure 4 N and Figure 5 G). Ubiquitination experiments further showed that Sa-EVs treatment reduced PD-L1 degradation ( Figure 4 O and Figure 5 H) together supported the enhanced stability of PD-L1. Given the immunosuppressive function of PD-L1, we used the OT-1 co-culture system to assess its biological effects ( Figure 4 P). YTN16 cells pretreated with Sa-EVs significantly attenuated CD8. + T cell cytotoxicity ( Figure 4 Q), and inhibited the secretion of IFN-γ and IL-2, a result obtained by ELISA ( Figure 4 R) and qRT-PCR ( Figure 4 S) was verified. In summary, *Streptococcus pharyngitis* EVs enhance PD-L1 stability by upregulating ZDHHC11, thereby inhibiting CD8. + T cell function ultimately drives immune escape from gastric cancer.

[0051] 4. S. anginosus Extracellular vesicles promote ZDHHC11-mediated PD-L1 palmitoylation and stability. Given the crucial role of ZDHHC11 as a palmitoyltransferase, we investigated... S. anginosus Does extracellular vesicle regulate PD-L1 palmitoylation? Liquid chromatography-mass spectrometry (LC-MS) analysis showed that PD-L1 is palmitoylated at the Cys272 site, and... S. anginosus After treatment with EVs, their palmitoylation level may increase ( Figure 6 A and Figure 6 B). This result was further validated in acyl-biotin exchange (ABE) experiments, and it was found that the palmitoylation inhibitor 2-bromopalmitate (2-BP) could reverse this effect. Figure 6 C). At the protein expression level, immunofluorescence showed that the palmitoylation inducer palmostatin B and EVs synergistically enhanced PD-L1 (C). Figure 6 D), while 2-BP inhibited PD-L1 upregulation induced by EVs ( Figure 6 E). More importantly, in doxycycline (Dox)-induced ZDHHC11 knockdown cell lines, S. anginosus EVs could no longer induce PD-L1 upregulation, confirming the ZDHHC11 dependence of this process. Figure 6 F). Additionally... S. anginosus EVs reduced PD-L1 ubiquitination in a ZDHHC11-dependent manner, indicating that ZDHHC11-mediated palmitoylation enhances PD-L1 stability. Figure 6 G). Functional experiments using the OT-1 co-culture system showed that... S. anginosus YTN16 cells pretreated with EVs significantly inhibited CD8. + T cell cytotoxicity, an effect that can be rescued by treatment with 2-bromopalmitic acid (2-BP). Figure 6 H). Consistently, ELISA and qRT-PCR showed S. anginosus EVs inhibited the production of IFN-γ and IL-2, and these effects were reversible upon administration of 2-BP. Figure 6 I and Figure 6 J). In summary, these data reveal the presence of Streptococcus pharyngitis. S. anginosus EVs promote PD-L1 palmitoylation mediated by ZDHHC11, which facilitates its stabilization and ultimately leads to immune escape.

[0052] 5. S. anginosus EV-derived SNF2 family proteins synergistically enhance ZDHHC11 transcription with the transcription factor TEAD1. To identify potential transcription factors regulating ZDHHC11, we used the JASPAR database for prediction. Analysis showed that, based on binding affinity scores, TEAD1, MEF2A, and IRF1 were the top-ranked candidate transcription factors. Figure 7 A). Functional validation in gastric cancer cell lines showed that only TEAD1 overexpression significantly upregulated ZDHHC11 mRNA levels in a dose-dependent manner, while MEF2A and IRF1 did not have this effect. Figure 7 B, Figure 8 A and Figure 8 B). Pan-cancer analysis using GEPIA showed that TEAD1 expression was elevated in gastric adenocarcinoma (STAD) tumors compared to normal tissue, and exhibited different patterns in other cancers (B). Figure 8 C). Notably, based on TCGA data, high expression of TEAD1 or ZDHHC11 is associated with poor survival in gastrointestinal cancers (STAD in gastric cancer, COAD in colon cancer, and READ in rectal adenocarcinoma). Figure 8 D and Figure 8 E). Furthermore, in the TCGA dataset, TEAD1 expression was positively correlated with ZDHHC11 in gastrointestinal cancers (STAD gastric cancer, COAD colon cancer, READ rectal adenocarcinoma, ESCA esophageal cancer, and LIHC hepatocellular carcinoma). Figure 8 F). Furthermore... S. anginosus EV administration increased ZDHHC11 expression but did not alter TEAD1 expression, which was confirmed by IHC staining. Figure 8 G. Chromatin immunoprecipitation (ChIP) experiments further confirmed that TEAD1 can directly bind to five specific sites in the ZDHHC11 promoter region (G). Figure 7 C and Figure 7 (D), indicating that TEAD1 is a transcriptional regulator of ZDHHC11.

