Effect of staphylococcus in tumor metastasis

Abstract

The invention relates to an effect of staphylococcus in a tumor metastasis process, in particular to application of a lactic acid transporter inhibitor or nucleic acid molecules for inhibiting lactic acid generation in staphylococcus in preparation of drugs or kits for treating tumors infected by staphylococcus.

Description

[0001] The present invention is a divisional application of the application with application number 202410763788.5, invention name “Role of Staphylococcus in Tumor Metastasis” and application date June 14, 2024. Technical Field

[0002] The invention belongs to the field of biotechnology, and in particular relates to application of Staphylococcus bacteria inhibitors in the treatment of lung cancer. Background Art

[0003] Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer deaths, of which lung adenocarcinoma (LUAD) is the most common pathological type of NSCLC. Most patients with lung adenocarcinoma are diagnosed at the advanced metastatic stage, which is also the main cause of their death [1-3]. Therefore, it is necessary to clarify the potential mechanisms that lead to the metastasis of lung adenocarcinoma.

[0004] At present, a large number of studies have attempted to analyze the potential causes of lung adenocarcinoma metastasis at the cellular level (epigenetics, transcriptomics, genomics), but the role of tumor microenvironment components outside the cells has rarely been reported. In early 2022, polymorphic microbiomes were added as one of the new "cancer hallmarks" [4], which shows that polymorphic microbiota has been widely accepted as an important part of tumor growth. These "intratumoral microbiota" can affect the biological behavior of tumor cells by regulating metabolism, immunity and other pathways [5,6]. Many functional experiments have shown that dysbiotic microbiome is associated with the progression of various tumors, such as colon cancer, pancreatic cancer, breast cancer, etc. [7-10], but there are few reports in lung cancer. The lung is the largest mucosal tissue organ in the human body and was considered sterile for a long time. In recent years, with the development of non-culture omics sequencing technologies, such as 16s rRNA sequencing and metagenomic sequencing, our understanding of the structure of the microbial community in the lower respiratory tract has been overturned. We found that lung tissue is actually colonized by a variety of microbial communities, but unlike the intestinal flora, the abundance of lung colonizing bacteria is low [11,12], so it has not attracted widespread attention from scholars. However, studies have shown that compared with the intestinal flora, symbiotic microorganisms in the lung tumor microenvironment can directly act on tumor cells, and their effects on the biological behavior of lung cancer cells are more significant. Therefore, exploring the role of potential microorganisms in the progression of lung adenocarcinoma may help develop new biomarkers and new treatment strategies, thereby improving the long-term prognosis of patients with lung adenocarcinoma.

[0005] Studies have shown that based on sequencing results of saliva, sputum, bronchoscopic alveolar lavage fluid, and lung cancer tissue specimens, lung cancer tissue exhibits a microbial signature spectrum that is distinct from that of normal tissue, and some of these species may even affect the patient’s disease-free survival (DFS)

[13] . For example, compared with normal tissue, Staphylococcus aureus ( Staphylococcus ), Streptococcus ( Streptococcus ) and Veillonella spp. Veillonella ) is more abundant in lung cancer tissues

[14] . In addition, some scholars have found that Mycobacterium tuberculosis is closely related to the occurrence and development of lung cancer. At the same time, relevant evidence has also been presented in mouse models: (1) Compared with SPF mice, TP53 / KRAS double mutations cannot induce lung cancer in germ-free mice and mice treated with antibiotics

[15] ; (2) Lung commensal flora can induce the formation of a local immunosuppressive microenvironment (recruiting neutrophils and γT cells) to assist the colonization of metastatic tumor cells in the lungs, and aerosolized inhalation of antibiotics can significantly reduce the number of lung metastases in mouse models

[16] . These research results confirm the role of lung commensal flora in promoting the development of lung cancer. However, most of the current research remains at the macroscopic feature level, such as analyzing the ecological structure imbalance of lung microorganisms and their association with clinical pathological parameters of patients through sequencing technology, and cannot answer the following two key questions: Is there a causal relationship between changes in the homeostasis of lung flora and the occurrence and development of lung cancer, and whether specific microorganisms can serve as potential targets for lung cancer treatment. Therefore, it is crucial to delve into the microscopic level such as specific strains and their carcinogenic mechanisms.

[0006] Based on this, this study combined microbial culture genomics technology, multi-group student signal analysis methods and functional experiments of tumor-derived organoid models to explore the impact of intratumoral colonization microorganisms on lung adenocarcinoma metastasis. Summary of the invention

[0007] The applicant first analyzed a large sample clinical cohort and found that perioperative infection significantly shortened the disease-free survival of patients with lung adenocarcinoma, suggesting the important role of microorganisms in promoting tumor progression. The applicant further integrated 16SrRNA sequencing and microbial culture genomics technology to focus on the genus Staphylococcus, which is likely to play an important role in the metastasis process as the dominant flora in lung adenocarcinoma tissue. At the same time, functional experimental exploration based on lung cancer organoid models and animal models further found that Staphylococcus infection can produce a large amount of lactic acid in the tumor microenvironment and activate the lactate transporter (MCT-1, etc.) of tumor cells, enhance lactate uptake capacity and increase the lactic acid level of total intracellular proteins, and ultimately promote the occurrence of epithelial-mesenchymal transition and the enhancement of tumor cell invasiveness. This effect can be blocked by the MCT-1 inhibitor AZD3965. This result suggests the important role of bacterial-derived lactic acid in the process of tumor metastasis, and provides new clues for the development of lung cancer treatment strategies based on suppressing the intratumoral microbial action network.

