Application of detection of enterococcus faecalis in liver cancer tissue in treatment of liver cancer

Abstract

The invention provides application of detection of enterococcus faecalis in liver cancer tissue in treatment of liver cancer. The invention finds that enterococcus faecalis is highly abundant in liver tumor tissues and is positively correlated with the pathogenesis of HCC (Hepatocellular Carcinoma). Enterococcus faecalis or an enterococcus faecalis conditioned medium promotes liver cancer cell proliferation, protein translation, cell migration and tumorigenesis. Animal experiments prove that colonization of enterococcus faecalis can promote in-vivo growth of liver cancer, and everolimus (an mTOR pathway inhibitor) administration can significantly inhibit the promotion effect. The colonization amount of the enterococcus faecalis can be used as a marker of a liver cancer clinical treatment strategy of colonization of the enterococcus faecalis.

Description

Technical Field

[0001] This invention relates to the field of cancer treatment, and in particular to the application of the detection of Enterococcus faecalis in liver cancer tissue in the treatment of liver cancer. Background Technology

[0002] Liver cancer (HCC) is one of the three leading causes of cancer death worldwide, with the highest mortality rate. Emerging data suggests that the gut microbiome plays a crucial role in the development and progression of cancer, including HCC. The gut microbiome can influence HCC through the gut-liver axis. Disruption of the gut barrier, alterations in bacterial metabolites, and chronic liver inflammation are associated with HCC progression.

[0003] The development of liver cancer is a complex, multi-step, and multifactorial process. Enterococcus faecalis is an important piece of the "puzzle," but by no means the sole cause. The tumor-resident microbiome in liver cancer is becoming increasingly important, but its influence is not yet fully characterized. We have discovered that Enterococcus faecalis is a resident microbiome in liver tumors. However, it remains unclear how tumor-resident Enterococcus faecalis affects tumor biological function or the tumor microenvironment. Extracellular vesicles (EVs) are well-known natural intercellular messengers involved in intra- and inter-species communication. EVs encapsulate many cell-specific molecules, such as proteins, DNA, RNA, peptidoglycans, and lipids. Tumor-colonized Enterococcus faecalis may utilize EVs to influence the biological activity of liver cancer cells. The EVs of tumor-resident Enterococcus faecalis have not yet been clearly characterized. For example, mechanistic studies of EV proteomics may reveal how Enterococcus faecalis-derived EVs influence tumorigenesis to promote liver tumor progression. Summary of the Invention

[0004] In this invention, we found that *Enterococcus faecalis* (EF) is highly abundant in liver tumor tissues and positively correlated with the pathogenesis of hepatocellular carcinoma (HCC). *E. faecalis* conditioned medium (EF-CM) promotes HCC cell proliferation, protein translation, cell migration, and tumorigenesis. Mechanistically, we found that EF-derived extracellular vesicles (EF-EVs) deliver the EF-Obg GTPase to activate host mTOR, thereby promoting HCC progression. The EF-Obg protein affects the mTOR pathway through its Ras-like G domain involved in GTP binding. EF-Obg and Rheb share striking homology at the G1 site of their G domains, and Rheb is a key positive regulator of mTOR. The *Enterococcus faecalis* Obg gene is crucial for *Enterococcus faecalis* to activate mTOR and promote HCC development; an engineered *Enterococcus faecalis* strain with Obg knockdown was developed based on the CRISPRi assay. Clinically, abundant EF-Obg protein expression is associated with enhanced mTOR activation, leading to poorer overall survival in HCC patients. Notably, treatment with the mTOR inhibitor everolimus was effective in an orthotopic model of Enterococcus faecalis colonized hepatocellular carcinoma, indicating that everolimus treatment is effective for hepatocellular carcinoma patients rich in Enterococcus faecalis.

[0005] In this study, we identified *Enterococcus faecalis* as a tumor-resident bacterium. *Enterococcus faecalis* can promote the growth of hepatocellular carcinoma cells. EF-EVs carry key molecules involved in mTOR signaling. Mechanistically, we found that the *Enterococcus faecalis* Obg GTPase (EF-Obg), delivered via extracellular vesicles, is homologous to the G1 site of Rheb, thereby activating the mTOR signaling pathway. Knockdown of the *Enterococcus faecalis* Obg gene expression with CRISPRi impairs the oncogenic role of *Enterococcus faecalis* in HCC. EF-Obg expression levels were associated with poor overall survival in HCC patients. Importantly, the mTOR inhibitor everolimus significantly inhibited tumor growth in an orthotopic hepatocellular carcinoma model exposed to *Enterococcus faecalis* colonization. In summary, our study found that *Enterococcus faecalis* is an important tumor-resident microorganism involved in the activation of oncogenic signaling pathways in cancer cells, and validated the causal relationship between EF-EV-derived Obg-GTPase and mTOR activation during hepatocellular carcinogenesis. These studies provide insights into the diagnosis of HCC exposed to tumor-resident bacteria Enterococcus faecalis and reveal the therapeutic potential of targeting mTOR with intratumoral Enterococcus faecalis to treat HCC. Attached Figure Description

[0006] Figure 1The diagram shows the composition of resident bacteria in liver tumors and normal tissues. (A) shows the principal coordinate analysis (PCoA) based on unweighted UniFrac analysis of DNA extracted from resident bacteria in tumors and normal tissues, with each point representing a single sample; (B) shows the relative abundance of resident microbiota in tumors and normal tissues at the target level based on 16S sequencing; (C) shows the relative abundance of *E. casseliflavus* in liver tumor tissues and normal tissues; and (D) shows the Spearman correlation analysis of the -ΔCt value of *E. casseliflavus* in HCC patients with serum PIVKA_II and AFP levels.