[0053] Next, to explore S. anginosus To understand the specific mechanism by which EVs regulate ZDHHC11, we used 4D label-free quantitative proteomics to characterize its protein composition and categorized it into multiple functional classes, including ribosomal proteins, ABC transporters, GTPases, transcription factors, and kinases. Figure 7 E). Among the 12 potential transcriptional regulators identified, the chromatin remodeling protein SNF2 is one of the most abundant components. Figure 7 F). Structural comparison shows, S. anginosus The derived SNF2 shares 55% similarity with human BRG1 in the ATP-binding domain. Figure 8H). Immunoprecipitation and proximity ligation assays (PLA) confirmed that... Sa -SNF2 can interact with TEAD1 in host cells ( Figure 7 G and Figure 7 H). Molecular docking and kinetic simulations further support the formation of a stable complex (binding energy: -10 kcal / mol; RMSF < 10 Å; RMSD equilibrium at ~11.2 Å). Figure 7 I, Figure 7 J and Figure 8 I). Functionally, Sa -SNF2 overexpression can increase the mRNA level of ZDHHC11 in various gastric cancer cells in a dose-dependent manner. Figure 7 K and Figure 8 J), and enhances TEAD1 recruitment on the ZDHHC11 promoter (J). Figure 7 L). Consistently, Sa -SNF2 can upregulate the expression of ZDHHC11 and PD-L1 proteins ( Figure 7 M and Figure 8 K), and this effect was reversed after ZDHHC11 was knocked down ( Figure 7 N and 8L). Acyl-biotin exchange (ABE) experiments showed that... Sa -SNF2-mediated PD-L1 palmitoylation is ZDHHC11-dependent. Figure 7 O), and can inhibit PD-L1 ubiquitination in a ZDHHC11-dependent manner ( Figure 7 P and Figure 8 M). In the OT-1 co-culture system Sa The immune escape phenotype driven by SNF2 (i.e., decreased CD8⁺ T cell cytotoxicity and reduced IFN-γ and IL-2 secretion) can be rescued by the palmitoylation inhibitor 2-BP. Figure 7 In summary, this study elucidates a novel immune regulatory pathway driven by pathogen vesicles: S. anginosus The SNF2 protein carried by EVs binds to the host transcription factor TEAD1 and synergistically activates the expression of ZDHHC11, thereby promoting the palmitoylation and stability of PD-L1, ultimately leading to immune escape.