[0008] The role of Staphylococcus in tumors has begun to emerge The present invention found that Staphylococcus aureus infection can induce the enhancement of lung cancer cell metastasis ability, among which Staphylococcus nepalensis ( Staphylococcus nepalensis, S.nepalensis ) and Staphylococcus capitis ( Staphylococcus capitis, S. capitis ) is particularly significant (see Example 2). In the prior art, studies on the interaction between bacteria and tumors usually directly use purchased or preserved strains to study the relationship between bacteria and tumors in vitro; although Staphylococci such as Staphylococcus nepalensis ( Staphylococcus nepalensis, S.nepalensis ) and Staphylococcus capitis ( Staphylococcus capitis, S.capitis ) is a strain known in the prior art, but the present invention is the first to discover Staphylococcus aureus, especially Staphylococcus nepalensis ( Staphylococcus nepalensis, S.nepalensis ) and Staphylococcus capitis ( Staphylococcus capitis, S.capitis ) survive in lung cancer tissues and are separated and cultured from lung cancer tissues through culture omics methods and used for subsequent research on their close relationship with cancer metastasis. Taking patient-derived strains as research objects is one of the important innovations of the present invention.

[0009] Staphylococcus Staphylococcus ) is a representative of Gram-positive bacteria, named because it is spherical and resembles grapes under a microscope. It is a common opportunistic pathogen that causes cross-infection in hospitals.

[0010] At the same time, most Staphylococcus bacteria can decompose glucose and produce organic acids such as lactic acid, acetic acid, and propionic acid

[19] . We found that Staphylococcus infection can produce a large amount of lactic acid in the microenvironment and create an acidic tumor microenvironment. The pH of normal tissue or blood is 7.4, while the pH of the microenvironment suitable for the survival of tumor cells is 5.6-7.0

[20] . Lactate has long been regarded as a metabolic waste of the anaerobic glycolysis pathway of cells. However, the latest studies have shown that lactate, as a signaling molecule in the tumor microenvironment, can reshape the biological behavior of tumor cells through various pathways, such as directly inhibiting PHD2 to increase the activity of HIF-1α transcription factor, inducing the production of reactive oxygen species (ROS), activating the NF-κB signaling pathway, and promoting the expression of VEGFR and angiogenesis. However, as a metabolite, the non-metabolism-related role of lactate in other physiological and pathological processes in the human body remains unknown. We found that upregulation of lactate levels can activate MCT1 and increase lactate uptake, thereby promoting the metastasis of lung adenocarcinoma, and this effect can be blocked by the MCT1 inhibitor AZD3965.

[0011] In summary, the present invention integrates multi-omics analysis strategies to further explore the relevant molecular mechanisms, providing experimental evidence to support the necessity of effective intervention in specific tumor microorganisms in clinical diagnosis and treatment practice, in order to delay the occurrence of metastasis and provide new ideas for the treatment of cancer patients such as lung adenocarcinoma.

[0012] In a specific embodiment of the present invention, the lactate transporter is also called monocarboxylate transporters (MCTs), which is part of the solute carrier 16 (SLC16) family and consists of 12 transmembrane helical structures with intracellular N-terminus and C-terminus and a large cytoplasmic loop between the 6th and 7th helical structures. It contains 14 members, among which MCT1-4 exhibits the transport of proton-coupled monocarboxylic acids, including pyruvate, lactate, ketone bodies and short-chain fatty acids (propionic acid and butyric acid).

[0013] The main function of MCT is to mediate the transmembrane transport of proton-coupled lactic acid and other monocarboxylic acid substances. The abnormal expression of MCT is closely related to the occurrence and development of various malignant tumors. For example, MCT-mediated lactate transport can also promote tumor angiogenesis and induce tumor immune tolerance.

[0014] In the present invention, only the metastasis-promoting effect of Staphylococcus aureus in lung cancer, such as lung adenocarcinoma, is verified by way of example. In fact, based on the existing pan-cancer research results, Staphylococcus aureus is widely present in a variety of human tumor tissues (PMID: 36565918, PMID: 37140927). Those skilled in the art can expect that Staphylococcus aureus, such as Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ) also has a metastasis-promoting effect in other tumors.

[0015] Specifically, the present invention provides the following technical solutions: 1. Use of a lactate transporter inhibitor or a nucleic acid molecule for inhibiting lactate production in Staphylococcus aureus in the preparation of a drug or a kit for treating a tumor infected by Staphylococcus aureus.

[0016] 2. The use according to item 1, wherein the sequence of the nucleic acid molecule that inhibits lactic acid production in Staphylococcus is as shown in SEQ ID NO: 1-2, 7-8.

[0017] 3. The use according to item 1, wherein the staphylococcus is Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ).

[0018] 4. The use according to item 1, wherein the lactate transporter is MCT-1; 5. The use according to item 1 or 4, wherein the lactate transporter inhibitor comprises a small molecule compound or an antibody targeting the lactate transporter; In a specific embodiment of the present invention, the lactate transporter inhibitor includes but is not limited to AZD3965 and AR-C155858, 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS).

[0019] 6. The use according to item 1, wherein the tumor is a solid tumor, including but not limited to lung cancer, esophageal cancer, gastric cancer, colorectal cancer, melanoma, liver cancer, breast cancer, prostate cancer, fibrosarcoma, bladder sarcoma or glioma, etc.

[0020] In a specific embodiment of the present invention, the lung cancer is lung adenocarcinoma or lung squamous cell carcinoma.

[0021] 7. A method for inhibiting metastasis of tumor cells infected with Staphylococcus aureus, comprising knocking out a lactic acid production gene in Staphylococcus aureus LqCy and Dh Alternatively, a lactate transporter inhibitor is administered to the tumor cells, wherein the method is for non-therapeutic purposes.

[0022] 8. The method according to item 7, wherein the staphylococcus is Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ).

[0023] 9. The method according to item 7, wherein the lactic acid production gene LqCy and Dh It was knocked out using Crisper / cas9 technology.