[0007] Figure 2 Figure 1 shows the enrichment of Enterococcus faecalis in liver cancer tissue and its promotion of liver cancer cell proliferation; where (A) shows the microbiota extracted from liver tumor tissue and normal tissue under 16S staining. rRNA amplicon sequencing (n=19), Faith_pd index showed α-diversity of microbial community between the two groups; (B) and (C) Spearman correlation analysis between relative abundance of bacteria in liver tumor tissue and clinical parameters; (D) shows the abundance of Enterococcus faecalis in liver tumor tissue and normal tissue detected by qPCR; (E) shows the Spearman correlation analysis results of Enterococcus faecalis abundance and serum PIVKA_II or AFP levels in HCC patients; (F) shows the H&E staining results of Enterococcus faecalis in liver tumor tissue and normal tissue detected by fluorescence in situ hybridization (FISH); (G) shows the growth curve of HCC cells after EF or EF-CM treatment as measured by CCK8; (H) shows the colony formation results of HCC cells after EF or EF-CM treatment; (I) shows the angiogenesis experiment results of HUVEC cells; (J) shows the migration experiment results of HUVEC cells.

[0008] Figure 3 Figure 1 shows the results of Enterococcus faecalis activating the mTOR signaling pathway and promoting hepatocellular carcinoma tumorigenesis. (A) shows the overlapping cancer-related pathway enrichment in Hep3B cells after EF and EF-CM treatment; (B) shows the GSEA plot of the treated mTOR signaling pathway, displaying NES and adjusted P values; (C) shows a heatmap displaying the mRNA levels of genes related to the mTOR signaling pathway after the specified treatment; (D) shows the protein levels and phosphorylation levels of mTOR, P70S6K, and 4EBP1 in HCC cells after the specified treatment, quantified as red numbers; (E) shows the translational activity of HCC cells after the specified treatment as shown by Western blot analysis against puromycin; (F) shows the results of antibiotic treatment in C57BL / 6 mice after gavage administration of 1×10⁻⁶ mcg. 8CFU (Enterococcus faecalis) or PBS (PBS) control were administered every other day for 4 weeks. Mice were subcutaneously injected with 1×10⁻⁶ CFU / PBS one week after the start of gavage. 6 Hepa1-6 cells, tumor volume, tumor weight, and representative tumor images; (G) C57BL / 6 mice were orally administered EF-CM (200 μL) or BHI every other day for 4 weeks after antibiotic treatment; one week after starting tube feeding, mice were subcutaneously injected with 1×10 6 Hepa1-6 cells. Tumor volume, tumor weight, and representative tumor images; (H) are representative images of Ki-67, p-mTOR, p-P70S6K, p-4EBP1, HIF1α, and CD31 immunohistochemical staining of tumor samples after specified treatment. Quantitative IHC staining is shown as a bar graph (right).

[0009] Figure 4 The images show that EF-EVs delivered via dynein-dependent endocytosis can promote the proliferation of hepatocellular carcinoma cells and activate mTOR. (A) shows transmission electron microscopy (TEM) images of Enterococcus faecalis (cross-section) and secreted EVs (red circles); (B) shows representative TEM images of EF-EVs and nanoparticle tracking analysis (NTA); (C) shows the growth curve of HCC cells treated with EF-EVs measured by the CCK8 assay; (D) shows the colony formation results of HCC cells treated with EF-EVs; (E) shows the angiogenesis results of HUVEC cells treated with EF-EVs; (F) shows the migration results of HUVEC cells treated with EF-EVs; (G) shows the phosphorylation levels of mTOR, P70S6K, and 4EBP1 proteins in HCC cells treated with EF-EVs, quantified as red numbers; (H) shows MHCC-97h cells treated with 20 μg / mL... Confocal micrographs of DiO-labeled EF-EVs incubated for different times; (I) shows confocal micrographs of MHCC-97h cells pretreated with specified inhibitors (chlorpromazine 5uM, cytochalasin D 2.5uM, Dynasore 40uM) for 1 hour, followed by co-incubation with 20ug / mL DiO-labeled EVs for 120 minutes; (J) Quantitative results of EVs-positive cells;

[0010] Figure 5Figures showing the correlation between EF-EVs promoting hepatocellular carcinoma and inducing pathological conditions in mice; (A) is a schematic diagram of the experimental protocol for orthotopic liver tumors treated with EF-EVs: after antibiotic treatment, C57BL / 6 mice were gavaged with EF-EVs (50ug) or PBS control every other day for 4 weeks. One week after the start of gavage, tumor blocks of Hepa1-6 cells expressing luciferase were transplanted into the mouse liver; (B) is a representative fluorescence image of mice 90 minutes after gavage with Cy7-labeled EF-EVs (right) or PBS (left); (C) and (D) show the bioluminescent images of the whole body and ex vivo liver of mice at the end of the experiment; (E) shows... Images of livers collected from the EF-EVs and PBS groups are shown, with blue dashed lines indicating tumor boundaries; tumor area is shown as a bar chart (right); (F) is a representative image of H&E staining on liver tissue sections (left), with the tumor area (right) shown as a bar chart; (G) shows the quantification of serum ALT, ALP, AFP, AST, ALB levels and DBIL / TBIL ratio in both groups of mice; (H) shows representative images of Ki-67, p-mTOR, pP70S6K, p-4EBP1, HIF1α, and CD31 immunohistochemical staining in tumor samples; quantification of IHC staining is shown as a bar chart (bottom image);