[0054] 6. S. anginosus EV-derived SNF2 regulates TEAD1-mediated transcription and BAF complex assembly through its conserved ATP-binding site. To investigate the functional conservation of bacterial SNF2 protein, we first compared its performance in various bacteria associated with gastric cancer (including Helicobacter pylori). Helicobacter pylori Fusobacterium nucleatum Fusobacterium nucleatum and melanin-producing Prevotella Prevotella melaninogenica The results showed that the full-length sequence similarity of SNF2 was low (<40%), indicating significant heterogeneity in its overall structure. Figure 9 A). However, we identified a region that is highly conserved across all species – the ATP-binding motif D_MGLGKT ( Figure 10 A). To verify the function of this motif, we constructed an SNF2 mutant (SNF2-A8 mut) in which the motif was replaced with alanine. Structural simulations performed using PyMOL clearly revealed the 3D structures of wild-type SNF2 (SNF2-WT), its ATP-binding site, and the mutant (SNF2-mut). Figure 10 B). Functional experiments confirmed that overexpression of wild-type SNF2 (SNF2-WT) significantly enhanced the transcription of ZDHHC11 in various gastric cancer cells (MKN1, AGS, and YTN16 cells), while this ability was completely lost in the mutant (SNF2-mut). Figure 10 C and Figure 9 B). Consistent with this, ChIP experiments showed that only SNF2-WT (not SNF2-mut) promoted the enrichment of TEAD1 on the ZDHHC11 promoter. Figure 10 D). Given that TEAD1 is a core transcription factor in the Hippo signaling pathway, we further examined... Sa -Does SNF2 regulate other downstream target genes of TEAD1? Results showed that TEAD1 overexpression upregulated multiple genes, including AXL, CTGF, CYR61, SNAI1, and SNAI3. Figure 10 E), this regulatory relationship is supported by correlations in the GEPIA database (TEAD1 is positively correlated with these downstream genes). Figure 9 C). Importantly, SNF2-WT (rather than SNF2-mut) also activates these genes, further demonstrating that their function depends on intact ATP-binding sites. Figure 10 F). Furthermore... Sa -SNF2-driven oncogenic phenotypes such as cell growth, colony formation, migration, and invasion were significantly reversed after AXL was silenced. Figure 10 G–10J, Figure 9 The above results indicate that... Sa -SNF2 is crucial for TEAD1-mediated activation of pro-tumor gene transcription through its conserved ATP-binding site.

[0055] Given that the BAF (BRG1 / BRM-associated factors) complex is a multi-protein chromatin remodeling mechanism in eukaryotic cells and is crucial for regulating transcriptional programs ( Figure 10 K), we discussed Sa Does SNF2 affect the assembly of the BAF complex? Gel filtration chromatography analysis showed that SNF2-WT enhanced the co-elution of core components such as BAF47, BAF60A, and ARID1A, while this phenomenon was not observed in the SNF2-mut group. Figure 10 L). These findings suggest Sa -SNF2 acts as a regulator of BAF complex kinetics in human cells.

[0056] 7. S. anginosus SNF2 derived from this source can serve as a tumor marker and target... Sa The -SNF2–ZDHHC11 axis can inhibit tumor growth and synergize with anti-PD-1 therapy. To investigate the carcinogenic effect of Sa-SNF2 in vivo, we subcutaneously injected YTN16 cells stably overexpressing pLVX-SNF2-HA into C57BL / 6 mice to establish a xenograft mouse model. Figure 11 A). The results showed that, compared with the pLVX-empty vector control group, Sa-SNF2 overexpression significantly increased tumor volume (A). Figure 11 B and Figure 11 C) and weight ( Figure 11 D). Using hybridoma technology, we prepared a monoclonal antibody against Sa-SNF2 ( Figure 12 A–12D was confirmed by immunohistochemical (IHC) staining of tissue sections, demonstrating its sensitivity and specificity. IHC analysis showed significantly enhanced signaling of both the HA tag and Sa-SNF2 in the overexpression group. Figure 11 E). Furthermore, tumors overexpressing Sa-SNF2 exhibited enhanced Ki-67 staining (a proliferation marker), decreased cleaved caspase-3 levels (an apoptosis marker), and upregulation of ZDHHC11 and PD-L1. Figure 11 E). These results indicate that SNF2 derived from S. anginosus EV promotes gastric cancer progression by activating the ZDHHC11–PD-L1 axis.

[0057] To assess its clinical significance, we performed IHC analysis on a tissue microarray (TMA) of 141 clinical gastric cancer and adjacent tissues. The results showed that tumor tissue contained… Sa -SNF2 expression was significantly higher than that in adjacent normal tissues ( Figure 11 F), and its high expression was associated with poorer overall survival and disease-free survival in patients (F). Figure 11 G), High, Low Sa The clinical characteristics of patients in the -SNF2 expression group are detailed in Table 1.