[0024] 10. The method according to item 9, wherein the primers used in the Crisper / cas9 are shown as SEQ ID NOs: 1-2, 7-8 respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1a to Figure 1g The microbial spectrum characteristics of the three groups of samples, PL, ML, and NML, are shown. Figure 1a The microbial composition is shown; Figure 1b Shows species diversity and richness; Figure 1c The richness overview at the genus level showed that the abundance of Staphylococcus was significantly higher in the PL and ML groups than in the NML group. Figure 1d The abundance distribution of Staphylococcus in various tumor types in the TCGA database is shown; Figure 1e It shows that the abundance of Staphylococcus in tumor tissues of the TCGA-LUAD database (a lung cancer imaging dataset known in the prior art) is significantly higher than that in normal tissues; Figure 1f Representative images of FISH detection of Staphylococcus abundance are shown; Figure 1g It was shown that high abundance of Staphylococcus aureus was significantly associated with poor prognosis in patients with lung adenocarcinoma.

[0026] Figures 2a to 2e It shows an overview of the results of the microbial culture group and the results of the low-throughput phenotypic screening experiment of 4 strains of Staphylococcus aureus. Figure 2a A flow chart of the microbiological culture group is shown; Figure 2b The information table and general summary of the 12 strains of human origin are shown; Figure 2c The invasive ability of A549 cells under co-culture of different strains is shown: the upper figure is the result of the scratch test; the lower figure is the result of the Transwell test; Figure 2d Bright field photographs of co-culture of Staphylococcus nepalensis, Staphylococcus capitis, and Escherichia coli with A549 cells are shown; Figure 2e The immunofluorescence results of A549 cells co-cultured with Staphylococcus nepalensis and Staphylococcus capitis are shown. Green is phalloidin representing the cytoskeleton, and blue is DAPI representing the cell nucleus. DNA is also shown as blue dots; a detailed zoom-in of Staphylococcus nepalensis and Staphylococcus capitis clustering around pseudopodia of A549 cells.

[0027] Figure 3a to Figure 3d The co-culture system of lung adenocarcinoma organoid model was established. Figure 3a Brightfield photographs of the lung adenocarcinoma organoid model are shown; Figure 3b A higher variant allele frequency (VAF) correlation analysis was shown; Figure 3c A high concordance analysis of copy number status was shown; Figure 3d The principal component analysis (PCA) of transcriptome data of the bacterial infection 7h, 14h group and control group is shown.

[0028] Figures 4a to 4e It was shown that Staphylococcus nepalensis infection promoted the expression of EMT-related genes. Figure 4a Representative images of the organoid model co-cultured with lung adenocarcinoma organoids are shown; Figure 4b The results of GSEA pathway enrichment analysis are shown; Figure 4c The enrichment of EMT signaling pathway is shown; Figure 4d The expression of Vimentin protein over time is shown; Figure 4e The figure shows that under the three modeling methods, infection with Staphylococcus nepalensis and Staphylococcus capitis enhanced the lung metastasis ability of LLC cells (upper figure) and the comparison of fluorescence values ​​between different groups (lower figure).

[0029] Figures 5a to 5k It shows that Staphylococcus nepalensis and Staphylococcus capitis infection create an acidic co-culture environment. Figure 5a The appearance color and pH value of the supernatant of the S. nepalensis infection group and the S. capitis infection group and the control group are shown; Figure 5b The results of the metabolic panel of the Staphylococcus nepalensis infection group and the Staphylococcus capitis infection group and the control group are shown, showing that the lactic acid content in the bacterial infection group was higher than that in the control group; Figure 5c The comparison of lactic acid content between the Staphylococcus nepalensis infection group and the Staphylococcus capitis infection group and the control group is shown; Figure 5d The expression of MCT1 at the mRNA and protein levels in the control and infected groups is shown; Figure 5e It showed that high expression of MCT1 was significantly associated with poor prognosis of TCGA-LUAD patients; Figure 5f The dynamic changes of intracellular / extracellular lactate levels in the co-culture system were shown; Figure 5g PCR validation was shown ldh / ddh Knockout effect; Figure 5h Shows Sc ddh- / ldh- The number of invadopodia formed by cells in the infected group was significantly reduced; Figure 5i Staphylococcus capitis ddh- / ldh- The invasion ability of lung adenocarcinoma cells in the infection group was significantly weaker than that in the Staphylococcus capitis infection group; Figure 5j Staphylococcus capitis ddh- / ldh- The expression of metastasis-related proteins in cells of the infection group was lower than that in the S. capitis infection group; Figure 5k Staphylococcus capitis ddh- / ldh- The number of lung metastases in the infected group was significantly less than that in the Staphylococcus capitis infected group.

[0030] Figures 6a to 6d It was shown that AZD3965 could reverse the enhanced invasion ability of lung adenocarcinoma induced by Staphylococcus aureus infection. Figure 6a It shows that MCT1 inhibitor blocked the enhanced metastatic phenotype caused by bacterial infection, where the left picture shows the results of the scratch assay and the right picture shows the results of the Transwell assay; Figure 6b The results of the Nodscid model showed that infection with Staphylococcus nepalensis and Staphylococcus capitis enhanced the lung metastasis and distant metastasis ability of the lung cancer organoid model, and this phenotype could be blocked by AZD3965. The fluorescence value comparison between different groups was also shown; Figure 6c It showed a clear colocalization of Staphylococci with lung metastases; Figure 6d It was shown that Staphylococci can be detected in distant metastatic tissues of mice. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0032] The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0033] Experimental Materials and Methods 1. Sample source inclusion criteria The inclusion criteria were as follows: (1) patients who underwent lobectomy or wedge resection and underwent mediastinal lymph node dissection or mediastinal lymph node biopsy; (2) patients who had not received neoadjuvant therapy before surgery; (3) patients whose postoperative pathology was stage IA invasive lung adenocarcinoma. The exclusion criteria were as follows: (1) patients who had dual primary tumors; (2) patients whose pathology was microinvasive adenocarcinoma; (3) patients with neuroendocrine differentiation; (4) patients who died of surgical complications; (5) patients whose imaging showed ground-glass components; and (6) patients whose recurrence status could not be assessed. A total of 282 patients were retrieved, and 132 of them met the inclusion and exclusion criteria. In addition, in order to have sufficient specimens for subsequent experimental verification, we further excluded 57 patients whose tumor diameter was less than 1 cm. Finally, a total of 75 patients were included in our cohort for subsequent data analysis and experimental verification.