[0011] Figure 6 This image depicts the altered gut microbiota environment in mice by Enterococcus faecalis extracellular vesicles (EF-EVs). (A) shows the 16S sequencing results of fecal matter from mice treated with either EF-EVs or PBS, with the Shannon index indicating α-diversity of the microbiota between the two groups. (B) represents the PCoA based on unweighted UniFrac analysis of fecal microbiota from both groups, with each point representing a single sample. (C) shows the relative abundance of fecal microbiota at the order level in samples from either EF-EVs or PBS-treated groups. (D) shows the relative abundance of fecal microbiota at the genus level in samples from either EF-EVs or PBS-treated groups. (E) is a heatmap showing the species distribution of fecal bacteria in samples from either EF-EVs or PBS-treated groups, with red boxes highlighting species significantly reduced in the EF-EVs group. (F) shows the taxa with the most significant differences between the two groups identified using linear discriminant analysis (LDA) (LDA>2 and p<0.05), with red boxes highlighting altered species reported to be associated with HCC.

[0012] Figure 7This indicates that EF-Obg, derived from EF-EVs, is a GTPase that interacts with mTOR. (A) shows the growth curve of MHCC97h cells after the corresponding treatment, measured using the CCK8 assay; (B) is the experimental design for identifying mTOR-bound proteins in EF-EVs, with four GTPases from EF-EVs identified by mTOR-IP mass spectrometry (right figure); (C) shows the protein structure and domains of GTPase-Obg visualized by PyMOL, with red arrows indicating GTP binding. (G1-G5) Sites; (D) Obg-mTOR interaction structure modeled by PyMOL; (E) Indication of in vitro co-IP assay to study the interaction between mTOR and Obg; protein products derived from the transcription and translation (TNT) system; (F) Indication of using HA-Obg-Myc exogenously expressed in 293T / MHCC-97H cells or using PET21a-HA-Obg-Myc expressed by bacteria (from E. coli BL21 cells) with Hep3B / MHC. Results of Co-IP assay using C-97H cell lysate mixture; (G) represents a representative image of the adjacent linkage assay (PLA) using anti-mTOR and Obg antibodies, with red signals indicating the interaction between mTOR and Obg, and cell nuclei stained with DAPI (blue signal); (H) shows the protein levels of phosphorylated mTOR, P70S6K, and 4EBP1 after overexpression of HA-Obg in the specified cells, quantified as red numbers; (I) shows the overexpression of Obg as measured by CCK8. (g) shows the growth curve of HCC cells; (J) shows representative tumor images, tumor volume, and tumor weight of C57BL / 6 female mice that were randomly divided and subcutaneously injected with Hepa1-6 cells overexpressing Obg (plvx-Obg) or control (plvx-vec); (K) shows representative images of tumor specimens with Ki-67, p-mTOR, pP70S6K, p-4EBP1, HIF1α, and CD31 immunohistochemical staining, and the quantitative IHC staining is shown as a bar graph (bottom image).

[0013] Figure 8 The image shows the results of proteomic analysis of extracellular vesicles of Enterococcus faecalis; (A) shows the results of Coomassie brilliant blue staining of protein bands of EF-EVs based on SDS-PAGE; (B) shows the KEGG annotation results of proteins identified from EF-EVs.

[0014] Figure 9 This indicates that the Obg gene is crucial for Enterococcus faecalis to activate mTOR and promote liver cancer development; (A) shows a comparison of the homology of GTP binding site 1 (G1) between Obg and Rheb; the aligned amino acid sequence in G1 is highlighted; (B) represents EF-Obg wild-type Obg. WTand the G1 site mutant Obg 8A Detailed nucleotide and amino acid sequences of the G1 site; (C) indicates the determination of mTOR and Obg by co-IP assay. WT Or Obg 8A Interactions between them; (D) indicates that the immunoblotting experiment showed Obg WT Or Obg 8A Phosphorylation level of mTOR after overexpression; (E) Schematic diagram of knockdown of Obg by CRISPR interference system; qPCR detection of Obg mRNA level in Enterococcus faecalis Obg gene knockdown strain (EF-dCas-Obg); (F) Volcano plot of RNA sequencing results showing gene expression difference in EF-dCas-Obg strain compared with EF-vec strain; the results indicate downregulation of Obg; (G) Growth curve of MHCC-97h cells corresponding to the treatment determined by CCK8 assay; (H) Immunoblotting experiment showing phosphorylation and total levels of mTOR, P70S6K, and 4EBP1 in HCC cells after the corresponding treatment; quantification is shown in red numbers (below); (I) Two weeks after antibiotic treatment, C57BL / 6 mice were orally administered EVs or PBS from EF-vec strain, EF-dCas-Obg strain, orally every other day for 3 weeks; one week after the start of gavage, mice were subcutaneously injected with 1×10 6 Hepa1-6 cells; representative tumor images, tumor volume and tumor weight; (J) are representative images of Ki-67, p-mTOR, pP70S6K, p-4EBP1, HIF1α and CD31 immunohistochemical staining of tumor specimens;

[0015] Figure 10 The results show the effects of engineered EF mutant strains (Obg gene knockdown strain, EF-dCas-Obg) on ​​growth rate, EV production, and biological function. (A) shows the calculated colony forming units (CFU) of EF-vec and EF-dCas-Obg strains after 12 hours of culture in BHI medium; (B) shows the nanoparticle tracking analysis results of EVs isolated from EF-vec and EF-dCas-Obg strains after normalization based on total bacterial content; (C) shows the protein concentration of EVs isolated from EF-vec and EF-dCas-Obg strains after normalization based on total bacterial content; (D) shows the gene ontology (GO) annotation results of differentially expressed genes between EF-vec and EF-dCas-Obg strains; and (E) shows the KEGG annotation results of differentially expressed genes between EF-vec and EF-dCas-Obg strains.