[0058] Table 1 Sa -Correlation between SNF2 expression and clinicopathological features of 141 gastric cancer cases

[0059] Next, we discussed Sa The role of the -SNF2–ZDHHC11 axis in the tumor immune microenvironment. Analysis of single-cell RNA sequencing (scRNA-seq) data from the TISCH database (http: / / tisch.comp-genomics.org / ) showed that in GC tissues, ZDHHC11 expression was higher in epithelial cells and malignant cells than in tumor-infiltrating lymphocytes (TILs). Figure 11 H). Using TIMER2.0 (<http: / / timer.cistrome.org / > In another scRNA-seq dataset, we observed a significant negative correlation between ZDHHC11 expression and CD8+ T cell infiltration. Figure 11 (I) suggests that ZDHHC11 may contribute to the formation of an immunosuppressive microenvironment. This finding is further supported by previous IHC staining of tumor sections, which showed that after gavage... S. anginosus , Sa- EVs or Sa Tumors with SNF2 overexpression significantly reduced CD8 expression. + T cell infiltration ( Figure 11 J), confirmed Sa- The SNF2–ZDHHC11 axis plays a crucial role in shaping the immunosuppressive microenvironment.

[0060] To evaluate the effect of ZDHHC11 inhibition combined with antiPD-1 therapy S. anginosus To investigate the effect of EVs on tumorigenesis, we treated tumor-bearing mice with a combination of 2-bromopalmitic acid (2-BP) and anti-PD-1 antibody. Figure 11 K). 2-BP treatment effectively reversed the effect caused by S. anginosus EVs induced tumor growth, accompanied by decreased levels of Ki-67, ZDHHC11, and PD-L1, and increased infiltration of cleaved caspase-3 and CD8⁺ T cells. Figure 11 More importantly, the combination therapy of 2-BP and anti-PD-1 antibody showed stronger tumor suppression and T-cell infiltration, with significantly better efficacy than monotherapy. Figure 11 O). In summary, this study clarifies S. anginosusSNF2 derived from EVs drives both tumor proliferation and immune escape by activating the ZDHHC11–PD-L1 pathway. Targeting this axis not only inhibits tumor growth but also synergizes with existing immune checkpoint blockade therapies, providing a promising combination strategy for gastric cancer treatment.

[0061] This study is the first to systematically elucidate that extracellular vesicles derived from *Streptococcus pharyngis* can be internalized by gastric cancer cells via a dynein-mediated endocytosis pathway, and subsequently... Sa The SNF2–ZDHHC11–PD-L1 signaling axis drives tumor progression and promotes immune escape. Conversely, blocking this pathway can effectively inhibit tumor growth and enhance tumor sensitivity to immune checkpoint blockade therapy.

[0062] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Streptococcus pharyngitis exovesicle endocytosis inhibitors in the preparation of gastric cancer for prevention or treatment of Streptococcus pharyngitis infection.

2. The endocytosis inhibitor of Streptococcus pharyngitis exovesicles is a dynein inhibitor.

3. The application as described in claim 2, characterized in that, The dynoprotein inhibitor is dynasore.

4. Application of quantitative detection reagents for SNF2 derived from Streptococcus pharyngitis exovesicles in the preparation of prognostic products for patients with gastric cancer infected with Streptococcus pharyngitis.

5. The application as described in claim 4, characterized in that, SNF2 originates from extracellular vesicles of Streptococcus pharyngitis.

6. The application as described in claim 4, characterized in that, The quantitative detection reagents for SNF2 are selected from reagents for the quantitative detection of SNF2 protein prepared based on enzyme-linked immunosorbent assay, Western blotting, and immunohistochemistry.

7. The application as described in claim 6, characterized in that, The quantitative detection reagent for SNF2 is a reagent for the quantitative detection of SNF2 protein prepared by immunohistochemistry.

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