[0034] 2. Co-culture methods and conditions A certain number of A549 cells (purchased from ATCC) or organoid models (for the method of obtaining the organoid model, please refer to the following "3. Establishment of lung adenocarcinoma organoid model") were collected, and the bacteria to be tested were added according to different MOI values ​​(1, 10, 50, 100, 500, etc.), and the concentration of gentamicin in the culture medium was set to 0, 0.025, 0.05, 0.1, 0.2, 0.4, 1 μg / ml, etc. The proliferation activity of cells and bacteria was continuously detected within 0-24 hours, wherein the cell proliferation activity was detected by the cell tilter method, and the bacterial proliferation activity was represented by a growth curve, and the optimal MOI value and gentamicin concentration were determined according to the intersection of the two result curves.

[0035] 3. Establishment of Lung Adenocarcinoma Organoid Model The method for constructing a lung adenocarcinoma organoid model can be found in prior art literature, such as PMID: 31694835, PMID: 31488816, PMID: 33846488.

[0036] 4. Crisper / Cas9 knockout LqCy (NCBI 77312680) and Dh (NCBI 77312717) gene (1) Preparation of gRNA and donor DNA fragments The present invention uses CRISPR / Cas9 system to perform Staphylococcus aureus LqCy and Dh Gene knockout, wherein the LqCy and Dh The genes are respectively the coding genes of L-lactate dehydrogenase LLDH and D-lactate dehydrogenase DLDH. First, the target gene sequence (gRNA- LqCy and gRNA- Dh , see SEQ ID NO: 1-2, 7-8 in Table 1) were designed using the CRISPR RGEN tool (http: / / www.rgenome.net / cas-designer / ), and then ligated into the linearized pGRB plasmid (purchased from Addgene plasmid #71539) after PCR amplification to construct a gRNA-expressing plasmid (pGRB- Ldh / Ddh ). Next, prepare Ldh / Ddh The missing donor DNA fragment (i.e., donorDNA- Ldh / Ddh ), using two pairs of primers (Ldh-UF / Ldh-UR, LqCy -DF / LqCy -DR Dh -UF / Dh -UR, Dh -DF / Dh -DR, see SEQ ID NO: 3-6, 9-12 in Table 1), and the two homology arms were generated by overlapping PCR.

[0037] (2) Gene knockout steps based on Crisper / cas9 method First, Staphylococcus chemical competent cells were prepared by the Cacl2 method, and the λ-Red plasmid (purchased from Addgen, catalog number 71541) was introduced into the Staphylococcus competent cells and 0.1 mM IPTG was added to induce the expression of λ-Red recombinase. Secondly, the pRedCas9 plasmid (Addgene plasmid # 71541) was introduced into the competent bacteria by electroporation, and the correct positive transformants were screened by colony PCR and the bacteria were amplified for the preparation of electrocompetent cells. Then, the gRNA-expressing plasmid pGRB- Ldh / Ddh and Ldh / Ddh Missing donor DNA fragment donorDNA- Ldh / Ddh Co-transformed into Staphylococcus aureus electrocompetent cells containing plasmid pREDCas9. After electroporation, the cells were plated on LB plates containing ampicillin and spectinomycin to confirm the presence of pGRB and pREDCas9, respectively. Finally, positive colonies were verified by colony PCR (see SEQ ID NO: 13, 14 in Table 1), and then the deletion of the target gene was confirmed by DNA sequencing. All DNA oligonucleotide sequences used in the present invention are listed in Table 1 below.

[0038] Table 1 Sequences involved in the process of Crisper / cas9 knockout of Ldh and Ddh genes

[0039] Example 1 Staphylococcus may play an important role in the metastasis of lung adenocarcinoma In this example, primary lesions (PL), metastatic (ML) and non-metastatic lymph node (NML) tissues of 13 patients with lung adenocarcinoma were collected and 16S rRNA sequencing analysis was performed to clarify the microbial profiles of the three groups of samples. The results showed: 1) There were significant differences in microbial composition and abundance among the PL, ML, and NML groups: Staphylococcus ( Staphylococcus ), Acinetobacter spp. Acinetobacter ) were enriched in the ML group, while the genus Acidioides ( Acidibacter ) were enriched in the NML group, while Bacillus ( Bacillus ) were widely present in the three groups of samples (Fig. 1a).

[0040] 2) The richness and diversity of microorganisms in the ML group were significantly lower than those in the PL and NML groups: The results of Alpha diversity analysis (wherein the Alpha diversity analysis is a conventional method in the prior art) showed that the species richness index (chao1 index), number of observed species (observed species), and microbial diversity index (shannonindex) of the ML group were lower than those of the other two groups ( Figure 1b ), indicating that during the metastasis process, most of the bacterial flora are lost due to various reasons (such as inability to tolerate blood shear stress, inability to adapt to the higher ROS levels during the metastasis process, etc.), and only a small amount of dominant flora participates in or even promotes the metastasis process.