[0016] Figure 11The data indicates that the EF-dCas-Obg strain altered the composition of the mouse gut microbiota; (A) represents the Faith_pd index showing the α diversity of the microbiota in the three groups; (B) represents the PCoA of the bacterial communities in the three groups based on weighted UniFrac analysis, with each point representing a single sample; (C) represents the relative abundance of the mouse fecal microbiota at the order level in the three groups; (D) shows the distribution of various bacterial families in the feces of the three groups, with the altered bacteria—Ackermaniaceae and Enterococciaceae—highlighted in red boxes; (E) represents the taxa with the most significant differences between the two groups (LDA>3) identified using linear discriminant analysis effect size, with the increased Ackermania spp. highlighted in red boxes.

[0017] Figure 12 The results indicate that high expression of EF-Obg in liver cancer tissue is associated with low survival, and that the mTOR inhibitor everolimus can be used for targeted therapy. (A) shows the relative expression levels of EF-Obg in liver tumor and normal tissues as determined by qPCR; (B) shows the protein levels of EF-Obg and p-mTOR in liver tumor and normal tissues as shown by Western blotting; N represents normal tissue, and T represents liver tumor tissue; (C) shows the levels of EF-Obg (using anti-EF-Obg antibody) and p-mTOR in liver tumor and normal tissues. Representative images of immunohistochemical staining in tissues; quantitative IHC staining is shown as a bar graph (right); (D) shows representative IHC staining images, indicating high and low expression of EF-Obg in human hepatocellular carcinoma and normal tissues in tissue microarray (TMA); matched p-mTOR staining is shown in the right figure; (E) shows the Kaplan-Meier survival curves (N=100) for the Obg high expression group and the Obg low expression group; (F) shows the results of antibiotic treatment in C57BL / 6 mice after gavage administration of 1×10⁻⁶ ozontally. 8 CFU fecal pellets or PBS control were administered every other day for 5 weeks; Hepa1-6 tumor blocks expressing luciferase were transplanted into mouse livers one week after the start of gavage; mice were administered everolimus or control via gavage daily for one week after tumor implantation; (G) shows representative images of tumor H&E staining and tumor region quantification (right); (H) shows serum levels of ALT, AST, AFP, ALP, ALB and the DBIL / TBIL ratio; (I) shows representative images of tumor specimens with p-mTOR, p-P70S6K, p4EBP1, HIF1α and CD31 immunohistochemical staining; quantification of IHC staining is shown as a bar graph (right).

[0018] Figure 13This diagram illustrates the preparation of anti-Obg antibodies. (A) shows the anti-Obg antibody production process; (B) shows the peptide sequence compared to the Obg immunogen; (C) shows the animal immunization and titer detection process; (D) shows the non-reduced (left) and reduced (right) SDS-PAGE electrophoresis results of the purified antibody; (E) shows a representative image of immunohistochemical staining of EF-Obg using the prepared anti-Obg antibody; IHC staining quantitative results are shown in a bar chart (below), scale bar = 25 μm. Detailed Implementation

[0019] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0020] 1. Enterococcus faecalis accumulates in liver cancer tissue and can promote the proliferation and migration of liver cancer cells. To investigate the role of colonizing microorganisms in liver tissue during the progression of hepatocellular carcinoma (HCC), we analyzed the microbial community composition of tumor tissue and adjacent normal tissue (as normal tissue) using 16S rRNA sequencing. Although principal coordinate analysis (PCoA) showed no significant differences in bacterial composition ( Figure 1 A and Figure 1 B), but we found reduced α diversity in liver tumor tissue (B), Figure 2 A). Furthermore, we observed that the abundance of several microbial phyla in hepatocellular carcinoma tissue correlated with clinical parameters. Notably, the abundance of Firmicutes_D was positively correlated with vitamin K deficiency or antagonist-induced protein-II (PIVKA_II), a tumor biomarker for hepatocellular carcinoma. The abundance of Enterococci, belonging to the Firmicutes_D phylum, was positively correlated with PIVKA_II and aspartate aminotransferase (AST) levels. Figure 2 B and Figure 2 (C) This indicates that Enterococci may be involved in promoting liver cancer. In practical applications, any commercially available product for detecting Enterococci can be used as a detection reagent for Enterococci, such as 16S rRNA gene sequencing reagents. The principle is to amplify the 16S rRNA gene and sequence it, then compare the sequence with a database for identification. Required reagents: universal 16S primers, PCR reagents, and sequencing library preparation kits. Whole-genome sequencing (WGS) works by sequencing the entire bacterial genome, providing the most comprehensive information for tracing the origin of bacteria and analyzing virulence and resistance genes. Required reagents: genomic DNA extraction kits and whole-genome sequencing library preparation kits.

[0021] To further define specific Enterococcal species associated with hepatocellular carcinoma, we used real-time PCR to quantify the abundance of several well-studied species, namely... E. casseliflavus , E.faecalis And tissue samples (n=40). The results showed that in all samples, E.faecium and E.mundtiwitin Almost undetectable. Enterococci are highly enriched in tumor tissue compared to normal tissue. Figure 2 D), and E. casseliflavus There was no significant difference. Figure 1 C). Further correlation analysis showed that Enterococcus faecalis, rather than... E. casseliflavus Abundance was significantly correlated with PIVKA_II / α-fetoprotein (AFP) levels. Figure 2 E and Figure 1 D). Under strictly aseptic conditions, the colonization of tumor-resident Enterococcus faecalis was confirmed by fluorescence in situ hybridization (FISH) and Enterococcus faecalis species-specific probes. Figure 2 F). In summary, these data suggest that Enterococcus faecalis is an enriched tumor-resident microbe in liver tumor tissue and is correlated with clinical parameters of HCC.

[0023] To determine the potential carcinogenic role of Enterococcus faecalis in human hepatocellular carcinoma (HCC), we performed co-culture experiments (infection) in a series of HCC cell lines to assess tumor cell growth. CCK8 and colony formation assays showed that Enterococcus faecalis infection at an MOI of 30 promoted HCC cell proliferation and colony formation. Figure 2 G and Figure 2 H). Enterococcus faecalis also promotes angiogenesis and migration of vascular endothelial cells (HUVECs). Figure 2 I and Figure 2 J). Since bacterial metabolites and secreted proteins may play an important role in these phenomena, we prepared a conditioned medium containing Enterococcus faecalis metabolites and secreted proteins (hereinafter referred to as EF-CM) to examine its effects on growth / migration. In fact, EF-CM also promoted the proliferation of HCC cells as well as angiogenesis and migration of HUVEC cells. Figure 2 G to Figure 2 These results indicate that Enterococcus faecalis and its conditioned medium can promote HCC cell growth and HUVEC cell migration.

[0024] 2. Enterococcus faecalis activates the mTOR signaling pathway in liver cancer and promotes HCC tumorigenesis.

[0025] To elucidate the exact mechanism of the effects of Enterococcus faecalis, we performed RNA sequencing analysis on Hep3B cells co-cultured with Enterococcus faecalis or treated with EF-CM. Gene set enrichment analysis (GSEA) showed that, compared with the control group, the mTOR pathway was enriched in both the Enterococcus faecalis infection group and the EF-CM treatment group. Figure 3 A and Figure 3B). Heatmap results showed that most mTOR downstream genes were upregulated after Enterococcus faecalis infection or EF-CM treatment. Figure 3 C). Immunoblotting experiments showed that both Enterococcus faecalis infection and EF-CM treatment could enhance the phosphorylation of mTOR and its downstream effectors (such as P70S6K and 4EBP1). Figure 3 D). Furthermore, anti-purinemycin immunoblotting analysis confirmed increased protein translation activity after treatment with Enterococcus faecalis or EF-CM. Figure 3 E). In summary, these findings suggest that Enterococcus faecalis and EF-CM activate the mTOR signaling pathway and promote protein synthesis.

[0026] To further characterize the functional role of Enterococcus faecalis in the development of hepatocellular carcinoma (HCC) in vivo, we conducted a subcutaneous tumorigenesis experiment using mouse Hepa1-6 hepatocellular carcinoma cells in C57BL / 6 mice. To eliminate the influence of the mice's own gut microbiota, the mice were fed antibiotics (doripene and vancomycin) for 2 weeks before transplantation of cancer cells. They were then treated with Enterococcus faecalis or EF-CM via gavage every other day. Figure 3 F and Figure 3 G), and measured mouse body weight and tumor volume until the end of the experiment. The results showed that treatment with Enterococcus faecalis or EF-CM promoted subcutaneous tumor growth in mice, resulting in larger tumor volume, increased tumor weight, and denser tumor vascular networks. Figure 3 F and Figure 3 G). Immunohistochemical staining showed a significant increase in the number of Ki-67 positive cells in tumors treated with Enterococcus faecalis and EF-CM, and also elevated levels of p-mTOR and its downstream phosphorylated proteins (including p-P70S6K, p-4EBP1, HIF1α, and CD31). Figure 3 In summary, these results indicate that Enterococcus faecalis can activate the mTOR pathway to promote liver cancer progression.

[0027] In this invention, the presence of Enterococcus faecalis in liver cancer tissue is sufficient to classify it as Enterococcus faecalis colonization-type liver cancer. Since Enterococcus faecalis can promote liver cancer development through mTOR, the presence of Enterococcus faecalis in liver cancer tissue allows for the use of mTOR inhibitors to suppress liver cancer development, thereby achieving the therapeutic effect on liver cancer.

[0028] 3. Extracellular vesicles of Enterococcus faecalis delivered via dydrin-dependent endocytosis can promote cell proliferation and activate the mTOR pathway in liver cancer cells.