[0041] 3) Staphylococcus is very likely to be involved in the metastasis of lung adenocarcinoma: Studies have shown that the bacterial flora involved in the metastasis process in the primary lesion can spread to distant organs along with tumor cells. Therefore, the present invention defines the bacterial genus that is common to the PL group and the ML group and significantly higher than the NML group as potential metastasis-related microorganisms. Analysis found that Staphylococcus was significantly enriched in the PL group and the ML group, but was less abundant in the NML group; in summary, the present invention preliminarily identified Staphylococcus as a microorganism associated with lung adenocarcinoma metastasis ( Figure 1c To further verify this hypothesis, we compared the abundance of Staphylococcus in different types of tumors in the TCGA database known in the prior art and found that its abundance was relatively high in the lung adenocarcinoma cohort ( Figure 1d ), and its abundance in tumor tissues is significantly higher than that in normal tissues ( Figure 1e). We then established clinical cohort 2 (paraffin tissue sections of 75 patients who underwent surgical treatment and were confirmed to have stage IA invasive lung adenocarcinoma by postoperative pathology. The average age of the cohort was 61.5 years, and the median follow-up time was 61 months. 57.3% of them were male, and the ECOG score of all people was 0). We analyzed the correlation between the abundance of Staphylococcus and the prognosis of patients. We detected the abundance of Staphylococcus by in situ fluorescence hybridization (FISH) and divided the bacteria into negative group (-), low abundance group (+) and high abundance group (++ to +++) according to the fluorescence intensity. Figure 1f ), the number of patients was 42 (57.5%), 20 (27.4%), and 11 (15.1%) respectively. The difference in disease-free survival (DFS) among the three groups was calculated by the Kaplan-Meier method known in the prior art, showing that the FISH high expression group (i.e., the high abundance group of Staphylococcus) had the shortest DFS of 30 months; the low expression group (i.e., the low abundance group of Staphylococcus) had a median DFS of 49 months; the median DFS of the high expression group was not reached (p<0.001), that is, the DFS of the patient group with high abundance of Staphylococcus was significantly shorter than that of the patient group with low abundance ( Figure 1g The inventors further performed multivariate COX regression analysis according to the conventional method in the prior art and showed that the abundance of Staphylococcus aureus was an independent predictor of DFS (p=0.003).

[0042] The above results indicate that Staphylococcus aureus is highly correlated with the recurrence and metastasis of lung adenocarcinoma.

[0043] Example 2 Patient-derived Staphylococcus infection induces morphological changes in lung adenocarcinoma cells and enhances their metastatic potential In this example, according to the above inclusion criteria (see "Sample Source Inclusion Criteria" in the "Experimental Materials and Methods" section for details), a total of 18 fresh ex vivo lung adenocarcinoma tissue samples were collected to design a culture method for lung cancer tissue (see the detailed process for details). Figure 2a ), the result is as follows: 1) The detection rate of bacterial culture in samples was 27.78%: Among the 18 patients, only 5 patients' tumor tissue samples could culture colonies, of which 12 strains were successfully isolated and cultured in this study (see Figure 2a ,b), of which 7 were Staphylococcus. Further, we analyzed and summarized the isolated strains in terms of phylum classification (Phyla), oxygen tolerance (O2 tolerance), microbial safety level (Risk group) and temperature of first growth. The results showed that the vast majority of bacteria belonged to the Firmicutes phylum and were facultative anaerobes, which may be related to the adaptation to the dynamic changes of gas conditions in the lung tumor microenvironment; the isolation and culture temperature was between 30-37°C, which was consistent with the characteristics of bacteria from human origin.

[0044] 2) Low-throughput phenotypic screening experiments focused on Staphylococcus nepalensis (S. nepalensis) Staphylococcus capitis (S. capitis) : In view of the fact that Staphylococcus aureus was identified as a lung adenocarcinoma metastasis-related microorganism in Example 1, we selected the four strains of Staphylococcus aureus (respectively S. nepalensis, S. capitis, S. warneri, S. hominis ) for downstream functional experiments. We co-cultured the four strains with the lung adenocarcinoma cell line A549 (purchased from ATCC) (see "Co-culture methods and conditions" in the "Experimental Materials and Methods" section), or used the co-culture supernatant instead of bacteria to perform low-throughput Transwell and scratch phenotype screening experiments (wherein the operation methods of the low-throughput Transwell and scratch phenotype screening experiments are all conventional techniques in the prior art). The results showed that compared with the control group (wherein the control group used Escherichia coli to co-culture with tumor cells), the four Staphylococcus strains all showed the ability to promote A549 cell migration, among which Staphylococcus nepalensis ( S. nepalensis ) and Staphylococcus capitis ( S. capitis ) is the most significant ( Figure 2c ), mainly in the following two aspects: compared with the other two strains of Staphylococcus aureus ( S.warneri and S.hominis )compared to, S. nepalensis and S. capitis Shows stronger ability to promote A549 cell migration; and S. nepalensis and S. capitis It showed stronger host cell adhesion ability and caused A549 cell polarity changes, leading to the formation of invadopodia, indicating enhanced cell invasion ability. This morphological change was not observed in the co-culture group with the other two Staphylococcus aureus strains ( Figure 2d ,e); Therefore, Staphylococcus nepalensis was selected in this study. S. nepalensis ) and Staphylococcus capitis ( S. capitis ) for subsequent functional experiments and molecular mechanism exploration, there have been no reports on the correlation between these two strains of bacteria and lung cancer.