[0029] Bacterial extracellular vesicles (EVs) are considered important mediators of bacterial-host interactions. Many spherical EVs can be observed on the surface of *Enterococcus faecalis* using transmission electron microscopy (TEM). Figure 4A). These EVs were separated by ultracentrifugation. Scanning electron microscopy and nanoparticle tracking analysis (NTA) showed that the separated EVs were mainly spherical vesicles with a diameter of approximately 160 nm, surrounded by a double membrane. Figure 4 B), which is consistent with the typical characteristics of EVs from Gram-positive bacteria. We hypothesize that Enterococcus faecalis EVs (hereinafter referred to as EF-EVs) play an important role in promoting HCC growth. To test this hypothesis, we treated HCC cells with EF-EVs at a concentration of 50 μg / mL. CCK8 and colony formation assays confirmed that EF-EVs can promote HCC cell proliferation. Figure 4 C and 4D). Furthermore, EF-EVs can also promote angiogenesis and migration of HUVEC cells (C and 4D). Figure 4 E and 4F). Immunoblotting assays showed that EF-EV treatment increased phosphorylation of mTOR and its downstream proteins P70S6K and 4EBP1 in HCC cells. Figure 4 G). These findings suggest that EF-EVs are potential effectors mediating mTOR activity in HCC.

[0030] EVs encapsulated in a lipid bilayer typically enter cells via endocytosis. To explore the specific mechanism of EF-EV endocytosis in HCC cells, we labeled EF-EVs with the lipophilic membrane dye 3,3'-dioctadecanocyanine perchlorate (DiO), which emits green fluorescence after entering the cell membrane. Treatment of MHCC-97h cells with DiO-labeled EF-EVs revealed significant green fluorescence within the cells after 2 hours of immunofluorescence, confirming that the EVs had been endocytosed. Figure 4 H). To further investigate the specific endocytic pathways that EVs depend on, we used inhibitors targeting different endocytic pathways: chlorpromazine (an inhibitor of clathrin-mediated endocytosis), Dynasore (an inhibitor of dynein-mediated endocytosis), and cytochalasin D (an inhibitor of actin-mediated endocytosis). Immunofluorescence results showed that Dynasore treatment significantly inhibited endocytosis of EF-EVs, while the other inhibitors had no significant inhibitory effect. Figure 4 I and Figure 4 Therefore, EF-EVs enter HCC cells via dynein-dependent endocytosis to exert their effects.

[0031] 4. EF-EVs promote liver cancer development in mice by stimulating mTOR signaling.

[0032] To verify the in vivo effects of EF-EVs, we established an orthotopic hepatocellular carcinoma model in C57BL / 6 mice. After two weeks of antibiotic pretreatment, the mice were randomly divided into two groups, receiving either EF-EVs or PBS by gavage every other day until the end of the experiment. Figure 5 A). To track EF-EVs in vivo, we pre-labeled EF-EVs with the fluorescent dye Cy7 and captured fluorescence images 90 minutes after gavage. The images showed that EF-EVs could be distributed in the mouse liver. Figure 5 B). Administration of EF-EVs promoted HCC progression in mice, as evidenced by stronger bioluminescent signals in the liver and larger in situ tumor volume. Figure 5 C- Figure 5 F). Furthermore, elevated levels of blood biochemical markers ALT, AST, and ALP indicate more severe liver damage. Figure 5 G). Immunohistochemical staining results also showed that EF-EVs gavage increased the levels of p-mTOR, p-P70S6K, p-4EBP1, HIF1α, and CD31 in tumor tissue, as well as the increase in Ki-67 positive cells. Figure 5 These results indicate that EF-EVs can promote HCC progression by activating the mTOR pathway.

[0033] To further investigate the effects of EF-EVs on the gut microbiota in mice, we collected mouse feces and performed 16S rRNA analysis. EF-EV treatment significantly reduced the overall abundance of the gut microbiota. Figure 6 A). Furthermore, the composition of the gut microbiota changed after EF-EV treatment ( Figure 6 B) Changes in the relative proportions of different bacterial populations may affect the balance and function of the gut microbiota. Figure 6 C and Figure 6 D). Notably, EF-EV treatment reduced the colonization of certain probiotics, such as Akkermansia, butyrate-producing Clostridium, and Bifidobacterium, bacteria known for their beneficial effects on gut health. In contrast, EF-EV treatment promoted the colonization of Enterococcus faecalis itself, as well as some harmful bacteria, such as Clostridium, Streptococcus, and Veillonella. Figure 6 E and Figure 6 F). These findings suggest that EF-EV treatment may have complex effects on the gut microbiota, potentially disrupting the balance between beneficial and harmful bacteria and altering the entire gut ecosystem. Such alterations could have significant implications for gut health and the host's immune response, thereby affecting liver tumorigenesis.

[0034] 5. The GTPase Obg derived from EF-EVs can interact with mTOR.

[0035] Bacterial-derived proteins, DNA, and RNA can interact with host cell molecules. To further identify effector molecules in EF-EVs, we treated MHCC-97h cells with EF-EVs that had been pretreated with nucleases to consume DNA and RNA, or heat-inactivated to denature proteins. Notably, heat-inactivated EVs lost their ability to promote HCC cell proliferation, while nuclease-treated EVs remained active. Figure 7 A). Based on these findings, we hypothesize that EF-EVs exert their carcinogenic effects through proteins. We then analyzed the protein composition of EF-EVs using proteomics and found that some enriched proteins were associated with ribosomes implicit in protein synthesis. Figure 8 A and 8B).

[0036] To further explore the regulatory mechanism of EF-EVs-mediated mTOR activation, we designed an mTOR co-IP mass spectrometry experiment. We overexpressed Flag-mTOR in HEK293T cells and then used anti-FlagM2 beads to pull mTOR-related proteins from EF-EV lysates. The eluted proteins were identified by mass spectrometry. A total of 547 proteins and peptides derived from EF-EVs were identified. Figure 7 B). We searched for any proteins that might activate mTOR. Considering that the GTPase Rheb belongs to the Ras family and activates mTORC1 located in lysosomes in mammalian cells, we hypothesized that similar GTPase proteins from EF-EVs might play a Rheb-like role in regulating the mTORC1 pathway to promote HCC cell growth. Several GTPase proteins from EF-EVs were identified as mTOR-related proteins. Among them, the spo0B-related GTP-binding protein (Obg) was particularly noteworthy. Obg proteins are TRAFAC (translation factor) class P-ring GTPases and are conserved in function and structure from bacteria to eukaryotes. Obg proteins are involved in basic cellular processes such as DNA replication and ribosome maturation, and in stress adaptation pathways such as sporulation. These characteristics are reminiscent of the role of mTOR. EF-Obg has an N-terminal domain, a non-conserved C-terminal domain, and a central G domain, which are common to all Obg proteins. Figure 7 C). The Obg protein possesses a Ras-like folded G domain and five conserved motifs responsible for GDP and GTP recognition and hydrolysis, similar to Ras or Rheb. Interestingly, structural modeling of molecular interactions predicts that EF-Obg will form a complex with mTOR, similar to Rheb. Figure 7D). To examine whether Obg binds directly to mTOR, we used a transcription and translation (TNT) system to exogenously express Obg and mTOR. The results showed that Obg binds directly to mTOR. Figure 7 E). We then further confirmed the binding / interaction of EF-Obg and mTOR in HEK293T and MHCC-97h cells (E). Figure 7 F). The direct in vivo binding between EF-Obg and mTOR was further demonstrated in adjacent-site connectivity assays. Figure 7 These data indicate that EF-Obg delivered by EVs interacts directly with mTOR.

[0037] 6. The E. faecalisObg gene is crucial for Enterococcus faecalis to activate mTOR and promote liver cancer development.

[0038] Immunoblotting experiments showed that EF-Obg can enhance mTOR pathway activation in a dose-dependent manner, as evidenced by increased phosphorylation of mTOR, p70S6K, and 4EBP1, thus demonstrating the role of EF-Obg in regulating mTOR. Figure 7 H). CCK8 assay showed that overexpression of EF-Obg promoted the proliferation of HCC cells (H). Figure 7 I). To further investigate the role of EF-Obg in mice, we prepared Hepa1-6 cells stably overexpressing EF-Obg. C57BL / 6 mice were randomly divided into two groups, receiving subcutaneous injections of either plvx-Obg or plvx-vec-infected Hepa1-6 cells, respectively. The results showed that EF-Obg overexpression promoted liver cancer progression (…). Figure 7 J). IHC staining of tumor tissue also revealed EF-Obg-mediated activation of the mTOR pathway. Figure 7 These results indicate that EV-derived EF-Obg activates the mTOR pathway to promote hepatocellular carcinoma tumorigenesis.

[0039] By comparing EF-Obg with the classic mTOR-activated GTPase Rheb, we found that Obg has a conserved amino acid sequence at the G1 site, as shown in PyMOL. Figure 9 A) The amino acid sequence of the G1 domain of human Rheb is GyrSVGKS, while the amino acid sequence of the G1 domain of EF-Obg is GfpSVGKS. Therefore, we hypothesize that EF-Obg may interact with mTOR through the G1 site. We then constructed a missense mutation at the G1 site of EF-Obg to obtain Obg. 8A mutant ( Figure 9 B). The co-IP experiment confirmed Obg 8A The mutant cannot bind to mTOR ( Figure 9 C). Consistently, Obg 8A The mutant impaired its ability to activate mTOR, while the wild-type Obg... WT Maintain its mTOR-enhancing activity ( Figure 9 D).

[0040] To verify the key role of the EF-Obg gene in activating mTOR and promoting hepatocellular carcinoma, we constructed an EF-Obg knockout strain, EF-dCas-Obg, using a CRISPR interference system. Figure 9 E). The sgRNA sequences targeting EF-Obg predicted by CHOPCHOP are shown in Table 1.

[0041] Table 1. Target sequences of Enterococcus faecalis Obg protein (EF-Obg) predicted by CHOPCHOP.

[0042]

[0043] qPCR showed decreased expression of Obg in Enterococcus faecalis EF-dCas-Obg. Figure 9 E). Furthermore, RNA sequencing confirmed the successful knockdown of Obg in the EF-dCas-Obg strain. Figure 9 F). Interestingly, the EF-dCas-Obg strain exhibited slow bacterial growth, indicating that Obg is an important gene for the growth of Enterococcus faecalis. Figure 10 A). NTA analysis showed that the concentration of EV particles secreted by the EF-dCas-Obg strain and the protein content within the EVs were decreased, while the EV vesicle size of the EF-dCas-Obg strain remained unchanged compared with the control Enterococcus faecalis (EF-vec). Figure 10 B and Figure 10 C). Therefore, knockdown of Obg affects a range of biological functions, including transmembrane transporter and fatty acid synthase activity, as evidenced by GO and KEGG analyses. Figure 10 D and 10E).