[0045] Example 3 Staphylococcus nepalensis ( S. nepalensis ) and Staphylococcus capitis ( S. capitis ) strain infection induces enhanced invasiveness of lung adenocarcinoma cells First, we further verified the initial screening results of Example 2: 1) Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ) infection induces polarity changes in lung adenocarcinoma cells: In this example, A549 cells (purchased from ATCC) were incubated with Escherichia coli (as a control group), Staphylococcus nepalensis, (S. nepalensis ) or Staphylococcus capitis ( S. capitis ) were co-cultured and tested, and the results were as follows: Under the bright field microscope, compared with the control group, A549 cells infected with Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ) Two hours after infection, obvious changes in cell polarity occurred, accompanied by the formation of a large number of pseudopodia, while there was no such change in the E. coli infection group ( Figure 2d ); The same phenomenon was observed in immunofluorescence experiments, with an increase in F-actin stress fibers in cells infected with Staphylococcus aureus ( Figure 2e ); This phenotypic change suggests that Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ) infection led to enhanced invasiveness of A549 cells. S. nepalensis ) or Staphylococcus capitis ( S. capitis ) gathered around the pseudopodia of A549 cells ( Figure 2e ), which strongly confirms the driving effect of bacteria on pseudopodia formation, but the specific mediating mechanism still needs further exploration.

[0046] Furthermore, we established a better in vitro model to explore the mechanism: 2) Establishment of lung adenocarcinoma organoid model: With reference to the known methods of the prior art (e.g. PMID: 31694835, PMID: 31488816, PMID: 33846488), the inventors successfully constructed a patient-derived lung cancer organoid model library (n=16), in which two lung adenocarcinoma organoids (named LC_05_O and LC_06_O, respectively) were constructed for the tumor cell sources of two patients. All organoids and tumor tissues of the corresponding sources have completed genome and transcriptome sequencing analysis. The analysis results show that the lung cancer organoid model basically maintains the phenotypic and genomic characteristics of tumor tissue (Figure 3a-c). Further, we explored the effect of different bacterial infection times on organoid gene expression (MOI=50). The results of PCA cluster analysis (where the specific method of PCA cluster analysis is the conventional steps of the prior art) showed that the gene expression profile of 7 hours of infection was not significantly different from that of the control group, but infection for 14 hours can cause significant changes in gene expression, so we set 14 hours as the critical point of co-culture time ( Figure 3d). The above shows that the lung adenocarcinoma organoid models LC_05_O and LC_06_O can truly reflect the characteristics of the original tumor tissue, so we chose them for subsequent functional studies, which provides a research model with a clear mutation background and more clinical translation value for the present invention.

[0047] 3) Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ) strain infection enhances the expression of EMT-related genes: Given the advantages of organoid models in presenting infection-related gene expression profiles, we S. nepalensis and S. capitis Transcriptome sequencing was performed on the two infected organoid models (representative images of organoids co-cultured with bacteria are shown in Figure 4a). The results showed: S. nepalensis Infection caused activation of common bacterial infection-related pathways in both organoid models, such as inflammation response and Nf-κb signaling pathway, reflecting the reliability of sample preparation and data analysis ( Figure 4b ); GSEA analysis (i.e., gene set enrichment analysis, the operation methods of which are conventional methods in the prior art) showed that the EMT (Epithelial mesenchymal transition) pathway of the two organoid models was significantly activated in the infection group (p<0.05) ( Figure 4c ); Since EMT is a signaling pathway that is significantly active during tumor metastasis, the above results verify that Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S.capitis ) The promoting effect of bacterial infection on tumor metastasis; Western blot analysis showed that S. nepalensis As the infection time increases, the expression of vimentin (i.e., Vimentin protein, the antibody used in the detection process was purchased from Proteintech 60330) gradually increases (the increased expression of Vimentin protein indicates enhanced tumor metastasis ability) Figure 4d ).

[0048] 4) Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S.capitis) strain infection enhances lung adenocarcinoma metastasis at the in vivo level: This part of the study was conducted at the two model levels of immunodeficient mice (Nod-scid) and non-immunodeficient mice (C57BL) (purchased from Vital River), and a comparative analysis was made as to whether there was a difference in the ability of the target strain to mediate the metastasis of lung adenocarcinoma cells under the two modeling pathways of the circulatory system and the respiratory system. The specific modeling methods are: (1) LLC mouse lung cancer cells (purchased from ATCC) infected with the target strain were injected into the tail vein of C57BL mice (purchased from Vital River); (2) The target strain was delivered to the lungs by intrapulmonary instillation to simulate its in situ living environment, and then the lung cancer cells were injected into the tail vein; (3) The lung cancer cells co-cultured with the target strain for a certain period of time were delivered to the lungs of mice to form tumors in situ. The results showed that under the three modeling pathways, S. nepalensis and S. capitis Infection significantly promoted the metastasis of mouse lung adenocarcinoma (Figure 4e).

[0049] The above results collectively confirmed that Staphylococcus infection can induce enhanced metastatic ability of lung adenocarcinoma cells.

[0050] Example 4 Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S.capitis ) strain secretes lactic acid to activate MCT1 expression and promote metastasis of lung adenocarcinoma The present invention found that compared with the control group (Escherichia coli infection group), S. nepalensis and S. capitis The culture supernatant of cells and organoid models in the bacterial infection group was significantly acidified ( Figure 5a , judged by the color change of phenol red indicator in the culture medium), the prior art literature discloses that Staphylococcus can decompose glucose and produce organic acids (lactic acid, acetic acid and propionic acid, etc.), therefore, we conducted further experimental verification and exploration: 1) Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S.capitis ) strain infection to create an acidic tumor microenvironment: First, we tested the pH values ​​of the supernatants of the two groups, and the results showed that the pH value of the co-culture group was significantly lower than that of the control group ( Figure 5a ); 2) Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S.capitis ) strain infection causes an increase in lactic acid content in the microenvironment and activates the uptake behavior of tumor cells: Furthermore, we detected the metabolites in the supernatant by metabolomics and found that the lactic acid content in the supernatant of the co-culture group was significantly higher than that of the control group ( Figure 5b ), which was further confirmed by the lactate test kit (purchased from Promega J5021) ( Figure 5c In addition, there were significant changes in metabolite composition between the staphylococcal infection and control groups, with significantly upregulated levels of organic acids and significantly downregulated levels of carnitine and amino acids in the infected organoids ( Figure 5b ). Evidence shows that in the presence of oxygen, Staphylococcus aureus can use D-glucose, D-fructose and other ingredients to produce acidic substances such as lactic acid.

[0051] Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S.capitis ) is of vital importance to the metastatic ability of tumor cells. Further analysis of transcriptome data in this study showed that lactate transporters (MCT1, etc.) in the bacterial infection group were significantly overexpressed ( Figure 5d ), TCGA-LUAD data (this database is a known database in the prior art) analysis showed that high expression of MCT1 is associated with poor prognosis in patients with lung adenocarcinoma ( Figure 5e ); Next, we explored the changes in lactate levels in the co-culture system. We first compared S.n and S.c The lactate production capacity in BHI (bacterial proliferation medium) and organoid culture medium was found to be: lactate levels gradually increased in a time-dependent manner ( Figure 5f ). At the same time, we compared the intracellular / extracellular lactate levels in the co-culture system and observed a similar trend: the extracellular lactate level began to increase 4 hours after the start of co-culture, while the intracellular lactate level began to increase after 12 hours ( Figure 5f ). This result indicates that tumor cells continuously take up bacteria-derived lactic acid.

[0052] 3) Knockout of lactate production genes ( Ldh , Dd h) Can weaken the ability of Staphylococcus aureus to induce lung adenocarcinoma metastasis: Lactic acid is a product of glycolysis and an energy source that can be exchanged between tumor cells. It is reported that lactate levels are associated with the aggressive biological behavior and poor prognosis of multiple tumor types (including lung cancer, melanoma, cervical cancer and ovarian cancer). This finding preliminarily suggests that lactate may play a key role in the process of promoting tumor cell metastasis mediated by Staphylococcus aureus. Lactic acid exists in two isomers: L- and D-lactic acid, respectively. Ldh (NCBI 77312680) and Ddh (NCBI 77312717). To further clarify the effect of lactate uptake on tumor cell phenotype, we knocked out the S.c Strains Ldh , Ddh ( S.c ddh- / ldh- )( Figure 5g ), and a series of phenotypic experiments were performed to observe the changes in its ability to induce metastasis of lung adenocarcinoma cells. The results showed that compared with the control group (non-knockout Ldh and Ddh of S.capitis strains) compared to S.c ddh- / ldh- The number of invadopodia formed by cells in the infected group was significantly reduced ( Figure 5h ),show S.c ddh- / ldh- The ability to mediate cytoskeletal remodeling was weakened, and the same trend was observed in scratch and transwell experiments ( Figure 5i ). Furthermore, through Western blot experiments, we observed that the metastasis-related proteins SNAI1 (the antibodies used in the detection process were purchased from Proteintech 13099) and VIM (the antibodies used in the detection process were purchased from Proteintech 13066) were expressed in S.c ddh- / ldh- The up-regulation degree in the infection group was significantly lower than that in the control group ( Figure 5j Finally, we verified the above results in animal models, namely, Ldh and Ddh of S.capitis strains (as control group) and knockout Ldh and Ddh of S.c ddh- / ldh- The strain was used to infect mice (purchased from Vital River Company). The results showed that compared with the control group, S.c ddh- / ldh- The number of lung metastases in the infected mouse model was significantly less than S.c Group( Figure 5k ).

[0053] 4) Inhibiting lactate uptake can reverse the enhanced metastatic phenotype caused by bacterial infection: The above experiments confirmed that curbing the production of bacteria-derived lactate can inhibit metastasis. Further, we explored from another level whether inhibiting lactate uptake can also reverse this phenotype.

[0054] Lactate uptake is usually carried out through lactate transporters, so lactate transporter inhibitors such as MCT1 inhibitors can inhibit lactate uptake. This patent explores the therapeutic potential of MCT1 inhibitors. Commonly used MCT1 inhibitors include: AZD3965, BAY-802, 7ACC2, etc. Among them, AZD3965 has been subjected to phase I clinical trials and has been proven to have certain anti-cancer effects and tolerable adverse events. Therefore, this study selected AZD3965 for efficacy exploration. We added the MCT1 inhibitor AZD3965 into S. nepalensis or S.capitis In the lung adenocarcinoma organoid culture system obtained in the above examples (the concentration was set to 1 μM according to the previous results), the results showed that inhibition of lactate uptake by AZD3965 could reverse the enhanced metastatic phenotype caused by infection with the target strain, which was specifically manifested in the weakening of scratch healing ability and migration ability ( Figure 6a ).

[0055] Next, we will S.n , S.c Co-cultured with lung cancer organoids and then injected into the tail vein of Nodscid mice (purchased from Vital River). The results showed that compared with the control group, bacterial infection significantly increased the number of lung metastases in mice ( Figure 6b ), which is consistent with the results of the C57BL mouse model described above. Interestingly, 2 / 3 S. n Mice in the infected group showed characteristics of abdominal metastasis, while this phenomenon was not observed in C57BL mice. This result suggests the involvement of the immune system in this process, or it may be due to differences between humanized and mouse preclinical models. The underlying mechanism needs further exploration. At the same time, AZD3965 can significantly inhibit bacteria-mediated lung metastasis. Next, we used the FISH method to detect the presence of Staphylococcus aureus in the lungs and metastatic lesions of mice, and we found that Staphylococcus aureus was significantly co-localized with metastatic tumor cells in the lungs ( Figure 6c ), in addition, the presence of bacteria was also observed in the metastatic lesion sections ( Figure 6d ), but no bacteria were observed in the surrounding normal tissues, indicating that bacteria were spread through the bloodstream along with metastatic tumor cells and promoted the metastasis process.