[0044] Further experiments confirmed that, compared with the EF-vec strain, the conditioned medium or EVs of the EF-dCas-Obg strain showed a weakened ability to promote HCC cell proliferation. Figure 9 G). Consistently, immunoblotting results showed that, compared with EF-vec strains, conditioned medium or EVs of EF-dCas-Obg impaired the activation of the mTOR signaling pathway. Figure 9 H). Compared with EF-vec, the effect of EVs from the EF-dCas-Obg strain on promoting the growth of mouse liver cancer was inhibited. Figure 9I). IHC staining of subcutaneous tumor sections showed that strains from EF-dCas-Obg lost their activation of the mTOR pathway, including decreased staining of p-mTOR, p-4EBP1, HIF1α, and CD31. Figure 9 We also collected mouse feces for 16S sequencing. The results showed that EVs of the EF-dCas-Obg strain altered the composition of the mouse gut microbiome. Figure 11 A- Figure 11 C). Notably, it led to the enrichment of Akkermansia, a well-known probiotic for liver disease (C). Figure 11 D and Figure 11 E) indicates that knocking out Obg alters the microbial composition in mice. Overall, these results allow us to establish a causal relationship between microbial dysbiosis and liver carcinogenesis, demonstrating that Obg gene knockout attenuates the role of Enterococcus faecalis in activating mTOR and promoting liver cancer development.

[0045] 7. The expression level of EF-Obg in liver cancer tissue is associated with poor prognosis in HCC patients.

[0046] To explore the clinical significance of our findings, we investigated the expression levels of EF-Obg in patient-derived liver tumor tissues and corresponding normal tissues. qPCR demonstrated elevated Obg gene expression in HCC. Figure 12 A). We used hybridoma technology to prepare a monoclonal antibody targeting the EF-Obg GMVAFRREKYVPD sequence. Figure 13 A- Figure 13 D). Sensitivity and specificity of monoclonal antibodies against EF-Obg in tissue sections of liver cancer models treated with Enterococcus faecalis, EF-CM, and EF-EVs ( Figure 3 H and Figure 5 This was verified on H). Figure 13 E). Notably, higher levels of EF-Obg were observed in tumor tissue compared to normal tissue, accompanied by elevated p-mTOR levels (E). Figure 12 B and Figure 12 C), which was evident in WB and IHC staining. IHC staining with this monoclonal antibody against EF-Obg in a cohort of 100 HCC and normal tissue specimens showed that patients with high EF-Obg expression had significantly lower overall survival and relapse-free survival compared to patients with low EF-Obg expression. Figure 12 D and Figure 12 E). Clinical association studies have shown that EF-Obg expression is significantly associated with the number of HCC tumors, tumor size, TNM (tumor, lymph node, metastasis) stage, and BCLC (Barcelona clinical hepatocellular carcinoma) stage (Table 2).

[0047] Table 2. Correlation between Enterococcus faecalis Obg protein expression and clinicopathological features in 100 patients with hepatocellular carcinoma.

[0048]

[0049]

[0050] 8. EF-mediated mTOR activation in liver cancer can be treated with the mTOR inhibitor everolimus.

[0051] Given that everolimus is a potent mTOR kinase inhibitor, we subsequently investigated whether this drug had a good inhibitory effect on hepatocellular carcinoma colonized by Enterococcus faecalis. We established an orthotopic mouse hepatocellular carcinoma model (Hepa1-6) in C57BL / 6 mice administered Enterococcus faecalis by gavage to study the therapeutic effect of everolimus. Figure 12 F). The results showed that everolimus significantly inhibited tumor growth and liver damage in the presence of Enterococcus faecalis colonization, while it had no significant therapeutic effect on liver cancer in the absence of Enterococcus faecalis colonization. Figure 12 G and Figure 12 H). IHC staining confirmed the effective inhibition of the mTOR pathway by everolimus in a mouse model of liver cancer colonized by Enterococcus faecalis. Figure 12 I). These data suggest that Enterococcus faecalis Obg is a potential biomarker and indicate that everolimus treatment, which inhibits the EF-Obg-mediated mTOR signaling axis, could be considered a treatment strategy for patients with Enterococcus faecalis colonizing hepatocellular carcinoma.

[0052] 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 reagents for detecting or identifying Enterococcus faecalis in liver cancer tissue in the preparation of indicators for the treatment of liver cancer patients.

2. The application as described in claim 1, characterized in that, The liver cancer mentioned is Enterococcus faecalis colonized liver cancer.

3. The application as described in claim 1, characterized in that, The treatment drug is an mTOR pathway inhibitor. In this application, based on the fact that Enterococcus faecalis promotes the development of liver cancer, the presence of Enterococcus faecalis in liver cancer tissue can indicate that the treatment drug for liver cancer is an mTOR inhibitor.

4. The application as described in claim 3, characterized in that, The drug is everolimus, which is used to inhibit the mTOR pathway.

5. Application of reagents for detecting or identifying Enterococcus faecalis in liver cancer tissue in the preparation of products for the prognosis of patients with Enterococcus faecalis colonized liver cancer.

6. Application of everolimus in the preparation of drugs for treating Enterococcus faecalis colonized liver cancer.

7. A drug for treating Enterococcus faecalis colonized liver cancer, characterized in that, Including mTOR pathway inhibitors.

8. The drug as described in claim 7, characterized in that, The mTOR pathway inhibitor is everolimus.

9. The detection results of Enterococcus faecalis in liver cancer tissue as a biomarker for the use of mTOR pathway inhibitors in liver cancer patients.

10. The application as described in claim 9, characterized in that, The mTOR pathway inhibitor is everolimus.

Images