[0056] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0057] References 1. Chen, Z., et al., Non-small-cell lung cancers: a heterogeneous set of diseases. Nat Rev Cancer, 2014. 14(8): p. 535-46. 2. Goldstraw, P., et al., The IASLC Lung Cancer Staging Project: Proposals for Revision of the TNM Stage Groupings in the Forthcoming (Eighth) Edition of the TNM Classification for Lung Cancer. J Thorac Oncol, 2016. 11(1): p. 39-51. 3. Zheng, R., et al., Cancer incidence and mortality in China, 2016. Journal of the National Cancer Center, 2022. 2(1): p. 1-9. 4. Hanahan, D., Hallmarks of Cancer: New Dimensions. Cancer Discov,2022. 12(1): p. 31-46. 5. Cullin, N., et al., Microbiome and cancer. Cancer Cell, 2021. 39(10): p. 1317-1341. 6. Nejman, D., et al., The human tumor microbiome is composed of tumor type-specific intracellular bacteria. Science, 2020. 368(6494): p. 973-980. 7. Apostolou, P., et al., Bacterial and fungal microflora in surgically removed lung cancer samples. J Cardiothorac Surg, 2011. 6: p. 137. 8. Fu, A., et al., Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer. Cell, 2022. 185(8): p. 1356-1372e26. 9. Laniewski, P., Z. E. Ilhan, and M. M. Herbst-Kralovetz, The microbiome and gynaecological cancer development, prevention and therapy. NatRev Urol, 2020. 17(4): p. 232-250. 10. Tsay, JJ, et al., Lower Airway Dysbiosis Affects Lung Cancer Progression. Cancer Discov, 2021. 11(2): p. 293-307. 11. Peters, B. A., et al., The lung microbiome, peripheral gene expression, and recurrence-free survival after resection of stage II non- small cell lung cancer. Genome Med, 2022. 14(1): p. 121. 12. Segal, L. N. and M. J. Blaser, A brave new world: the lung microbiota in an era of change. Ann Am Thorac Soc, 2014. 11 Suppl 1(Suppl 1):p. S21-7. 13. Gowing, S. D., et al., Gram-positive pneumonia augments non-small cell lung cancer metastasis via host toll-like receptor 2 activation. Int JCancer, 2017. 141(3): p. 561-571. 14. Liu, N.N., et al., Microbiome dysbiosis in lung cancer: from composition to therapy. NPJ Precis Oncol, 2020. 4(1): p. 33. 15. Greathouse, K.L., et al., Interaction between the microbiome and TP53 in human lung cancer. Genome Biol, 2018. 19(1): p. 123. 16. Le Noci, V., et al., Modulation of Pulmonary Microbiota by Antibiotic or Probiotic Aerosol Therapy: A Strategy to Promote Immunosurveillance against Lung Metastases. Cell Rep, 2018. 24(13): p. 3528-3538. 17. Wang, Y., et al., Staphylococcus aureus induces COX-2-dependent proliferation and malignant transformation in oral keratinocytes. J OralMicrobiol, 2019. 11(1): p. 1643205. 18. Sakamoto, R., et al., Existence of Staphylococcus aureus correlates with the progression of extramammary Paget's disease: potential involvement of interleukin-17 and M2-like macrophage polarization. Eur JDermatol, 2021. 31(1): p. 48-54. 19. Heim, CE, et al., Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection. Nat Microbiol, 2020. 5(10): p. 1271-1284. 20. Ippolito, L., et al., Lactate: A Metabolic Driver in the Tumour Landscape. Trends Biochem Sci, 2019. 44(2): p. 153-166. 21. Zhang, D., et al., Metabolic regulation of gene expression by histone lactylation. Nature, 2019. 574(7779): p. 575-580. 22. Yu, J., et al., Histone lactylation drives oncogenesis by facilitating m(6)A reader protein YTHDF2 expression in ocular melanoma. Genome Biol, 2021. 22(1): p. 85.

Claims

1. Use of a lactate transporter inhibitor or a nucleic acid molecule that inhibits lactate production in Staphylococcus aureus in the preparation of a drug or a kit for treating a tumor infected by Staphylococcus aureus.

2. The use according to claim 1, wherein The sequences of the nucleic acid molecules that inhibit lactic acid production in Staphylococcus are shown in SEQ ID NOs: 1-2, 7-8.

3. The use according to claim 1, wherein The staphylococcus is Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ).

4. The use according to claim 1, wherein The lactate transporter is MCT-1.

5. The use according to claim 1 or 4, wherein The lactate transporter inhibitor includes a small molecule compound or an antibody targeting the lactate transporter.

6. The use according to claim 1, wherein The tumor is a solid tumor, including but not limited to lung cancer, esophageal cancer, gastric cancer, colorectal cancer, melanoma, liver cancer, breast cancer, prostate cancer, fibrosarcoma, bladder sarcoma or glioma.

7. A method for inhibiting metastasis of tumor cells infected with Staphylococcus aureus, comprising knocking out a lactic acid production gene in Staphylococcus aureus LqCy and Dh Alternatively, a lactate transporter inhibitor is administered to the tumor cells, wherein the method is for non-therapeutic purposes.

8. The method according to claim 7, wherein the staphylococcus is Staphylococcus nepalensis ( S. nepalensis ) or Staphylococcus capitis ( S. capitis ).

9. The method according to claim 7, wherein the lactic acid production gene LqCy and Dh It was knocked out using Crisper / cas9 technology.

10. The method according to claim 9, wherein: The primers used in the Crisper / cas9 are shown in SEQ ID NOs: 1-2, 7-8, respectively.

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